Improved Production of Induced Pluripotent Stem Cells Using Dot Pattern Culture Plates.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

Identifying robust and reliable volatile organic compounds in human sebum for biomarker discovery.

Sebum from sebaceous glands is a rich source of volatile organic compounds (VOCs) that can readily be sampled non-invasively from the surface of skin. The VOC profiles of sebum can then be used to obtain information regarding different medical conditions including diabetes and Parkinson's Disease. However, the effects of sampling approaches and environmental factors on sebum VOC profiles are not established and the confident attribution of VOCs to disease states needs to be free of extraneous influences such as sampling materials and preparatory conditions. Here, we investigated a more standardised skin swab sampling approach for profiling sebum VOCs from healthy human subjects using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Using a standard GC-MS method for the chemical analysis of sebum swabs, a surprisingly high number of VOCs originate from 'blank' medical swab material alone (up to 74 VOCs) and from the ambient environment (up to 29 VOCs) based on control experiments. We found that heat-treatment of medical swabs prior to GC-MS reduced the number of VOCs detected from 'blank' swabs and improved the reproducibility of VOC profiling, however significant VOC absorption can still occur from environmental exposure to ambient air. VOCs identified in 'blank' swabs consisted predominantly of hydrocarbons, esters, and silicon-based compounds and depended strongly on the material used (cotton and polyester-rayon). Environmental VOCs found to absorb to swabs from the ambient air during sampling included 1-butylheptyl-benzene and hexadecanoic acid methyl ester as well as exogenous VOCs such as isopropyl palmitate and isopropyl myristate. In contrast, sebum VOCs consisted primarily of esters, alcohols, ketones, and aldehydes. 23 and 18 VOCs were identified in sebum collected using polyester-rayon and cotton-based medical swabs, respectively, with 14 VOCs common to both swabs. The effect of subject bathing prior to sebum sampling had minimal impact on the VOC profiles. However, individual differences owing to external factors such as skin type, diet, and exercise will likely influence sebum production. This study highlights the importance of using rigorous controls in sebum sampling, and recommendations are provided for future research involving sebum VOC analysis. For example, the use of sebum sample replicates across multiple days, and the use of control swabs during sample collection is required to confirm the origin and reliability of sebum VOCs. It is anticipated that these recommendations in conjunction with a library of well-established VOCs from medical swabs will further strengthen biomarker identification resulting from sebum VOC analysis.

A versatile toolbox for studying cortical physiology in primates.

Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments. MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

Importance of 2,4,6-Trichloroanisole (TCA) as an odorant in the emissions from anaerobically stabilized dewatered biosolids.

Odours from stabilized biosolids after anaerobic digestion of wastewater sludge can cause local community impact. Apart from the well-known odorants such as sulfur compounds, contributions from other volatile organic compounds (VOCs) to nuisance odours is limited. The presence of compounds with low odour detection thresholds (ODTs) at low concentrations, can present challenges for analytical identification. Thirty-six biosolids samples were taken after anaerobic stabilisation and dewatering at a wastewater treatment plant, Sydney, Australia. Biosolid cake samples were stored outside in loosely covered trays under aerobic conditions, however without interactions with soil microorganisms as it would be in reality. All biosolids cake samples were analysed over a period of 35 days. Emissions were collected onto Tenax TA sorbent tubes using a U.S. EPA flux hood method at storage days 1, 3, 7, 10, 14, 21 and 35. Gas chromatography (GC) coupled with mass spectrometer detector (MSD) and an olfactory detection port (ODP) was used to identify a musty/moldy/earthy type odorant in the biosolids emissions as 2,4,6-trichloroanisole (TCA). Measured odour intensities, classified on a scale from 1 to 4, and odour characters were specified by three ODP assessors. TCA was identified in all biosolid cake emissions. The measured odour intensities of the TCA did not significantly alter as the biosolids were aged, however varied between biosolids cakes. Due to its odour intensity, 85% frequency of detection and its low ODT, which is orders of magnitudes lower than sulfur compounds, TCA should be considered as a potential odorant of concern in biosolids emissions.

Emissions of volatile sulfur compounds (VSCs) throughout wastewater biosolids processing.

Volatile sulfur compounds (VSCs) are important contributors to nuisance odours from the processing of wastewater sludge and biosolids. However, emission characteristics are difficult to predict as they vary between sites and are likely to be affected by biosolids processing configuration and operation. VSC emissions from biosolids throughout 6 wastewater treatment plants (WWTPs) in Sydney, Australia were examined in this study. H2S was the VSC found at the highest concentrations throughout the WWTPs, with concentrations ranging from 7 to 39,000µg/m3. Based on odour activity values (OAVs), H2S was typically also the most dominant odorant. However, methyl mercaptan (MeSH) was also found to be sensorially important in the biosolids storage areas given its low odour detection threshold (ODT). High concentrations of VOSCs such as MeSH in the storage areas were shown to potentially interfere with H2S measurements using the Jerome 631-X H2S sensor and these interferences should be investigated in more detail. The VSC composition of emissions varied throughout biosolids processing as well as between the different WWTPs. The primary sludge and biosolids after dewatering and during storage, were key stages producing nuisance odours as judged by the determination of OAVs. Cluster analysis was used to group sampling locations according to VSC emissions. These groups were typically the dewatered and stored biosolids, primary and thickened primary sludge, and waste activated sludge (WAS), thickened WAS, digested sludge and centrate. Effects of biosolids composition and process operation on VSC emissions were evaluated using best subset regression. Emissions from the primary sludge were dominated by H2S and appeared to be affected by the presence of organic matter, pH and Fe content. While volatile organic sulfur compounds (VOSCs) emitted from the produced biosolids were shown to be correlated with upstream factors such as Fe and Al salt dosing, anaerobic digestion and dewatering parameters.

Distribution and sensorial relevance of volatile organic compounds emitted throughout wastewater biosolids processing.

A diverse range of volatile organic compounds (VOCs) are emitted from wastewater biosolids processing. Odorous emissions are predominately made up of volatile sulfur compounds (VSCs) which are typically the only odorants measured. However, a range of VOCs are known to contribute to malodours yet previous studies often overlook the contribution of VOCs in comparison with VSCs. This study aims to evaluate how emissions are affected by different biosolids processing configurations, and if any non-sulfur VOCs should be included in odour measurement and management. Non-sulfur VOCs emitted from biosolids throughout six wastewater treatment plants in the Sydney, Australia region were measured at six locations on average twice each week over 2-3weeks at each site. Variations in types of VOCs emitted throughout and between the sites were assigned to differences in WWTP processing configurations, plant operation and variations in industrial and municipal flows to the sewer network, referred to as sewer catchments. The presence of VOCs is likely due to biotic generation as well as industrial or residential additions to the sewer network. The dewatered and stored biosolids samples had the highest levels of VOC emissions. Sensorially important odorants were p-cresol and butanoic acid, based on the frequency of detection and odour activity values. Other compounds with a high risk of nuisance impacts were trimethylamine, indole and phenol emitted from the dewatered and stored biosolids, and volatile fatty acids from the anaerobic digester inlet and outlet at one particular site. The findings show that non-sulfur VOCs should be added to odorant monitoring campaigns at WWTPs. Identification of VOCs as sensorially important odorants opens opportunities for the more efficient management of nuisance odours, through targeted odour control or process improvement.

Odorous volatile organic compound (VOC) emissions from ageing anaerobically stabilised biosolids.

Opportunities for the beneficial re-use of biosolids are limited by nuisance odour emissions. Volatile organic compounds (VOCs) from anaerobically stabilised biosolids were measured to identify compounds that could contribute to the overall odour character of nuisance emissions. Flux hood sampling and chemical analysis were used to identify VOCs emitted from biosolids as they were stored in ambient conditions. Compounds emitted varied as the biosolid cakes were stored for a period of 50 days. VOCs detected in the biosolids are likely to occur from catchment sources as well as abiotic and biotic generation in the wastewater processing and the biosolids as they are stored. Odour activity values (OAVs) were used to compare odorants. Trimethylamine was the only VOC detected that exceeded the sulfur compounds in terms of OAVs. Other compounds such as limonene, ethyl methyl benzene and acetic acid were detected at concentrations exceeding their olfactory detection limits, however at lower OAVs than sulfur compounds.

Fate of antibiotics during municipal water recycling treatment processes.

Municipal water recycling processes are potential human and environmental exposure routes for low concentrations of persistent antibiotics. While the implications of such exposure scenarios are unknown, concerns have been raised regarding the possibility that continuous discharge of antibiotics to the environment may facilitate the development or proliferation of resistant strains of bacteria. As potable and non-potable water recycling schemes are continuously developed, it is imperative to improve our understanding of the fate of antibiotics during conventional and advanced wastewater treatment processes leading to high-quality water reclamation. This review collates existing knowledge with the aim of providing new insight to the influence of a wide range of treatment processes to the ultimate fate of antibiotics during conventional and advanced wastewater treatment. Although conventional biological wastewater treatment processes are effective for the removal of some antibiotics, many have been reported to occur at 10-1000 ng L(-1) concentrations in secondary treated effluents. These include beta-lactams, sulfonamides, trimethoprim, macrolides, fluoroquinolones, and tetracyclines. Tertiary and advanced treatment processes may be required to fully manage environmental and human exposure to these contaminants in water recycling schemes. The effectiveness of a range of processes including tertiary media filtration, ozonation, chlorination, UV irradiation, activated carbon adsorption, and NF/RO filtration has been reviewed and, where possible, semi-quantitative estimations of antibiotics removals have been provided.

The application of membrane bioreactors as decentralised systems for removal of endocrine disrupting chemicals and pharmaceuticals.

The concentrations of some important endocrine disrupting chemicals and pharmaceuticals after various stages of wastewater treatment were investigated. The endocrine disrupting chemicals included natural and synthetic estrogenic and androgenic steroids. The pharmaceuticals included a series of sulfonamide antibiotics and trimethoprim. The removal efficiency of a membrane bioreactor (MBR) was investigated and compared with a conventional activated sludge (CAS) system. Samples were analysed by liquid chromatography tandem mass spectrometry. Results showed that the MBR and CAS systems effectively removed steroidal estrogens and androgens, but only partially eliminated the target antibiotics from wastewater. The MBR was shown to be more effective than the CAS system which was possibly attributed to the high solid retention time and concentration of biosolids in the MBR. The results highlight the potential wider application of MBRs for the removal of trace chemical contaminants in wastewater and their potential for use as decentralised wastewater treatment systems.

Fate and levels of steroid oestrogens and androgens in waste stabilisation ponds: quantification by liquid chromatography-tandem mass spectrometry.

The capacity for removing wastewater-borne endocrine disrupting chemicals (EDCs) was investigated for two wastewater treatment plants (WWTPs) incorporating waste stabilisation ponds (WSPs) as the principal treatment technology. Samples were analysed for a number of steroidal oestrogens and androgens using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Removal efficiency for steroid androgens was high for both WWTPs (93-100%) but WSP treatment was observed to be less effective for removing steroid oestrogens, particularly oestriol.

Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators.

The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered feeding time.

Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.

In mammals, circadian oscillators exist not only in the suprachiasmatic nucleus, which harbors the central pacemaker, but also in most peripheral tissues. It is believed that the SCN clock entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Here we show that temporal feeding restriction under light-dark or dark-dark conditions can change the phase of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of cyclic gene expression in the SCN unaffected. Hence, changes in metabolism can lead to an uncoupling of peripheral oscillators from the central pacemaker. Sudden large changes in feeding time, similar to abrupt changes in the photoperiod, reset the phase of rhythmic gene expression gradually and are thus likely to act through a clock-dependent mechanism. Food-induced phase resetting proceeds faster in liver than in kidney, heart, or pancreas, but after 1 wk of daytime feeding, the phases of circadian gene expression are similar in all examined peripheral tissues.