Improved measurement of the rotor temperature in analytical ultracentrifugation

Sedimentation velocity is a classical method for measuring the hydrodynamic, translational friction coefficient of biological macromolecules. In a recent study comparing various analytical ultracentrifuges, we showed that external calibration of the scan time, radial magnification, and temperature is critically important for accurate measurements (Anal. Biochem. 440 (2013) 81–95). To achieve accurate temperature calibration, we introduced the use of an autonomous miniature temperature logging integrated circuit (Maxim Thermochron iButton) that can be inserted into an ultracentrifugation cell assembly and spun at low rotor speeds. In the current work, we developed an improved holder for the temperature sensor located in the rotor handle. This has the advantage of not reducing the rotor capacity and allowing for a direct temperature measurement of the spinning rotor during high-speed sedimentation velocity experiments up to 60,000 rpm. We demonstrated the sensitivity of this approach by monitoring the adiabatic cooling due to rotor stretching during rotor acceleration and the reverse process on rotor deceleration. Based on this, we developed a procedure to approximate isothermal rotor acceleration for better temperature control.     

Modification and miniaturization of Thermochron iButtons for surgical implantation into small animals

Thermochron iButtons are being used increasingly by animal physiologists to measure long-term patterns of body temperature in reptiles, birds and mammals. Typically, iButtons are surgically implanted into the intraperitoneal cavity where they measure and store body temperature together with the date and time from an onboard real-time clock. In 16-bit resolution, the DS1922L iButton can store a total of 4,096 data points over pre-determined sampling intervals. iButtons have proved invaluable in measuring patterns of torpor and hibernation in animals larger than 70 g. Weighing around 3.5 g after potting with wax, iButtons are too heavy and large to implant into animals smaller than 70 g because their weight exceeds 5% of the animal’s total body weight. This paper describes how the stainless steel canister housing the DS 1922L iButton battery and circuit board can be removed to reduce the weight of the components to 1.49 g after waxing (ready for implantation) without compromising the function or battery life of the iButton. The modified iButton can be implanted into animals as small as 20–30 g. Calibration data revealed an offset of ca. 1°C on average, confirming that iButtons must be calibrated prior to implantation.     

CONFINE-MAS: a magic-angle spinning NMR probe that confines the sample in case of a rotor explosion

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called “rotor crash” involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe (“CONFINE-MAS” probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid.     

Features of the circadian rhythm of skin temperature in eight- to nine-year-old children and young adults

The age-related features of the circadian rhythm of skin temperature were studied using the Termochron iButton method in two age groups of subjects: eight-to nine-year-old boys and girls and young men and women aged 20–22 years. For this purpose, the temperature was monitored for 48 h at a recording interval of 10 min. The following authentic chronobiological differences were revealed: the temperature mesor was higher in the girls than in the boys and in the young men it was higher than in the young women. The circadian rhythm’s amplitude was higher in the boys and the young women. Studies of the chronological values in different 24-h periods showed that the average level of temperature at night was lower in all subjects. The amplitude in the adults did not differ in different 24-h periods, whereas it was higher in the children at night.     

Thermocron iButton and iBBat temperature dataloggers emit ultrasound

Thermocron iButton dataloggers are widely used to measure thermal microclimates experienced by wild animals. The iBBat is a smaller version of the datalogger, also commercially available, that is used to measure animal skin or core body temperatures when attached externally or surgically implanted. Field observations of bats roosting under a bridge suggested that bats avoided locations with iButtons. A heterodyne bat detector revealed that the dataloggers emitted ultrasound which was detectable from a distance of up to 30 cm. We therefore recorded and quantified the acoustic properties [carrier frequency (Hz) and root mean square sound pressure level (dB SPL)] of iButton and iBBat dataloggers. All units emitted a 32.9 kHz pure tone that was readily picked up with a time expansion bat detector at a distance of 1 cm, and most were detected at a distance of 15 cm. The maximum amplitude of iButton dataloggers was 46.5 dB SPL at 1.0 cm—a level within the range of auditory sensitivity for most small mammals. Wrapping iButtons in plastic insulation severely attenuated the amplitude of ultrasound. Although there was a statistically significant reduction in rates of warming and cooling with insulation, this effect was small and we suggest that insulation may be a viable solution to eliminate unwanted ultrasonic noise in instances when small delays in thermal response dynamics are not a concern. We recommend behavioural studies to assess if the electronic signals emitted by iButtons are disturbing to small mammals.     

Ways to measure body temperature in the field

Body temperature (T_b) represents one of the key parameters in ecophysiological studies with focus on energy saving strategies. In this study we therefore comparatively evaluated the usefulness of two types of temperature-sensitive passive transponders (LifeChips and IPTT-300) and one data logger (iButton, DS1922L) mounted onto a collar to measure T_b in the field. First we tested the accuracy of all three devices in a water bath with water temperature ranging from 0 to 40 °C. Second, we evaluated the usefulness of the LifeChips and the modified iButtons for measuring T_b of small heterothermic mammals under field conditions. For this work we subcutaneously implanted 14 male edible dormice (Glis glis) with transponders, and equipped another 14 males with data loggers to simultaneously record T_b and oxygen consumption with a portable oxygen analyzer (Oxbox). In one individual we recorded T_b with both devices and analyzed recorded T_b patterns.LifeChips are able to measure temperature within the smallest range from 25 to 40 °C with an accuracy of 0.07±0.12 °C. IPTT-300 transponders measured temperature between 10 and 40 °C, but accuracy decreased considerably at values below 30 °C, with maximal deviations of nearly 7 °C. An individual calibration of each transponder is therefore needed, before using it at low T_bs. The accuracy of the data logger was comparatively good (0.12±0.25 °C) and stable over the whole temperature range tested (0–40 °C). In all three devices, the repeatability of measurements was high.LifeChip transponders as well as modified iButtons measured T_b reliably under field conditions. Simultaneous T_b-recordings in one edible dormouse with an implanted LifeChip and a collar-mounted iButton revealed that values of both measurements were closely correlated. Taken together, we conclude that implanted temperature-sensitive transponders represent an appropriate and largely non-invasive method to measure T_b also under field conditions.     

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton–proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.     

Use of centrifugal force in the study of xylem cavitation

Two methods were evaluated for using centrifugal force to measurethe occurrence of cavitation as a function of negative pressuresin xylem. The general protocol was to measure the hydraulicconductivity of xylem segments (stem or root pieces) beforeand after centring them on a centrifuge rotor and spinning themabout their long axis to generate negative xylem pressure. Thepercentage decrease in conductivity from the initial to finalmeasurement was used to quantify the embolism resulting fromcavitation during spinning. In one approach, segments were spunwith their ends exposed to air. This method could only be usedwhen xylem conduits were much shorter than the segment. Resultsfrom an angiosperm (Betula occidentalis) and a gymnosperm (AblesIasiocarpa) corresponded to previous observations of embolismcaused by air dehydration where negative pressure was measuredwith the pressure chamber. Results also agreed with embolismcaused by injection of air into the xylem, in support of theair-seeding hypothesis for cavitation. In a second approach,segments were spun in a rotor designed to keep the segment endsimmersed in water during spinning. This gave the same resultsas for non-immersed segments. Immersing the segment ends allowedmeasurements on any material, regardless of conduit length,as demonstrated for roots of B. occidentalis. The chief advantageof the centrifugal force method is the rapidity and precisionwith which any desired xylem pressure can be imposed. Key words: Cavitation, embolism, drought stress, water relations, water transport     

Features of the circadian rhythm of temperature of the skin at children of 8-9 years and young men and girls

Problem of the present work was studying age features circadian rhythm of temperature of a skin with application of a method "Termochron iButton". Day-night rhythm of a skin temperature at two age-grades was investigated: boys and girls of 8-9 years and young men and girls of 20-22 years. For this purpose monitoring temperature during 48 hours with an interval of registration 10 minutes has been lead. Are revealed authentic chronobiological differences: mesor temperatures above at girls, than at boys, and at young men, above, than at girls. Amplitude of a circadian rhythm is above at boys and at girls. Researches chronobiological parameters in the different terms of day have shown, that an average level of temperature at night below at all examinees. The amplitude at adult people in the different terms of day does not differ, while at children it above in the night term.     

Does waterproofing Thermochron iButton dataloggers influence temperature readings?

Miniature Thermochron iButton dataloggers have transformed the ways in which researchers collect thermal data. However, one important limitation is that these dataloggers are not waterproof, which can lead to device failure and loss of data under field conditions. Several methods have been used to increase their water resistance, but no study to date has investigated whether any of these techniques affects the accuracy of temperature readings. Waterproofing potentially could affect the accuracy of iButtons by biasing temperatures or altering rates of warming and cooling. We compared temperature profiles of unmodified Thermochron iButtons (model DS1921G) to iButtons that we coated with a clear plastic dip (designed to coat tool handles) to determine whether this waterproof coating affects the accuracy of temperatures they record. We also compared temperatures recorded by uncoated and coated iButtons that were embedded within physical models that mimic frog body temperatures. Finally, we used our field data to test whether coating iButtons with plastic prevents failure of dataloggers during fieldwork. Although we found statistically significant differences between the temperatures recorded by uncoated and coated iButtons, and also between uncoated and coated iButtons embedded in frog models, these effects were relatively small (0–1.3 °C). We also found that coating iButtons with plastic reduced the likelihood of device failure under field conditions (from 8.3% to 0%). We conclude that coating Thermochron iButtons with plastic is an affordable and reliable method of waterproofing dataloggers that prevents device failure and data loss with minimal influence on temperature readings.     

Method for the preparation of stabile microencapsulated lactic acid bacteria

Summary A method to produce viable and stabile dry microorganisms for food and agricultural purposes was developed. Spray-dried, freeze-dried or liquid culture concentrates of lactic acid-producing bacteria were mixed with various bulking agents to form a homogeneous wet granulation having a water content of 35–60% (w/w). The wet granulation was extruded through a dye onto a spinning plate (350–500 rpm) of a spheronizing device which resulted in the formation of discrete spherical particles. After forming spheres, the aggregate cell particles, both coated and uncoated, were dried to a moisture level of 5–10% using a temperature below the microorganism's optimum growth temperature. The coated and uncoated products were stored at different temperatures and periodically sampled to determine stability. Uncoated cell particles were more stabile at 4°C than at 22°C for 76 days. While both coated (with sodium alginate or carboxymethyl-cellulose) and uncoated particles showed similar stability at 4°C, at higher storage temperatures the applied coating improved the storage stability of the culture particles.     

Energy costs of the predation strategy of the web-spinning spider Lepthyphantes zimmermanni bertkau (Linyphiidae)

Summary The energy costs of the predation strategy of the web-spinning spider Lypthyphantes zimmermanni were investigated in the laboratory. The standard respiratory costs associated with the stationary aspect of the strategy were estimated by means of a Gilson respirometer run at the different temperatures prevailing month by month in the beech woodland litter layer which comprises the spider's natural habitat. Respiration rate is related to weight by an exponent with a mean value of 0.7398. The Q ₁₀ of respiration rate is 2.41 between 5°C and 10°C and 1.97 between 10°C and 15°C. The energy costs of producing a web comprise the active respiratory costs associated with the locomotory activity involved in spinning a web together with the energy value of the silk used in the web manufacture. The former were evaluated by allowing a spider to spin a web in a respirometer, subtracting the calculated standard respiratory energy costs for a spider of equivalent weight and multiplying by a correction factor for web size. The relationship between spider weight and area of web produced was established in the laboratory. The respiratory cost of spinning a web is effectively constant with temperature at 724.46·10^-3 J for an adult (4 mg) spider. The energy value of spider silk was estimated by means of a bomb calorimeter and found to be 17,435 J g^-1. The energy content of the silk of a single adult's web is 1.16 J, giving energy cost of web production of 1.88 J at all temperatures.     

Analytical techniques for cell fractions. XII. A multiple-cuvet rotor for a new microanalytical system

A new microanalytical system has been developed that employs a rotor containing 15 cuvets spinning past a beam of light. The signal is displayed on an oscilloscope, and a peak is observed continuously for each cuvet. Standards, samples, and reagents are placed in a central loading disc, and all solutions moved out into the cuvets by centrifugal force. Minimum volume to fill the cuvets is 200 λ. By using the Weichselbaum biuret reagent for proteins, 15 analyses may be completed in as little as 30 sec after the rotor is started. The data are obtained photographically.     

Ad hoc instrumentation methods in ecological studies produce highly biased temperature measurements

In light of global climate change, ecological studies increasingly address effects of temperature on organisms and ecosystems. To measure air temperature at biologically relevant scales in the field, ecologists often use small, portable temperature sensors. Sensors must be shielded from solar radiation to provide accurate temperature measurements, but our review of 18 years of ecological literature indicates that shielding practices vary across studies (when reported at all), and that ecologists often invent and construct ad hoc radiation shields without testing their efficacy. We performed two field experiments to examine the accuracy of temperature observations from three commonly used portable data loggers (HOBO Pro, HOBO Pendant, and iButton hygrochron) housed in manufactured Gill shields or ad hoc, custom‐fabricated shields constructed from everyday materials such as plastic cups. We installed this sensor array (five replicates of 11 sensor‐shield combinations) at weather stations located in open and forested sites. HOBO Pro sensors with Gill shields were the most accurate devices, with a mean absolute error of 0.2°C relative to weather stations at each site. Error in ad hoc shield treatments ranged from 0.8 to 3.0°C, with the largest errors at the open site. We then deployed one replicate of each sensor‐shield combination at five sites that varied in the amount of urban impervious surface cover, which presents a further shielding challenge. Bias in sensors paired with ad hoc shields increased by up to 0.7°C for every 10% increase in impervious surface. Our results indicate that, due to variable shielding practices, the ecological literature likely includes highly biased temperature data that cannot be compared directly across studies. If left unaddressed, these errors will hinder efforts to predict biological responses to climate change. We call for greater standardization in how temperature data are recorded in the field, handled in analyses, and reported in publications.     

High-resolution magic angle spinning (1)H NMR spectroscopy of intact liver and kidney: optimization of sample preparation procedures and biochemical stability of tissue during spectral acquisition

High-resolution magic angle spinning (MAS) (1)H NMR spectroscopy has been used to investigate the biochemical composition of whole rat renal cortex and liver tissue samples. The effects of a number of sample preparation procedures and experimental variables have been investigated systematically in order to optimize spectral quality and maximize information recovery. These variables include the effects of changing the sample volume in the MAS rotor, snap-freezing the samples, and the effect of organ perfusion with deuterated saline solution prior to MAS NMR analysis. Also, the overall biochemical stability of liver and kidney tissue MAS NMR spectra was investigated under different temperature conditions. We demonstrate improved resolution and line shape of MAS NMR spectra obtained from small spherical tissue volume (12 microl) rotor inserts compared to 65 microl cylindrical samples directly inserted into the MAS rotors. D(2)O saline perfusion of the in situ afferent vascular tree of the tissue immediately postmortem also improves line shape in MAS NMR spectra. Snap-freezing resulted in increased signal intensities from alpha-amino acids (e.g., valine) in tissue together with decreases in renal osmolytes, such as myo-inositol. A decrease in triglyceride levels was observed in renal cortex following stasis on ice and in the MAS rotor (303 K for 4 h). This work indicates that different tissues have differential metabolic stabilities in (1)H MAS NMR experiments and that careful attention to sample preparation is required to minimize artifacts and maintain spectral quality.     

Characterization of the separation properties of the beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12–35 μm diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at each collection rotor speed) and a long collection method (10–15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

Band-selective 13C Homonuclear 3D Spectroscopy for Solid Proteins at High Field with Rotor-synchronized Soft Pulses

We demonstrate improved 3D ¹³C–¹³C–¹³C chemical shift correlation experiments for solid proteins, utilizing band-selective coherence transfer, scalar decoupling and homonuclear zero-quantum polarization transfer. Judicious use of selective pulses and a z-filter period suppress artifacts with a two-step phase cycle, allowing higher digital resolution in a fixed measurement time. The novel correlation of C^ali–C^ali–CX (C^ali for aliphatic carbons, CX for any carbon) reduces measurement time by an order of magnitude without sacrificing digital resolution. The experiment retains intensity from side-chain carbon resonances whose chemical shift dispersion is critical to minimize spectral degeneracy for large proteins with a predominance of secondary structure, such as β-sheet rich fibrillar proteins and α-helical membrane proteins. We demonstrate the experiment for the β1 immunoglobulin binding domain of protein G (GB1) and fibrils of the A30P mutant of α-synuclein, which is implicated in Parkinson’s disease. Selective pulses of duration comparable the rotor period give optimal performance, but must be synchronized with the spinning in non-trivial ways to minimize chemical shift anisotropy recoupling effects. Soft pulses with a small bandwidth-duration product are best for exciting the ~70 ppm bandwidth required for aliphatic-only dimensions.     

Overwinter body temperature patterns in captive jerboas (<Emphasis Type="Italic">Jaculus orientali</Emphasis>s): influence of sex and group

The jerboa (Jaculus orientalis) has been described in the past as a hibernator, but no reliable data exist on the daily and seasonal rhythmicity of body temperature (T _b). In this study, T _b patterns were determined in different groups of jerboas (isolated males and females, castrated males and grouped animals) maintained in captivity during autumn and winter, and submitted to natural variations of light and ambient temperature (T _a). T _b and T _a variations were recorded with surgically implanted iButton temperature loggers at 30-min intervals for two consecutive years. About half (6/13) of isolated female jerboas hibernated with a T _b < 33°C, with hibernation bouts interspersed with short periods of normothermy from November to February. Hibernation bout durations were longer (4–5 days) than those of normothermia phases (1–4 days). During hibernation, the minimum T _b was low (T _bmin ~10.7°C). In contrast, one of the 12 isolated males showed short hibernation bouts of ca. 2 days late in the hibernation season, February–March. The males had T _bmin values of 15.1°C. In contrast to predictions, no castrated males hibernated. When jerboas were grouped, females and males exhibited concomitant torpor bouts. In males, the longest bouts were observed during the late hibernation season. These data suggest complex regulation of hibernation in jerboas.     

Assessment of Circadian Rhythms of Both Skin Temperature and Motor Activity in Infants During the First 6 Months of Life

The authors developed a method useful for home measurement of temperature, activity, and sleep rhythms in infants under normal-living conditions during their first 6 mos of life. In addition, parametric and nonparametric tests for assessing circadian system maturation in these infants were compared. Anthropometric parameters plus ankle skin temperature and activity were evaluated in 10 infants by means of two data loggers, Termochron iButton (DS1291H, Maxim Integrated Products, Sunnyvale, CA) for temperature and HOBO Pendant G (Hobo Pendant G Acceleration, UA-004-64, Onset Computer Corporation, Bourne, MA) for motor activity, located in special baby socks specifically designed for the study. Skin temperature and motor activity were recorded over 3 consecutive days at 15 days, 1, 3, and 6 mos of age. Circadian rhythms of skin temperature and motor activity appeared at 3 mos in most babies. Mean skin temperature decreased significantly by 3 mos of life relative to previous measurements (p?=?.0001), whereas mean activity continued to increase during the first 6 mos. For most of the parameters analyzed, statistically significant changes occurred at 3–6 mos relative to 0.5–1 mo of age. Major differences were found using nonparametric tests. Intradaily variability in motor activity decreased significantly at 6 mos of age relative to previous measurements, and followed a similar trend for temperature; interdaily stability increased significantly at 6 mos of age relative to previous measurements for both variables; relative amplitude increased significantly at 6 mos for temperature and at 3 mos for activity, both with respect to previous measurements. A high degree of correlation was found between chronobiological parametric and nonparametric tests for mean and mesor and also for relative amplitude versus the cosinor-derived amplitude. However, the correlation between parametric and nonparametric equivalent indices (acrophase and midpoint of M5, interdaily stability and Rayleigh test, or intradaily variability and P₁/P_ultradian) despite being significant, was lower for both temperature and activity. The circadian function index (CFI index), based on the integrated variable temperature-activity, increased gradually with age and was statistically significant at 6 mos of age. At 6 mos, 90% of the infants' rest period coincided with the standard sleep period of their parents, defined from 23:00 to 07:00?h (dichotomic index I?<?O; when I?<?O?=?100%, there is a complete coincidence between infant nocturnal rest period and the standard rest period), whereas at 15 days of life the coincidence was only 75%. The combination of thermometry and actimetry using data loggers placed in infants' socks is a reliable method for assessing both variables and also sleep rhythms in infants under ambulatory conditions, with minimal disturbance. Using this methodological approach, circadian rhythms of skin temperature and motor activity appeared by 3 mos in most babies. Nonparametric tests provided more reliable information than cosinor analysis for circadian rhythm assessment in infants. (Author correspondence: elvirada@um.es)