Fungal spore fragmentation as a function of airflow rates and fungal generation methods

The aim of this study was to characterise and quantify the fungal fragment propagules derived and released from several fungal species (Penicillium, Aspergillus niger and Cladosporium cladosporioides) using different generation methods and different air velocities over the colonies. Real time fungal spore fragmentation was investigated using an Ultraviolet Aerodynamic Particle Sizer (UVASP) and a Scanning Mobility Particle Sizer (SMPS). The study showed that there were significant differences (p < 0.01) in the fragmentation percentage between different air velocities for the three generation methods, namely the direct, the fan and the fungal spore source strength tester (FSSST) methods. The percentage of fragmentation also proved to be dependant on fungal species. The study found that there was no fragmentation for any of the fungal species at an air velocity ≤ 0.4 m/s for any method of generation. Fluorescent signals, as well as mathematical determination also showed that the fungal fragments were derived from spores. Correlation analysis showed that the number of released fragments measured by the UVAPS under controlled conditions can be predicted on the basis of the number of spores, for Penicillium and Aspergillus niger, but not for Cladosporium cladosporioides. The fluorescence percentage of fragment samples was found to be significantly different to that of non-fragment samples (p < 0.0001) and the fragment sample fluorescence was always less than that of the non-fragment samples. Size distribution and concentration of fungal fragment particles were investigated qualitatively and quantitatively, by both UVAPS and SMPS, and it was found that the UVAPS was more sensitive than the SMPS for measuring small sample concentrations, and the results obtained from the UVAPS and SMAS were not identical for the same samples.

Real time detectionof airborne fungal spores and investigations into their dynamics in indoor air

Concern regarding the health effects of indoor air quality has grown in recent years, due to the increased prevalence of many diseases, as well as the fact that many people now spend most of their time indoors. While numerous studies have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly understood and very few studies have focused on fungal spore dynamics in indoor environments. Consequently, this work investigated the dynamics of fungal spores in indoor air, including fungal spore release and deposition, as well as investigating the mechanisms involved in the fungal spore fragmentation process. In relation to the investigation of fungal spore dynamics, it was found that the deposition rates of the bioaerosols (fungal propagules) were in the same range as the deposition rates of nonbiological particles and that they were a function of their aerodynamic diameters. It was also found that fungal particle deposition rates increased with increasing ventilation rates. These results (which are reported for the first time) are important for developing an understanding of the dynamics of fungal spores in the air. In relation to the process of fungal spore fragmentation, important information was generated concerning the airborne dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation. The results obtained from these investigations into the dynamics of fungal propagules in indoor air significantly advance knowledge about the fate of fungal propagules in indoor air, as well as their deposition in the respiratory tract. The need to develop an advanced, real-time method for monitoring bioaerosols has become increasingly important in recent years, particularly as a result of the increased threat from biological weapons and bioterrorism. However, to date, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only commercially available instrument capable of monitoring and measuring viable airborne micro-organisms in real-time. Therefore (for the first time), this work also investigated the ability of the UVAPS to measure and characterise fungal spores in indoor air. The UVAPS was found to be sufficiently sensitive for detecting and measuring fungal propagules. Based on fungal spore size distributions, together with fluorescent percentages and intensities, it was also found to be capable of discriminating between two fungal spore species, under controlled laboratory conditions. In the field, however, it would not be possible to use the UVAPS to differentiate between different fungal spore species because the different micro-organisms present in the air may not only vary in age, but may have also been subjected to different environmental conditions. In addition, while the real-time UVAPS was found to be a good tool for the investigation of fungal particles under controlled conditions, it was not found to be selective for bioaerosols only (as per design specifications). In conclusion, the UVAPS is not recommended for use in the direct measurement of airborne viable bioaerosols in the field, including fungal particles, and further investigations into the nature of the micro-organisms, the UVAPS itself and/or its use in conjunction with other conventional biosamplers, are necessary in order to obtain more realistic results. Overall, the results obtained from this work on airborne fungal particle dynamics will contribute towards improving the detection capabilities of the UVAPS, so that it is capable of selectively monitoring and measuring bioaerosols, for which it was originally designed. This work will assist in finding and/or improving other technologies capable of the real-time monitoring of bioaerosols. The knowledge obtained from this work will also be of benefit in various other bioaerosol applications, such as understanding the transport of bioaerosols indoors.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

In Vitro and In Vivo Validation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction

We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.

Development and characterization of fluorescent pH sensors based on porous silica and hydrogel support matrices

The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes. The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes. Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm). Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".

Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α TubulinV⃞

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

New percentage body fat prediction equations for Japanese females

Anthropometry is a simple and cost-efficient method for the assessment of body composition. However prediction equations to estimate body composition using anthropometry should be ‘population-specific’. Most popular body composition prediction equations for Japanese females were proposed more than 40 years ago and there is some concern regarding their usefulness in Japanese females living today. The aim of this study was to compare percentage body fat (%BF) estimated from anthropometry and dual energy x-ray absorptiometry (DXA) to examine the applicability of commonly used prediction equations in young Japanese females. Body composition of 139 Japanese females aged between 18 and 27 years of age (BMI range: 15.1–29.1 kg/m2) was measured using whole-body DXA (Lunar DPX-LIQ) scans. From anthropometric measurements %BF was estimated using four equations developed from Japanese females. The results showed that the traditionally employed prediction equations for anthropometry significantly (p<0.01) underestimate %BF of young Japanese females and therefore are not valid for the precise estimation of body composition. New %BF prediction equations were proposed from the DXA and anthropometry results. Application of the proposed equations may assist in more accurate assessment of body fatness in Japanese females living today.

A class E resonant inverter for use as electronic fluorescent lamp ballast

Class E Resonant Inverters are theoretically capable of delivering any power to a load and achieve 100% efficiency at any frequency of operation. In practice efficiency in the “high 90's” can be achieved into megahertz frequencies regardless of inverter output powers. The topology also allows the manipulation of output power through sub-optimal operation, with a negligible efficiency penalty. The 24W inverter discussed in this paper was specifically designed to harness the benefits, and discuss the shortcomings, of the Class E topology for use in the growing market for portable, battery powered lighting. It exhibits a peak recorded power efficiently of over 98%, and a conservatively measured efficiency of 95% across a range of dimming settings.

Associations of body mass index and waist circumference with: energy intake and percentage energy from macronutrients, in a cohort of Australian children

Background It is evident from previous research that the role of dietary composition in relation to the development of childhood obesity remains inconclusive. Several studies investigating the relationship between body mass index (BMI), waist circumference (WC) and/or skin fold measurements with energy intake have suggested that the macronutrient composition of the diet (protein, carbohydrate, fat) may play an important contributing role to obesity in childhood as it does in adults. This study investigated the possible relationship between BMI and WC with energy intake and percentage energy intake from macronutrients in Australian children and adolescents. Methods Height, weight and WC measurements, along with 24 h food and drink records (FDR) intake data were collected from 2460 boys and girls aged 5-17 years living in the state of Queensland, Australia. Results Statistically significant, yet weak correlations between BMI z-score and WC with total energy intake were observed in grades 1, 5 and 10, with only 55% of subjects having a physiologically plausible 24 hr FDR. Using Pearson correlations to examine the relationship between BMI and WC with energy intake and percentage macronutrient intake, no significant correlations were observed between BMI z-score or WC and percentage energy intake from protein, carbohydrate or fat. One way ANOVAs showed that although those with a higher BMI z-score or WC consumed significantly more energy than their lean counterparts. Conclusion No evidence of an association between percentage macronutrient intake and BMI or WC was found. Evidently, more robust longitudinal studies are needed to elucidate the relationship linking obesity and dietary intake.