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1.  Exploring the Genetic Basis of Adaptation to High Elevations in Reptiles: A Comparative Transcriptome Analysis of Two Toad-Headed Agamas (Genus Phrynocephalus) 
PLoS ONE  2014;9(11):e112218.
High elevation adaptation offers an excellent study system to understand the genetic basis of adaptive evolution. We acquired transcriptome sequences of two closely related lizards, Phrynocephalus przewalskii from low elevations and P. vlangalii from high elevations. Within a phylogenetic framework, we compared their genomic data along with green anole, chicken and Chinese softshell turtle, and identified candidate genes and functional categories that are potentially linked to adaptation to high elevation environments. More than 100 million sequence reads were generated for each species via Illumina sequencing. A de novo assembly produced 70,919 and 62,118 transcripts for P. przewalskii and P. vlangalii, respectively. Based on a well-established reptile phylogeny, we detected 143 positively selected genes (PSGs) along the P. vlangalii lineage from the 7,012 putative orthologs using a branch-site model. Furthermore, ten GO categories and one KEGG pathway that are over-represented by PSGs were recognized. In addition, 58 GO categories were revealed to have elevated evolutionary rates along the P. vlangalii lineage relative to P. przewalskii. These functional analyses further filter out PSGs that are most likely involved in the adaptation process to high elevations. Among them, ADAM17, MD, and HSP90B1 likely contributed to response to hypoxia, and POLK likely contributed to DNA repair. Many other candidate genes involved in gene expression and metabolism were also identified. Genome-wide scan for candidate genes may serve as the first step to explore the genetic basis of high elevation adaptation. Detailed comparative study and functional verification are needed to solidify any conclusions. High elevation adaptation requires coordinated changes in multiple genes that involve various physiological and biochemical pathways; we hope that our genetic studies will provide useful directions for future physiological or molecular studies in reptiles as well as other poikilothermic species.
PMCID: PMC4227734  PMID: 25386640
2.  Thermal porosity analysis of croscarmellose sodium and sodium starch glycolate by differential scanning calorimetry 
AAPS PharmSciTech  2003;4(4):531-538.
The aim of the study was to demonstrate the applicability of differential scanning calorimetry (DSC) on porosity analysis for cellulose and starch. Croscarmellose sodium (CCS) and sodium starch glycolate (SSG) were allowed to sorb moisture in 85%, 90%, 95%, and 100% relative humidity (RH) at 40°C for 24 hours. The pretreated samples were then subjected to DSC running temperature ranging from 25°C to −50°C at a cooling rate of 10°C/min. The cooling traces of water crystallization, if present, were transformed to porosity distribution via capillary condensation using Kelvin's equation. The porosity analysis of CCS and SSG was also done using nitrogen adsorption as a reference method. It was found that sorbed water could not be frozen (in cases of 85% and 90% RH) until the moisture content exceeded a cutoff value (in cases of 95% and 100% RH). The nonfreezable moisture content was referred to tightly bound, plasticizing water, whereas the frozen one may be attributed to loosely bound water condensation in pore structure of CCS and SSG surfaces. Not only capillary condensation but also the tightly bound, nonfreezable monolayer water lying along the inner pores of the surface contributed to porosity determination. Good agreement with less than 5% deviation of mean pore size was observed when the results were compared with nitrogen adsorption. The narrower pore size distributions, however, were obtained because of the limitations of the technique. It was concluded that pore analysis by DSC could be successful. Further research needs to be done to account for limitations and to extend the applicability of the technique.
PMCID: PMC2750660  PMID: 15198562
thermoporometry; differential scanning calorimetry (DSC); croscarmellose sodium (CCS); sodium starch glycolate (SSG)
3.  "Reticular" and "Areticular" Nissl Bodies in Sympathetic Neurons of a Lizard 
Sympathetic ganglia of the horned lizard, Phrynosoma cornutum, were fixed in OsO4 and imbedded in methacrylate. Thin sections were cut for electron microscopy. Some adjacent thick sections were cut for light microscopy and were stained in acidified, dilute thionine both before and after digestion by RNase. In the light microscope two types of Nissl bodies are found, both removable by RNase: (1) a deep, diffuse, indistinctly bounded, metachromatic variety, and (2) a superficial, dense, sharply delimited, orthochromatic sort. Electron microscopically, the former ("reticular" Nissl bodies) corresponds to the granulated endoplasmic reticular structure of Nissl material previously described by others, whereas the latter ("areticular" Nissl bodies) comprises compact masses of particles of varying internal density and devoid of elements of endoplasmic reticulum. The constituent particles of the areticular Nissl material are 4 to 8 x the diameter of single ribonucleoprotein granules of the reticular Nissl substance and seem, near zones of junction with the reticular type, to arise by clustering of such granules with subsequent partial dispersion of the substance of the granules into an added, less dense material. It is suggested that the observed orthochromasia of the areticular Nissl substance is due to accumulation of a large amount of protein bound to RNA and, further, that these Nissl bodies may represent storage depots of RNA and protein.
PMCID: PMC2229759  PMID: 13673051
4.  Scaling up nanoscale water-driven energy conversion into evaporation-driven engines and generators 
Nature Communications  2015;6:7346.
Evaporation is a ubiquitous phenomenon in the natural environment and a dominant form of energy transfer in the Earth's climate. Engineered systems rarely, if ever, use evaporation as a source of energy, despite myriad examples of such adaptations in the biological world. Here, we report evaporation-driven engines that can power common tasks like locomotion and electricity generation. These engines start and run autonomously when placed at air–water interfaces. They generate rotary and piston-like linear motion using specially designed, biologically based artificial muscles responsive to moisture fluctuations. Using these engines, we demonstrate an electricity generator that rests on water while harvesting its evaporation to power a light source, and a miniature car (weighing 0.1 kg) that moves forward as the water in the car evaporates. Evaporation-driven engines may find applications in powering robotic systems, sensors, devices and machinery that function in the natural environment.
Harvesting energy from evaporation is constrained by the limited transport kinetics of materials and the slowly changing humidity of the environment. Chen et al. follow hierarchical design strategies to overcome these problems and create engines that start and run when placed at air-water interfaces.
PMCID: PMC4490384  PMID: 26079632
5.  Food and water security issues in Russia II: Water security in general population of Russian Arctic, Siberia and Far East, 2000–2011 
International Journal of Circumpolar Health  2013;72:10.3402/ijch.v72i0.22646.
Poor state of water supply systems, shortage of water purification facilities and disinfection systems, low quality of drinking water generally in Russia and particularly in the regions of the Russian Arctic, Siberia and Far East have been defined in the literature. However, no standard protocol of water security assessment has been used in the majority of studies.
Study design and methods
Uniform water security indicators collected from Russian official statistical sources for the period 2000–2011 were used for comparison for 18 selected regions in the Russian Arctic, Siberia and Far East. The following indicators of water security were analyzed: water consumption, chemical and biological contamination of water reservoirs of Categories I and II of water sources (centralized – underground and surface, and non-centralized) and of drinking water.
Water consumption in selected regions fluctuated from 125 to 340 L/person/day. Centralized water sources (both underground and surface sources) are highly contaminated by chemicals (up to 40–80%) and biological agents (up to 55% in some regions), mainly due to surface water sources. Underground water sources show relatively low levels of biological contamination, while chemical contamination is high due to additional water contamination during water treatment and transportation in pipelines. Non-centralized water sources are highly contaminated (both chemically and biologically) in 32–90% of samples analyzed. Very high levels of chemical contamination of drinking water (up to 51%) were detected in many regions, mainly in the north-western part of the Russian Arctic. Biological contamination of drinking water was generally much lower (2.5–12%) everywhere except Evenki AO (27%), and general and thermotolerant coliform bacteria predominated in drinking water samples from all regions (up to 17.5 and 12.5%, correspondingly). The presence of other agents was much lower: Coliphages – 0.2–2.7%, Clostridia spores, Giardia cysts, pathogenic bacteria, Rotavirus – up to 0.8%. Of a total of 56 chemical pollutants analyzed in water samples from centralized water supply systems, 32 pollutants were found to be in excess of hygienic limits, with the predominant pollutants being Fe (up to 55%), Cl (up to 57%), Al (up to 43%) and Mn (up to 45%).
In 18 selected regions of the Russian Arctic, Siberia and Far East Category I and II water reservoirs, water sources (centralized – underground, surface; non-centralized) and drinking water are highly contaminated by chemical and biological agents. Full-scale reform of the Russian water industry and water security system is urgently needed, especially in selected regions.
PMCID: PMC3860331  PMID: 24350065
water security; drinking water; centralized; non-centralized water sources; chemical; biological contamination; pollutants; bacteria; spores; cysts; virus; pesticides; metals; Russian Arctic
6.  Measuring and Modeling the Effect of Surface Moisture on the Spectral Reflectance of Coastal Beach Sand 
PLoS ONE  2014;9(11):e112151.
Surface moisture is an important supply limiting factor for aeolian sand transport, which is the primary driver of coastal dune development. As such, it is critical to account for the control of surface moisture on available sand for dune building. Optical remote sensing has the potential to measure surface moisture at a high spatio-temporal resolution. It is based on the principle that wet sand appears darker than dry sand: it is less reflective. The goals of this study are (1) to measure and model reflectance under controlled laboratory conditions as function of wavelength () and surface moisture () over the optical domain of 350–2500 nm, and (2) to explore the implications of our laboratory findings for accurately mapping the distribution of surface moisture under natural conditions. A laboratory spectroscopy experiment was conducted to measure spectral reflectance (1 nm interval) under different surface moisture conditions using beach sand. A non-linear increase of reflectance upon drying was observed over the full range of wavelengths. Two models were developed and tested. The first model is grounded in optics and describes the proportional contribution of scattering and absorption of light by pore water in an unsaturated sand matrix. The second model is grounded in soil physics and links the hydraulic behaviour of pore water in an unsaturated sand matrix to its optical properties. The optical model performed well for volumetric moisture content 24% ( 0.97), but underestimated reflectance for between 24–30% ( 0.92), most notable around the 1940 nm water absorption peak. The soil-physical model performed very well ( 0.99) but is limited to 4% 24%. Results from a field experiment show that a short-wave infrared terrestrial laser scanner ( = 1550 nm) can accurately relate surface moisture to reflectance (standard error 2.6%), demonstrating its potential to derive spatially extensive surface moisture maps of a natural coastal beach.
PMCID: PMC4226492  PMID: 25383709
7.  Bacteriological assessment of urban water sources in Khamis Mushait Governorate, southwestern Saudi Arabia 
Urban water sources of Khamis Mushait Governorate, southwestern Saudi Arabia, were studied to assess their bacteriological characteristics and suitability for potable purposes. A cross-sectional epidemiological method was adopted to investigate the four main urban water sources (i.e. bottled, desalinated, surface, and well water). These were sampled and examined between February and June 2007.
A total of 95 water samples from bottled, desalinated, surface, and well water were collected randomly from the study area using different gathering and analysing techniques. The bacteriological examination of water samples included the most probable number of presumptive coliforms, faecal coliforms, and faecal streptococci (MPN/100 ml). The results showed that the total coliform count (MPN/100 ml) was not detected in any samples taken from bottled water, while it was detected in those taken from desalinated, surface, and well water: percentages were 12.9, 80.0, and 100.0, respectively. Faecal coliforms were detected in desalinated, surface, and well water, with percentages of 3.23, 60.0 and 87.88, respectively. About 6.45% of desalinated water, 53.33% of surface water, and 57.58% of well water was found positive for faecal streptococci. Colonies of coliforms were identified in different micro-organisms with various percentages.
Water derived from traditional sources (wells) showed increases in most of the investigated bacteriological parameters, followed by surface water as compared to bottled or desalinated water. This may be attributed to the fact that well and surface water are at risk of contamination as indicated by the higher levels of most bacteriological parameters. Moreover, well water is exposed to point sources of pollution such as septic wells and domestic and farming effluents, as well as to soil with a high humus content. The lower bacteriological characteristics in samples from bottled water indicate that it is satisfactory for human drinking purposes. Contamination of desalinated water that is the main urban water source may occur during transportation from the desalination plant or in the house reservoir of the consumer. Improving and expanding the existing water treatment and sanitation systems is more likely to provide safe and sustainable sources of water over the long term. Strict hygienic measures should be applied to improve water quality and to avoid deleterious effects on public health, by using periodical monitoring programmes to detect sewage pollution running over local hydrological networks and valleys.
PMCID: PMC2672944  PMID: 19302710
8.  A new device for 100 per cent humidification of inspired air 
Critical Care  2000;4(1):54-60.
A new humidifier for use during mechanical ventilation in endotracheally intubated patients is described and tested. The humidifier is based on a heat-moisture exchanger, which absorbs the expired heat and moisture and releases it into the inspired air. External heat and water are then added at the patient side of the heat-moisture exchanger, so that the inspired gas should reach 100% humidity (44 mg/l) at 37°C. In bench tests using constant and decelerating inspiratory flow and minute volumes of 3–25 l the device gave an absolute humidity of 41–44 mg/l, and it reduced the amount of water consumed in eight mechanically ventilated patients compared with a conventional active humidifier. During a 24-h test period there was no water condensation in the ventilator tubing with the new device.
Devices for active humidification of the inspired air in mechanically ventilated patients cause water condensation in the ventilator tubing, which may become contaminated or interfere with the function of the ventilator. The present study describes and tests the performance of a new humidifier, which is designed to eliminate water condensation.
To test the performance of the new humidifier at different ventilator settings in a lung model, and to compare this new humidifier with a conventional active humidifier in ventilator-treated critically ill patients.
Materials and methods:
The humidifier (Humid-Heat; Louis Gibeck AB, Upplands Väsby, Sweden) consists of a supply unit with a microprocessor and a water pump, and a humidification device, which is placed between the Y-piece and the endotracheal tube. The humidification device is based on a hygroscopic heat-moisture exchanger (HME), which absorbs the expired heat and moisture and releases it into the inspired gas. External heat and water are then added to the patient side of the HME, so the inspired gas should reach 100% humidity at 37°C (44 mg H2O/l air). The external water is delivered to the humidification device via a pump onto a wick and then evaporated into the inspired air by an electrical heater. The microprocessor controls the water pump and the heater by an algorithm using the minute ventilation (which is fed into the microprocessor) and the airway temperature measured by a sensor mounted in the flex-tube on the patient side of the humidification device.
The performance characteristics were tested in a lung model ventilated with a constant flow (inspiratory:expiratory ratio 1:2, rate 12–20 breaths/min and a minute ventilation of 3–25 l/min) or with a decelerating flow (inspiratory:expiratory ratio 1:2, rate 12–15 breaths/min and a minute ventilation of 4.7–16.4 l/min). The device was also tested prospectively and in a randomized order compared with a conventional active humidifier (Fisher & Paykel MR730, Auckland, New Zealand) in eight mechanically ventilated, endotracheally intubated patients in the intensive care unit. The test period with each device was 24 h. The amount of fluid consumed and the amount of water in the water traps were measured. The number of times that the water traps were emptied, changes of machine filters, the suctions and quality of secretions, nebulizations, and the amount of saline instillations and endotracheal tube obstruction were recorded. In order to evaluate increased expiratory resistance due to the device, the airway pressure was measured at the end of a prolonged end-expiratory pause at 1 h of use and at the end of the test, and was compared with the corresponding pressure before the experiment. The body temperature of the patient was measured before and after the test of each device.
Both with constant flow and decelerating flow, the Humid-Heat gave an absolute humidity of 41–44 mgH2O/l at 37°C, with the lower level at the highest ventilation. In the patients, both Humid-Heat and the conventional active humidifier (MR730) maintained temperatures, indicating that they provided the intended heat and moisture to the inspired air. With both devices, the body temperature was maintained during the test period. There was no difference in the amount of secretions, the quality of the secretions and the frequency of suctions, saline instillations or nebulizations between the test periods with the two devices. There was no endotracheal tube obstruction, and after 1 h of use and at the end of the test no increased airway resistance was found with either device. When the MR730 was used, however, the water traps needed to be emptied six to 14 (mean eight) times (total amount of fluid in the traps was 100–300 ml) and the machine filters were changed two to six (mean four) times due to an excessive amount of condensed water with flow obstruction. No condensation of water was found in the tubing with the Humid-Heat. The water consumption was 23–65 ml/h (mean 30 ml/h) with the MR730 and 4–8 ml/h (mean 6 ml/h) with the Humid-Heat (P < 0.0008). The same relations were found when the water consumption was corrected for differences in minute ventilation.
The new humidifier, the Humid-Heat, gave an absolute humidity of 41–44 mg/l at 37°C in the bench tests. The tests in ventilated patients showed that the device was well tolerated and that condensation in the tubing was eliminated. There was no need to empty water traps. The test period was too short to evaluate whether the new device had any other advantages or disadvantages compared with conventional humidifiers.
PMCID: PMC29037  PMID: 11056746
airway humidification; heated humidifier; intensive care; mechanical ventilation
9.  Brochosomal coats turn leafhopper (Insecta, Hemiptera, Cicadellidae) integument to superhydrophobic state 
Leafhoppers (Insecta, Hemiptera, Cicadellidae) actively coat their integuments with brochosomes, hollow proteinaceous spheres of usually 200–700 nm in diameter, with honeycombed walls. The coats have been previously suggested to act as a water-repellent and anti-adhesive protective barrier against the insect's own exudates. We estimated their wettability through contact angle (CA) measurements of water, diiodomethane, ethylene glycol and ethanol on detached wings of the leafhoppers Alnetoidia alneti, Athysanus argentarius and Cicadella viridis. Intact brochosome-coated integuments were repellent to all test liquids, except ethanol, and exhibited superhydrophobicity, with the average water CAs of 165–172°, and the apparent surface free energy (SFE) estimates not exceeding 0.74 mN m−1. By contrast, the integuments from which brochosomes were removed with a peeling technique using fluid polyvinylsiloxane displayed water CAs of only 103–129° and SFEs above 20 mN m−1. Observations of water-sprayed wings in a cryo-scanning electron microscope confirmed that brochosomal coats prevented water from contacting the integument. Their superhydrophobic properties appear to result from fractal roughness, which dramatically reduces the area of contact with high-surface-tension liquids, including, presumably, leafhopper exudates.
PMCID: PMC3574307  PMID: 23235705
brochosome; surface; insect; superhydrophobicity; wettability; contact mechanics
10.  Assessment of Evapotranspiration and Soil Moisture Content Across Different Scales of Observation 
Sensors (Basel, Switzerland)  2008;8(1):70-117.
The proper assessment of evapotranspiration and soil moisture content are fundamental in food security research, land management, pollution detection, nutrient flows, (wild-) fire detection, (desert) locust, carbon balance as well as hydrological modelling; etc. This paper takes an extensive, though not exhaustive sample of international scientific literature to discuss different approaches to estimate land surface and ecosystem related evapotranspiration and soil moisture content. This review presents: (i)a summary of the generally accepted cohesion theory of plant water uptake and transport including a shortlist of meteorological and plant factors influencing plant transpiration;(ii)a summary on evapotranspiration assessment at different scales of observation (sap-flow, porometer, lysimeter, field and catchment water balance, Bowen ratio, scintillometer, eddy correlation, Penman-Monteith and related approaches);(iii)a summary on data assimilation schemes conceived to estimate evapotranspiration using optical and thermal remote sensing; and(iv)for soil moisture content, a summary on soil moisture retrieval techniques at different spatial and temporal scales is presented.
Concluding remarks on the best available approaches to assess evapotranspiration and soil moisture content with and emphasis on remote sensing data assimilation, are provided.
PMCID: PMC3681150
Evapotranspiration; soil moisture content; plant; field; landscape - regional scales; remote sensing
11.  Effect of rainfall patterns on soil surface CO2 efflux, soil moisture, soil temperature and plant growth in a grassland ecosystem of northern Ontario, Canada: implications for climate change 
BMC Ecology  2002;2:10.
The effect of rainfall patterns on soil surface CO2 efflux, soil moisture, soil temperature and plant growth was investigated in a grassland ecosystem of northern Ontario, Canada, where climatic change is predicted to introduce new precipitation regimes. Rain shelters were established in a fallow field consisting mainly of Trifolium hybridum L., Trifolium pratense L., and Phleum pratense L. Daytime ambient air temperatures within the shelters increased by an average of 1.9°C similar to predicted future increases in air temperatures for this region. To simulate six precipitation regimes which cover the maximum range to be expected under climate change, a portable irrigation system was designed to modify the frequency of monthly rainfall events with a constant delivery rate of water, while maintaining contemporary average precipitation volumes. Controls consisted of blocks irrigated with frequencies and total monthly precipitation consistent with the 25 year average rainfall for this location.
Seasonal soil moisture correlated with soil surface CO2 efflux (R = 0.756, P < 0.001) and above ground plant biomass (R = 0.447, P = 0.029). By reducing irrigation frequency, soil surface CO2 efflux decreased by 80%, P < 0.001, while soil moisture content decreased by 42%, P < 0.001.
Manipulating the number of precipitation events and inter-rainfall intervals, while maintaining monthly rainfall averages impacted CO2 efflux and plant growth. Even with monthly rainfall averages that are similar to contemporary monthly precipitation averages, decreasing the number of monthly rainfall events reduced soil surface CO2 efflux and plant growth through soil moisture deficits. Although many have speculated that climate change will increase ecosystem productivity, our results show that a reduction in the number of monthly rainfall events while maintaining monthly averages will limit carbon dynamics.
PMCID: PMC137609  PMID: 12445327
12.  Evaluating Humidity Recovery Efficiency of Currently Available Heat and Moisture Exchangers: A Respiratory System Model Study 
Clinics (Sao Paulo, Brazil)  2009;64(6):585-590.
To evaluate and compare the efficiency of humidification in available heat and moisture exchanger models under conditions of varying tidal volume, respiratory rate, and flow rate.
Inspired gases are routinely preconditioned by heat and moisture exchangers to provide a heat and water content similar to that provided normally by the nose and upper airways. The absolute humidity of air retrieved from and returned to the ventilated patient is an important measurable outcome of the heat and moisture exchangers’ humidifying performance.
Eight different heat and moisture exchangers were studied using a respiratory system analog. The system included a heated chamber (acrylic glass, maintained at 37°C), a preserved swine lung, a hygrometer, circuitry and a ventilator. Humidity and temperature levels were measured using eight distinct interposed heat and moisture exchangers given different tidal volumes, respiratory frequencies and flow-rate conditions. Recovery of absolute humidity (%RAH) was calculated for each setting.
Increasing tidal volumes led to a reduction in %RAH for all heat and moisture exchangers while no significant effect was demonstrated in the context of varying respiratory rate or inspiratory flow.
Our data indicate that heat and moisture exchangers are more efficient when used with low tidal volume ventilation. The roles of flow and respiratory rate were of lesser importance, suggesting that their adjustment has a less significant effect on the performance of heat and moisture exchangers.
PMCID: PMC2705149  PMID: 19578664
Heat and moisture exchangers; Humidity; Temperature; Mechanical ventilation
13.  Influence of Surface Characteristics on the Stability of Cryptosporidium parvum Oocysts 
Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.
PMCID: PMC165138  PMID: 12839749
14.  Microbiological Aspects of Ethylene Oxide Sterilization 
Applied Microbiology  1970;19(1):157-162.
An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 ± 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed.
PMCID: PMC376629  PMID: 4984365
15.  Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation 
BMC Biotechnology  2010;10:57.
Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.
First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT) under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.
Water sorption leads to aggregation, inactivation, and structural changes of α-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.
PMCID: PMC2924255  PMID: 20696067
16.  Three-Dimensionally Preserved Integument Reveals Hydrodynamic Adaptations in the Extinct Marine Lizard Ectenosaurus (Reptilia, Mosasauridae) 
PLoS ONE  2011;6(11):e27343.
The physical properties of water and the environment it presents to its inhabitants provide stringent constraints and selection pressures affecting aquatic adaptation and evolution. Mosasaurs (a group of secondarily aquatic reptiles that occupied a broad array of predatory niches in the Cretaceous marine ecosystems about 98–65 million years ago) have traditionally been considered as anguilliform locomotors capable only of generating short bursts of speed during brief ambush pursuits. Here we report on an exceptionally preserved, long-snouted mosasaur (Ectenosaurus clidastoides) from the Santonian (Upper Cretaceous) part of the Smoky Hill Chalk Member of the Niobrara Formation in western Kansas, USA, that contains phosphatized remains of the integument displaying both depth and structure. The small, ovoid neck and/or anterior trunk scales exhibit a longitudinal central keel, and are obliquely arrayed into an alternating pattern where neighboring scales overlap one another. Supportive sculpturing in the form of two parallel, longitudinal ridges on the inner scale surface and a complex system of multiple, superimposed layers of straight, cross-woven helical fiber bundles in the underlying dermis, may have served to minimize surface deformation and frictional drag during locomotion. Additional parallel fiber bundles oriented at acute angles to the long axis of the animal presumably provided stiffness in the lateral plane. These features suggest that the anterior torso of Ectenosaurus was held somewhat rigid during swimming, thereby limiting propulsive movements to the posterior body and tail.
PMCID: PMC3217950  PMID: 22110629
17.  Ca2+ cycling in cardiomyocytes from a high-performance reptile, the varanid lizard (Varanus exanthematicus) 
The varanid lizard possesses one of the largest aerobic capacities among reptiles with maximum rates of oxygen consumption that are twice that of other lizards of comparable sizes at the same temperature. To support this aerobic capacity, the varanid heart possesses morphological adaptations that allow the generation of high heart rates and blood pressures. Specializations in excitation-contraction coupling may also contribute to the varanids superior cardiovascular performance. Therefore, we investigated the electrophysiological properties of the l-type Ca2+ channel and the Na+/Ca2+ exchanger (NCX) and the contribution of the sarcoplasmic reticulum to the intracellular Ca2+ transient (Δ[Ca2+]i) in varanid lizard ventricular myocytes. Additionally, we used confocal microscopy to visualize myocytes and make morphological measurements. Lizard ventricular myocytes were found to be spindle-shaped, lack T-tubules, and were ∼190 μm in length and 5–7 μm in width and depth. Cardiomyocytes had a small cell volume (∼2 pL), leading to a large surface area-to-volume ratio (18.5), typical of ectothermic vertebrates. The voltage sensitivity of the l-type Ca2+ channel current (ICa), steady-state activation and inactivation curves, and the time taken for recovery from inactivation were also similar to those measured in other reptiles and teleosts. However, transsarcolemmal Ca2+ influx via reverse mode Na+/Ca2+ exchange current was fourfold higher than most other ectotherms. Moreover, pharmacological inhibition of the sarcoplasmic reticulum led to a 40% reduction in the Δ[Ca2+]i amplitude, and slowed the time course of decay. In aggregate, our results suggest varanids have an enhanced capacity to transport Ca2+ through the Na+/Ca2+ exchanger, and sarcoplasmic reticulum suggesting specializations in excitation-contraction coupling may provide a means to support high cardiovascular performance.
PMCID: PMC2803631  PMID: 19812356
sarcoplasmic reticulum; electrophysiology; excitation-contraction coupling; l-type Ca channel; Na+/Ca2+ exchanger
18.  Colonization patterns of soil microbial communities in the Atacama Desert 
Microbiome  2013;1:28.
The Atacama Desert is one of the driest deserts in the world and its soil, with extremely low moisture, organic carbon content, and oxidizing conditions, is considered to be at the dry limit for life.
Analyses of high throughput DNA sequence data revealed that bacterial communities from six geographic locations in the hyper-arid core and along a North-South moisture gradient were structurally and phylogenetically distinct (ANOVA test for observed operating taxonomic units at 97% similarity (OTU0.03), P <0.001) and that communities from locations in the hyper-arid zone displayed the lowest levels of diversity. We found bacterial taxa similar to those found in other arid soil communities with an abundance of Rubrobacterales, Actinomycetales, Acidimicrobiales, and a number of families from the Thermoleophilia. The extremely low abundance of Firmicutes indicated that most bacteria in the soil were in the form of vegetative cells. Integrating molecular data with climate and soil geochemistry, we found that air relative humidity (RH) and soil conductivity significantly correlated with microbial communities’ diversity metrics (least squares linear regression for observed OTU0.03 and air RH and soil conductivity, P <0.001; UniFrac PCoA Spearman’s correlation for air RH and soil conductivity, P <0.0001), indicating that water availability and salt content are key factors in shaping the Atacama soil microbiome. Mineralization studies showed communities actively metabolizing in all soil samples, with increased rates in soils from the southern locations.
Our results suggest that microorganisms in the driest soils of the Atacama Desert are in a state of stasis for most of the time, but can potentially metabolize if presented with liquid water for a sufficient duration. Over geological time, rare rain events and physicochemical factors potentially played a major role in selecting micro-organisms that are most adapted to extreme desiccating conditions.
PMCID: PMC3971613  PMID: 24451153
Soil microbial communities; Extreme environment; Arid soil; Atacama Desert; Desertification; High-throughput 16S rRNA sequencing
19.  Geographical Variation in Body Size and Sexual Size Dimorphism in an Australian Lizard, Boulenger's Skink (Morethia boulengeri) 
PLoS ONE  2014;9(10):e109830.
Ecogeographical rules help explain spatial and temporal patterns in intraspecific body size. However, many of these rules, when applied to ectothermic organisms such as reptiles, are controversial and require further investigation. To explore factors that influence body size in reptiles, we performed a heuristic study to examine body size variation in an Australian lizard, Boulenger's Skink Morethia boulengeri from agricultural landscapes in southern New South Wales, south-eastern Australia. We collected tissue and morphological data on 337 adult lizards across a broad elevation and climate gradient. We used a model-selection procedure to determine if environmental or ecological variables best explained body size variation. We explored the relationship between morphology and phylogenetic structure before modeling candidate variables from four broad domains: (1) geography (latitude, longitude and elevation), (2) climate (temperature and rainfall), (3) habitat (vegetation type, number of logs and ground cover attributes), and (4) management (land use and grazing history). Broad phylogenetic structure was evident, but on a scale larger than our study area. Lizards were sexually dimorphic, whereby females had longer snout-vent length than males, providing support for the fecundity selection hypothesis. Body size variation in M. boulengeri was correlated with temperature and rainfall, a pattern consistent with larger individuals occupying cooler and more productive parts of the landscape. Climate change forecasts, which predict warmer temperature and increased aridity, may result in reduced lizard biomass and decoupling of trophic interactions with potential implications for community organization and ecosystem function.
PMCID: PMC4206273  PMID: 25337999
20.  Hydrologic Remote Sensing and Land Surface Data Assimilation 
Sensors (Basel, Switzerland)  2008;8(5):2986-3004.
Accurate, reliable and skillful forecasting of key environmental variables such as soil moisture and snow are of paramount importance due to their strong influence on many water resources applications including flood control, agricultural production and effective water resources management which collectively control the behavior of the climate system. Soil moisture is a key state variable in land surface–atmosphere interactions affecting surface energy fluxes, runoff and the radiation balance. Snow processes also have a large influence on land-atmosphere energy exchanges due to snow high albedo, low thermal conductivity and considerable spatial and temporal variability resulting in the dramatic change on surface and ground temperature. Measurement of these two variables is possible through variety of methods using ground-based and remote sensing procedures. Remote sensing, however, holds great promise for soil moisture and snow measurements which have considerable spatial and temporal variability. Merging these measurements with hydrologic model outputs in a systematic and effective way results in an improvement of land surface model prediction. Data Assimilation provides a mechanism to combine these two sources of estimation. Much success has been attained in recent years in using data from passive microwave sensors and assimilating them into the models. This paper provides an overview of the remote sensing measurement techniques for soil moisture and snow data and describes the advances in data assimilation techniques through the ensemble filtering, mainly Ensemble Kalman filter (EnKF) and Particle filter (PF), for improving the model prediction and reducing the uncertainties involved in prediction process. It is believed that PF provides a complete representation of the probability distribution of state variables of interests (according to sequential Bayes law) and could be a strong alternative to EnKF which is subject to some limitations including the linear updating rule and assumption of jointly normal distribution of errors in state variables and observation.
PMCID: PMC3675527
Remote sensing; Soil Moisture; Snow; Data Assimilation
21.  Evo-Devo of Amniote Integuments and Appendages 
Integuments form the boundary between an organism and the environment. The evolution of novel developmental mechanisms in integuments and appendages allows animals to live in diverse ecological environments. Here we focus on amniotes. The major achievement for reptile skin is an adaptation to the land with the formation of a successful barrier. The stratum corneum enables this barrier to prevent water loss from the skin and allowed amphibian/reptile ancestors to go onto the land. Overlapping scales and production of β-keratins provide strong protection. Epidermal invagination led to the formation of avian feather and mammalian hair follicles in the dermis. Both adopted a proximal - distal growth mode that maintains endothermy. Feathers form hierarchical branches which produce the vane that makes flight possible. Recent discoveries of feathered dinosaurs in China inspire new thinking on the origin of feathers. In the laboratory, epithelial - mesenchymal recombinations and molecular mis-expressions were carried out to test the plasticity of epithelial organ formation. We review the work on the transformation of scales into feathers, conversion between barbs and rachis, and the production of “chicken teeth”. In mammals, tilting the balance of the BMP pathway in K14 noggin transgenic mice alters the number, size and phenotypes of different ectodermal organs, making investigators rethink the distinction between morpho-regulation and pathological changes. Models on the evolution of feathers and hairs from reptile integuments are discussed. A hypothetical Evo-Devo space where diverse integument appendages can be placed according to complex phenotypes and novel developmental mechanisms is presented.
PMCID: PMC4386668  PMID: 15272390
feather; scale; hair; evolution; development; Mesozoic; skin appendage
22.  Silage produces biofuel for local consumption 
In the normal process of bioethanol production, biomass is transported to integrated large factories for degradation to sugar, fermentation, and recovery of ethanol by distillation. Biomass nutrient loss occurs during preservation and degradation. Our aim was to develop a decentralized ethanol production system appropriate for farm or co-operative level production that uses a solid-state fermentation method for producing bio-ethanol from whole crops, provides cattle feed, and produces no wastes. The idea is to incorporate traditional silage methods with simultaneous saccharification and fermentation. Harvested, fresh biomass is ensiled with biomass-degrading enzymes and yeast. Multiple parallel reactions for biomass degradation and ethanol and lactic acid production are induced in solid culture in hermetically sealed containers at a ranch. After fermentation, ethanol is collected on site from the vapor from heated fermented products.
The parallel reactions of simultaneous saccharification and fermentation were induced efficiently in the model fermentation system. In a laboratory-scale feasibility study of the process, 250 g of freshly harvested forage rice with 62% moisture was treated with 0.86 filter paper units/g dry matter (DM) of cellulase and 0.32 U/g DM of glucoamylase. After 20 days of incubation at 28°C, 6.4 wt.% of ethanol in fresh matter (equivalent to 169 g/kg DM) was produced. When the 46 wt.% moisture was gathered as vapor from the fermented product, 74% of the produced ethanol was collected. Organic cellular contents (such as the amylase and pronase degradable fractions) were decreased by 63% and organic cell wall (fiber) content by 7% compared to silage prepared from the same material.
We confirmed that efficient ethanol production is induced in nonsterilized whole rice plants in a laboratory-scale solid-state fermentation system. For practical use of the method, further study is needed to scale-up the fermentation volume, develop an efficient ethanol recovery method, and evaluate the fermentation residue as an actual cattle feed.
PMCID: PMC3224754  PMID: 22040609
23.  Species delineation using Bayesian model-based assignment tests: a case study using Chinese toad-headed agamas (genus Phrynocephalus) 
Species are fundamental units in biology, yet much debate exists surrounding how we should delineate species in nature. Species discovery now requires the use of separate, corroborating datasets to quantify independently evolving lineages and test species criteria. However, the complexity of the speciation process has ushered in a need to infuse studies with new tools capable of aiding in species delineation. We suggest that model-based assignment tests are one such tool. This method circumvents constraints with traditional population genetic analyses and provides a novel means of describing cryptic and complex diversity in natural systems. Using toad-headed agamas of the Phrynocephalus vlangalii complex as a case study, we apply model-based assignment tests to microsatellite DNA data to test whether P. putjatia, a controversial species that closely resembles P. vlangalii morphologically, represents a valid species. Mitochondrial DNA and geographic data are also included to corroborate the assignment test results.
Assignment tests revealed two distinct nuclear DNA clusters with 95% (230/243) of the individuals being assigned to one of the clusters with > 90% probability. The nuclear genomes of the two clusters remained distinct in sympatry, particularly at three syntopic sites, suggesting the existence of reproductive isolation between the identified clusters. In addition, a mitochondrial ND2 gene tree revealed two deeply diverged clades, which were largely congruent with the two nuclear DNA clusters, with a few exceptions. Historical mitochondrial introgression events between the two groups might explain the disagreement between the mitochondrial and nuclear DNA data. The nuclear DNA clusters and mitochondrial clades corresponded nicely to the hypothesized distributions of P. vlangalii and P. putjatia.
These results demonstrate that assignment tests based on microsatellite DNA data can be powerful tools for distinguishing closely related species and support the validity of P. putjatia. Assignment tests have the potential to play a significant role in elucidating biodiversity in the era of DNA data. Nonetheless, important limitations do exist and multiple independent datasets should be used to corroborate results from assignment programs.
PMCID: PMC2904330  PMID: 20579368
24.  Diversity of functional microornamentation in slithering geckos Lialis (Pygopodidae) 
The skin of geckos is covered with countless microscopic protuberances (spines). This surface structure causes low wettability to water. During evolution, representatives of the recent gekkotan clade Pygopodidae started slithering on the ground. This manner of locomotion affected limb reduction resulting in a snake-like body. Regarding abrasion and frictional properties, a surface covered with gekkotan spines is a topography that hampers the snake-like locomotion mode. Using scanning electron microscopy, we investigated the shed skins of two pygopodid lizards, Lialis jicari (Papua snake lizard) and Lialis burtonis (Burton's legless lizard), in order to show epidermal adaptations to limbless locomotion. Our data showed that Pygopodidae differ from their relatives not only anatomically, but also in their epidermal microstructure. Scales of L. jicari have five different structural patterns on various body regions. Ventral scales have nanoridges, similar to those found on the ventralia of snakes. Surfaces of scales covering the jaw bones, have flattened spine-like microstructures that might be an adaptation to reduce abrasion. Dorsal scales have oblong microscopic bulges covered with nanoridges. Spines cover the undersides and the interstices of scales over the entire body of both species and in L. jicari also the top of dorsal head scales. Our measurements of surface wettability (surface free energy) show superhydrophobic properties of the spiny surfaces in comparison with the other microstructural patterns of other body parts.
PMCID: PMC3813336  PMID: 24107533
Pygopodidae; microstructure; skin; Gekkota; water repellency; microornamentation
25.  Evaluation of the predicted error of the soil moisture retrieval from C-band SAR by comparison against modelled soil moisture estimates over Australia 
Remote Sensing of Environment  2012;120(2):188-196.
The Sentinel-1 will carry onboard a C-band radar instrument that will map the European continent once every four days and the global land surface at least once every twelve days with finest 5 × 20 m spatial resolution. The high temporal sampling rate and operational configuration make Sentinel-1 of interest for operational soil moisture monitoring. Currently, updated soil moisture data are made available at 1 km spatial resolution as a demonstration service using Global Mode (GM) measurements from the Advanced Synthetic Aperture Radar (ASAR) onboard ENVISAT. The service demonstrates the potential of the C-band observations to monitor variations in soil moisture. Importantly, a retrieval error estimate is also available; these are needed to assimilate observations into models. The retrieval error is estimated by propagating sensor errors through the retrieval model.
In this work, the existing ASAR GM retrieval error product is evaluated using independent top soil moisture estimates produced by the grid-based landscape hydrological model (AWRA-L) developed within the Australian Water Resources Assessment system (AWRA). The ASAR GM retrieval error estimate, an assumed prior AWRA-L error estimate and the variance in the respective datasets were used to spatially predict the root mean square error (RMSE) and the Pearson's correlation coefficient R between the two datasets. These were compared with the RMSE calculated directly from the two datasets. The predicted and computed RMSE showed a very high level of agreement in spatial patterns as well as good quantitative agreement; the RMSE was predicted within accuracy of 4% of saturated soil moisture over 89% of the Australian land mass. Predicted and calculated R maps corresponded within accuracy of 10% over 61% of the continent. The strong correspondence between the predicted and calculated RMSE and R builds confidence in the retrieval error model and derived ASAR GM error estimates.
The ASAR GM and Sentinel-1 have the same basic physical measurement characteristics, and therefore very similar retrieval error estimation method can be applied. Because of the expected improvements in radiometric resolution of the Sentinel-1 backscatter measurements, soil moisture estimation errors can be expected to be an order of magnitude less than those for ASAR GM. This opens the possibility for operationally available medium resolution soil moisture estimates with very well-specified errors that can be assimilated into hydrological or crop yield models, with potentially large benefits for land-atmosphere fluxes, crop growth, and water balance monitoring and modelling.
► The error of the existing ENVISAT ASAR GM soil moisture product was assessed. ► Independent top soil moisture estimates produced by the AWRA model were used. ► A high quality of the ASAR GM error estimate was demonstrated. ► The identical error model was proposed for the potential Sentinel-1 product.
PMCID: PMC3587384  PMID: 23483015
Synthetic Aperture Radar (SAR); Soil moisture; ASAR GM; Error evaluation; Australia; Australian Water Resources Assessment System (AWRA)

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