Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections.

The implementation of ion mobility spectrometry (IMS) in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflows has become a valuable tool for improving compound annotation in metabolomics analyses by increasing peak capacity and by adding a new molecular descriptor, the collision cross section (CCS). Although some studies reported high repeatability and reproducibility of CCS determination and only few studies reported good interplatform agreement for small molecules, standardized protocols are still missing due to the lack of reference CCS values and reference materials. We present a comparison of CCS values of approximatively one hundred lipid species either commercially available or extracted from human plasma. We used three different commercial ion mobility technologies from different laboratories, drift tube IMS (DTIMS), travelling wave IMS (TWIMS) and trapped IMS (TIMS), to evaluate both instrument repeatability and interlaboratory reproducibility. We showed that CCS discrepancies of 0.3% (average) could occur depending on the data processing software tools. Moreover, eleven CCS calibrants were evaluated yielding mean RSD below 2% for eight calibrants, ESI Low concentration tuning mix (Tune Mix) showing the lowest RSD (< 0.5%) in both ion modes. Tune Mix calibrated CCS from the three different IMS instruments proved to be well correlated and highly reproducible (R2 > 0.995 and mean RSD ≤ 1%). More than 90% of the lipid CCS had deviations of less than 1%, demonstrating high comparability between techniques, and the possibility to use the CCS as molecular descriptor. We highlighted the need of standardized procedures for calibration, data acquisition, and data processing. This work demonstrates that using harmonized analytical conditions are required for interplatform reproducibility for CCS determination of human plasma lipids.

Impact of Source Conditions on Collision Cross Section Determination by Trapped Ion Mobility Spectrometry.

Collision cross section (CCS) values determined in ion mobility-mass spectrometry (IM-MS) are increasingly employed as additional descriptors in metabolomics studies. CCS values must therefore be reproducible and the causes of deviations must be carefully known and controlled. Here, we analyzed lipid standards by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to evaluate the effects of solvent and flow rate in flow injection analysis (FIA), as well as electrospray source parameters including nebulizer gas pressure, drying gas flow rate, and temperature, on the ion mobility and CCS values. The stability of ion mobility experiments was studied over 10 h, which established the need for a delay-time of 20 min to stabilize source parameters (mostly pressure and temperature). Modifications of electrospray source parameters induced shifts of ion mobility peaks and even the occurrence of an additional peak in the ion mobility spectra. This behavior could be essentially explained by ion-solvent cluster formation. Changes in source parameters were also found to impact CCS value measurements, resulting in deviations up to 0.8%. However, internal calibration with the Tune Mix calibrant reduced the CCS deviations to 0.1%. Thus, optimization of source parameters is essential to achieve a good desolvation of lipid ions and avoid misinterpretation of peaks in ion mobility spectra due to solvent effects. This work highlights the importance of internal calibration to ensure interoperable CCS values, usable in metabolomics annotation.

Quantitative determination of the full switching cycle of photochromic fluorescent proteins.

In this study, we develop a general analytical model of the photochromism of fluorescent proteins and apply it to spectroscopic measurements performed on six different labels. Our approach provides quantitative explanations for phenomena such as the existence of positive and negative switching, limitations in the photochromism contrast, and the fact that initial switching cycles may differ from subsequent ones. It also allows us to perform the very first measurement of all four isomerization quantum yields involved in the switching process.

A re-calibration procedure for interoperable lipid collision cross section values measured by traveling wave ion mobility spectrometry.

Collision cross sections (CCS) have been described as relevant molecular descriptors in metabolomics and lipidomics analyses for ascertaining compound identity. Ion mobility spectrometry (IMS) allows to determine CCS with different techniques, such as drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS) or trapped ion mobility spectrometry (TIMS). In contrast with DTIMS where CCS can be obtained directly with measured drift times and mathematical relationship, TWIMS and TIMS techniques require an additional step of calibration to obtain CCS values. However, literature reports significantly disparate CCS values depending on the calibrant used (often more than 10%), as no consensus has been reached to define a universal CCS reference standard or harmonized calibration procedure. Therefore, publicly available CCS databases cannot be regarded as readily interoperable and exchangeable. Here, we performed a comprehensive evaluation of 11 distinct CCS calibrants in a traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) instrument. We showed that, using lipids from plasma as model compounds, CCS determination drastically fluctuates from one calibrant to the other with up to 25% differences, which precludes direct CCS comparison. Using the large panel of calibration curves generated, we showed that any CCS value can be efficiently re-calibrated relatively to the calibration curve made with the widely used Tune Mix solution whatever the calibration procedure originally used. The re-calibrated CCS values for each calibrant constitute a database which allows to correct any deviation on lipid CCS values whatever the calibrant originally used. Resulting corrected CCS values from plasma lipids were thus efficiently matched to those previously reported in the literature (with deviations<2%). Therefore, this work shows that unique and comparable CCS values can be obtained upon re-calibration relatively to Tune Mix CCS values, while also paving the way for the establishment of a universal CCS database of various metabolite or lipid classes.

Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification.

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

Utilization Impact of Cost-Sharing Elimination for Preventive Care Services: A Rapid Review.

Consumer cost-sharing has been shown to diminish utilization of preventive services. Recent efforts, including provisions within the Affordable Care Act, have sought to increase use of preventive care through elimination of cost-sharing for clinically indicated services. We conducted a rapid review of the literature to determine the impact of cost-share elimination on utilization of preventive services. Searches were conducted in PubMed, Scopus, and CINAHL Complete databases as well as in grey literature. A total of 35 articles were included in qualitative synthesis and findings were summarized for three clinical service categories: cancer screenings, contraceptives, and additional services. Impacts of cost-sharing elimination varied depending on clinical service, with a majority of findings showing increases in use. Studies that included socioeconomic status reported that those who were financially vulnerable incurred substantial increases in utilization. Future investigations on additional clinical services are warranted as is research to better elucidate populations who most benefit from cost-sharing elimination.

An oligomeric switch controls the Mrr-induced SOS response in E. coli.

Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.

Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure.

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

The Timetree of Prokaryotes: New Insights into Their Evolution and Speciation.

The increasing size of timetrees in recent years has led to a focus on diversification analyses to better understand patterns of macroevolution. Thus far, nearly all studies have been conducted with eukaryotes primarily because phylogenies have been more difficult to reconstruct and calibrate to geologic time in prokaryotes. Here, we have estimated a timetree of 11,784 'species' of prokaryotes and explored their pattern of diversification. We used data from the small subunit ribosomal RNA along with an evolutionary framework from previous multi-gene studies to produce three alternative timetrees. For each timetree we surprisingly found a constant net diversification rate derived from an exponential increase of lineages and showing no evidence of saturation (rate decline), the same pattern found previously in eukaryotes. The implication is that prokaryote diversification as a whole is the result of the random splitting of lineages and is neither limited by existing diversity (filled niches) nor responsive in any major way to environmental changes.

Effect of temperature on thermal acclimation in growing pigs estimated using a nonlinear function.

Ninety-six Large White growing barrows were used to determine the effect of temperature on thermoregulatory responses during acclimation to increased ambient temperature. Pigs were exposed to 24°C for 10 d and thereafter to a constant temperature of 24, 28, 32, or 36°C for 20 d. The study was conducted in a climate-controlled room at the INRA experimental facilities in Guadeloupe, French West Indies. Relative humidity was kept constant at 80% throughout the experimental period. Rectal temperature, cutaneous temperature, and respiratory rate were measured [breaths per minute (bpm)] 3 times daily (0700, 1200, and 1800 h) every 2 or 3 d during the experiment. The thermal circulation index (TCI) was determined from rectal, cutaneous, and ambient temperature measurements. Changes in rectal temperature, respiratory rate, TCI, and ADFI over the duration of exposure to hot temperatures were modeled using nonlinear responses curves. Within 1 h of exposure to increased temperature, rectal temperature and respiratory rate increased by 0.46°C/d and +29.3 bpm/d, respectively, and ADFI and TCI decreased linearly by 44.7 g•d(-2)•kg(-0.60) and 1.32°C/d, respectively until a first breakpoint time (td(1)). This point marked the end of the short-term heat acclimation phase and the beginning of the long-term heat acclimation period. The td(1) value for ADFI was greater at 28°C than at 32 and 36°C (2.33 vs. 0.31 and 0.26 d, respectively, P < 0.05), whereas td(1) for the TCI increase was greater at 36°C than at 28 and 32°C (1.02 vs. 0.78 and 0.67 d, respectively; P < 0.05). For rectal temperature and respiratory rate responses, td(1) was not influenced by temperature (P > 0.05) and averaged 1.1 and 0.89 d, respectively. For respiratory rate and rectal temperature, the long-term heat acclimation period was divided in 2 phases, with a rapid decline for both variables followed by a slight decrease (P < 0.05). These 2 phases were separated by a second threshold day (td(2)). For rectal temperature, td(2) increased significantly with temperature (1.60 vs. 5.16 d from 28 to 36°C; P < 0.05). After td(2), the decline in rectal temperature during the exposure to thermal challenge was not influenced by temperature, suggesting that the magnitude of heat stress would affect thermoregulatory responses only at the beginning of the long-term heat acclimation period. The inclusion of random effects in the nonlinear model showed that whatever the temperature considered, interindividual variability of thermoregulatory responses would exist.

Effects of the level of sugarcane molasses on growth and carcass performance of Caribbean growing pigs reared under a ground sugarcane stalks feeding system.

The influence of the level of sugarcane (SC) molasses on growth performance, carcass traits, and meat quality in Creole (CR) growing pigs fed with ground sugarcane stalks (GCS)-based diet was studied in a mixed farming system context. The aim of the study was to optimize the growth performance of CR pigs with SC-molasses as an energy source in this unconventional feeding. A total of 32 CR pigs were used from 30 to 60 kg of body weight (BW). The experimental dietary treatments consisted of four levels of inclusion of SC-molasses (200, 400, 600, and 800 g DM/d/pig) into a GCS diet, for diets 1, 2, 3 and 4 respectively. The GCS allowance was based on live BW (170 g/kg BW/d) and the diets were supplemented with a soya-bean meal supplement (350 g/d of a 49.2% CP and 16.6 MJ DE/kg). All the pigs were slaughtered at 60 kg BW. Increasing the level of molasses did not affect (p > 0.05) average BW gain (254 g/d), CP intake (154 g/d) and sugar extraction rate from the total ration (85%). A gradual inclusion of molasses in a GCS-based diet did not affect the carcass and meat quality of CR pigs. In conclusion, molasses supplementation does not allow the increase of growth performance in GCS fed pigs.

Effect of temperature level on thermal acclimation in Large White growing pigs.

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.

Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate.

Sixty-two multiparous Large White sows were used to determine the effect of dietary fiber level on lactation performance according to season under conditions of a humid tropical climate. This experiment was conducted in Guadeloupe (West French Indies, lat 16 degrees N, long 61 degrees W) between October 1999 and January 2001. Two seasons were distinguished a posteriori from climatic measurements parameters continuously recorded in the farrowing room. During the warm season, ambient temperature and relative humidity averaged 25 degrees C and 86.8%, respectively. The corresponding values for the hot season were 27.5 degrees C and 83.5%. Experimental diets fed during lactation were a control diet (C; 14% neutral detergent fiber) and a high-fiber diet (HF; 20% neutral detergent fiber) obtained by substitution of wheat middlings by wheat bran. The two diets were formulated to provide the same ratios between essential amino acids and lysine and between lysine and net energy. No interaction between season and diet composition was found for all criteria studied. Over the 28-d lactation, average daily feed intake (ADFI) was lower and body weight loss was higher (P < 0.001) during the hot season compared to the warm season (3,447 vs 4,907 g/d and 33 vs 17 kg, respectively). The number of stillborn piglets was higher (P < 0.05) during the hot season than during the warm season (2.0 vs 1.1 piglets, respectively). Litter growth rate and mean BW of piglets at weaning were reduced (P < 0.01) during the hot season vs the warm season (2.1 vs 2.3 kg/d and 7.7 vs 8.3 kg, respectively). The ADFI was similar for both diets and digestible energy (DE) intake tended to be lower (P = 0.06) with the HF diet (54.9 vs 59.3 MJ of DE/d for C sows) in relation with its lower DE concentration. The body weight loss was greater (P < 0.01) for HF sows than for C sows (30 vs 21 kg). Compared with the C diet, the HF diet increased (P < 0.05) litter growth rate and piglet body weight at weaning (2.3 vs 2.1 kg/d and 8.3 vs 7.7 kg/d for HF vs C, respectively). Season and diet composition did not affect the weaning-to-estrus interval. In conclusion, the hot season in humid tropical climates, which combines high levels of temperature and humidity, has a major negative effect on the performance of lactating sows.