The effects of simulated transportation conditions on the core body and extremity temperature, blood physiology, and behavior of white-strain layer pullets.

Transportation of poultry is stressful. The transportation of broilers has been well studied, while the transportation of layer pullets from rearing to laying facilities has not been thoroughly evaluated. This experiment aimed to establish the effects of temperature (T)/RH combinations and duration (D) of transport, via a 5 × 2 factorial arrangement of simulated transport conditions using 5 T/RH combinations (21°C with 30% RH [21/30], 21°C with 80% RH [21/80], 30°C with 30% RH [30/30], 30°C with 80% RH [30/80], and -15°C with uncontrolled RH [-15]), and 2 exposure D (4 or 8 h). Pullets (18-19 wk; n = 240) were obtained from 3 commercial farms (N = 3 farms). Pretreatment, birds were orally administered a miniature data logger to record core body temperature (CBT), an initial blood sample was taken (5 birds/replicate), and initial foot T was recorded. Behavior during exposure was video recorded. Following exposure, a final blood sample was taken (analyzed for heterophil to lymphocyte ratio, partial pressure of CO2, total CO2, bicarbonate, and glucose), birds were slaughtered, and data loggers were retrieved. Data were analyzed as a randomized complete block design via Proc Mixed (SAS 9.4) and significance was declared at P ≤ 0.05. There were no interactions observed for the T/RH and D combinations throughout the study. The CBT and foot T were lowest in pullets exposed to -15 compared with all other treatments. Foot T was also highest in pullets exposed to 30/80 compared with -15, 21/30, and 21/80. There was no impact of T/RH on pullet blood physiology. Activity and thermoregulatory behaviors were impacted by the T/RH combinations. Pullets exposed to 30/30 and 30/80 spent the most time panting. Pullets exposed to 30/80 also spent the least amount of time motionless. Duration had minor impacts on pullet CBT, blood physiology, and behavior. These data indicate that as a response to thermal stress, layer pullets were successful at implementing mechanisms to maintain homeostasis.

The effects of simulated transport conditions on the muscle tissue characteristics of white-strain layer pullets.

The objective of this study was to evaluate the effects of temperature (T)/relative humidity (RH) combinations and exposure duration (D) on the muscle tissue characteristics of layer pullets during simulated transport. While layer pullets are not processed for meat, muscle physiology can be used as an indicator to assess welfare. Pullets (n = 240) were randomly assigned to 1 of 5 T/RH combinations (-15°C uncontrolled RH [-15], 21°C 30%RH [21/30], 21°C 80%RH [21/80], 30°C 30%RH [30/30], and 30°C 80%RH [30/80]) and 2 D (4 or 8 h) in a 5 x 2 factorial arrangement (3 replications). Birds were weighed before exposure, crated (density 45.5 kg/m2) and exposed to the conditions above. After exposure, birds were weighed (live shrink calculated) and slaughtered using a small-scale facility. Postslaughter, carcasses were eviscerated, and an initial pH was obtained from the right breast and thigh. Final breast and thigh pH and color values (lightness [L∗], yellowness [b∗], and redness [a∗]) were obtained 30 h postslaughter. Left breast muscles were frozen and analyzed for thaw and cook loss 4 wk postslaughter. Data were analyzed as a randomized complete block design via ANOVA (Proc Mixed; SAS 9.4), with farm of origin as block. Differences were considered significant when P ≤ 0.05. Live shrink (kg) was higher for pullets exposed to 30/30 and 30/80 compared with those exposed to 21/80 (P = 0.04) and for pullets exposed for 8 h compared with 4 h (P < 0.01). Breast muscle thaw loss (%) was higher in pullets exposed for 4 h compared with 8 h (P = 0.01). Breast and thigh muscle a∗ were higher for pullets exposed to 30/30 compared with 21/30 (P = 0.02). Thigh muscle b∗ was lower for pullets exposed to -15 compared with 21/80 (P = 0.05). Breast b∗ was higher for pullets exposed for 8 h compared with 4 h (P = 0.04). The results from this study demonstrates that increasing exposure D had minor effects on pullet muscle characteristics. In addition, layer pullets coped well with thermal stressors associated with simulated transport.

Author Correction: Constraining the rise of oxygen with oxygen isotopes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Constraining the rise of oxygen with oxygen isotopes.

After permanent atmospheric oxygenation, anomalous sulfur isotope compositions were lost from sedimentary rocks, demonstrating that atmospheric chemistry ceded its control of Earth's surficial sulfur cycle to weathering. However, mixed signals of anoxia and oxygenation in the sulfur isotope record between 2.5 to 2.3 billion years (Ga) ago require independent clarification, for example via oxygen isotopes in sulfate. Here we show <2.31 Ga sedimentary barium sulfates (barites) from the Turee Creek Basin, W. Australia with positive sulfur isotope anomalies of ∆33S up to + 1.55‰ and low δ18O down to -19.5‰. The unequivocal origin of this combination of signals is sulfide oxidation in meteoric water. Geochemical and sedimentary evidence suggests that these S-isotope anomalies were transferred from the paleo-continent under an oxygenated atmosphere. Our findings indicate that incipient oxidative continental weathering, ca. 2.8-2.5 Ga or earlier, may be diagnosed with such a combination of low δ18O and high ∆33S in sulfates.

Mesophilic microorganisms build terrestrial mats analogous to Precambrian microbial jungles.

Development of Archean paleosols and patterns of Precambrian rock weathering suggest colonization of continents by subaerial microbial mats long before evolution of land plants in the Phanerozoic Eon. Modern analogues for such mats, however, have not been reported, and possible biogeochemical roles of these mats in the past remain largely conceptual. We show that photosynthetic, subaerial microbial mats from Indonesia grow on mafic bedrocks at ambient temperatures and form distinct layers with features similar to Precambrian mats and paleosols. Such subaerial mats could have supported a substantial aerobic biosphere, including nitrification and methanotrophy, and promoted methane emissions and oxidative weathering under ostensibly anoxic Precambrian atmospheres. High C-turnover rates and cell abundances would have made these mats prime locations for early microbial diversification. Growth of landmass in the late Archean to early Proterozoic Eons could have reorganized biogeochemical cycles between land and sea impacting atmospheric chemistry and climate.

Authigenic iron oxide proxies for marine zinc over geological time and implications for eukaryotic metallome evolution.

Here, we explore enrichments in paleomarine Zn as recorded by authigenic iron oxides including Precambrian iron formations, ironstones, and Phanerozoic hydrothermal exhalites. This compilation of new and literature-based iron formation analyses track dissolved Zn abundances and constrain the magnitude of the marine reservoir over geological time. Overall, the iron formation record is characterized by a fairly static range in Zn/Fe ratios throughout the Precambrian, consistent with the shale record (Scott et al., 2013, Nature Geoscience, 6, 125-128). When hypothetical partitioning scenarios are applied to this record, paleomarine Zn concentrations within about an order of magnitude of modern are indicated. We couple this examination with new chemical speciation models to interpret the iron formation record. We present two scenarios: first, under all but the most sulfidic conditions and with Zn-binding organic ligand concentrations similar to modern oceans, the amount of bioavailable Zn remained relatively unchanged through time. Late proliferation of Zn in eukaryotic metallomes has previously been linked to marine Zn biolimitation, but under this scenario the expansion in eukaryotic Zn metallomes may be better linked to biologically intrinsic evolutionary factors. In this case, zinc's geochemical and biological evolution may be decoupled and viewed as a function of increasing need for genome regulation and diversification of Zn-binding transcription factors. In the second scenario, we consider Archean organic ligand complexation in such excess that it may render Zn bioavailability low. However, this is dependent on Zn-organic ligand complexes not being bioavailable, which remains unclear. In this case, although bioavailability may be low, sphalerite precipitation is prevented, thereby maintaining a constant Zn inventory throughout both ferruginous and euxinic conditions. These results provide new perspectives and constraints on potential couplings between the trajectory of biological and marine geochemical coevolution.

Role of extracellular polymeric substances in the surface chemical reactivity of Hymenobacter aerophilus, a psychrotolerant bacterium.

Bacterial surface layers, such as extracellular polymeric substances (EPS), are known to play an important role in metal sorption and biomineralization; however, there have been very few studies investigating how environmentally induced changes in EPS production affect the cell's surface chemistry and reactivity. Acid-base titrations, cadmium adsorption assays, and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the surface reactivities of Hymenobacter aerophilus cells with intact EPS (WC) or stripped of EPS (SC) and purified EPS alone. Linear programming modeling of titration data showed SC to possess functional groups corresponding to phosphoryl (pKa approximately 6.5), phosphoryl/amine (pKa approximately 7.9), and amine/hydroxyl (pKa approximately 9.9). EPS and WC both possess carboxyl groups (pKa approximately 5.1 to 5.8) in addition to phosphoryl and amine groups. FT-IR confirmed the presence of polysaccharides and protein in purified EPS that can account for the additional carboxyl groups. An increased ligand density was observed for WC relative to that for SC, leading to an increase in the amount of Cd adsorbed (0.53 to 1.73 mmol/liter per g [dry weight] and 0.53 to 0.59 mmol/liter per g [dry weight], respectively). Overall, the presence of EPS corresponds to an increase in the number and type of functional groups on the surface of H. aerophilus that is reflected by increased metal adsorption relative to that for EPS-free cells.

A cytosolic trans-activation domain essential for ammonium uptake.

Polytopic membrane proteins are essential for cellular uptake and release of nutrients. To prevent toxic accumulation, rapid shut-off mechanisms are required. Here we show that the soluble cytosolic carboxy terminus of an oligomeric ammonium transporter from Arabidopsis thaliana serves as an allosteric regulator essential for function; mutations in the C-terminal domain, conserved between bacteria, fungi and plants, led to loss of transport activity. When co-expressed with intact transporters, mutants inactivated functional subunits, but left their stability unaffected. Co-expression of two inactive transporters, one with a defective pore, the other with an ablated C terminus, reconstituted activity. The crystal structure of an Archaeoglobus fulgidus ammonium transporter (AMT) suggests that the C terminus interacts physically with cytosolic loops of the neighbouring subunit. Phosphorylation of conserved sites in the C terminus are proposed as the cognate control mechanism. Conformational coupling between monomers provides a mechanism for tight regulation, for increasing the dynamic range of sensing and memorizing prior events, and may be a general mechanism for transporter regulation.

Development and use of fluorescent nanosensors for metabolite imaging in living cells.

To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.

A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants.

A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

Interlaboratory comparison of autoradiographic DNA profiling measurements: precision and concordance.

Knowledge of the expected uncertainty in restriction fragment length polymorphism (RFLP) measurements is required for confident exchange of such data among different laboratories. The total measurement uncertainty among all Technical Working Group for DNA Analysis Methods laboratories has previously been characterized and found to be acceptably small. Casework cell line control measurements provided by six Royal Canadian Mounted Police (RCMP) and 30 U.S. commercial, local, state, and Federal forensic laboratories enable quantitative determination of the within-laboratory precision and among-laboratory concordance components of measurement uncertainty typical of both sets of laboratories. Measurement precision is the same in the two countries for DNA fragments of size 1000 base pairs (bp) to 10,000 bp. However, the measurement concordance among the RCMP laboratories is clearly superior to that within the U.S. forensic community. This result is attributable to the use of a single analytical protocol in all RCMP laboratories. Concordance among U.S. laboratories cannot be improved through simple mathematical adjustments. Community-wide efforts focused on improved concordance may be the most efficient mechanism for further reduction of among-laboratory RFLP measurement uncertainty, should the resources required to fully evaluate potential cross-jurisdictional matches become burdensome as the number of RFLP profiles on record increases.

Expression of a wheat ADP-glucose pyrophosphorylase gene during development of normal and water-stress-affected anthers.

In wheat (Triticum aestivum L.), water deficit during meiosis in the microspore mother cells (MMCs) induces pollen abortion, resulting in the failure of fertilization and a reduction in grain set. In stressed plants, meiosis in MMCs proceeds normally but subsequent pollen development is arrested. Unlike normal pollen grains, which accumulate starch during the late maturation phase, stress-affected anthers contain pollen grains with little or no starch. Stress also alters the normal distribution of starch in the anther wall and connective tissue. To determine how starch biosynthesis is regulated within the developing anthers of stressed plants, we studied the expression of ADP-glucose pyrophosphorylase (AGP), which catalyzes the rate limiting step of starch biosynthesis. Two partial-length cDNAs corresponding to the large subunit of AGP were amplified by RT-PCR from anther RNA, and used as probes to monitor AGP expression in developing anthers of normal and water-stressed plants. These clones, WAL1 and WAL2, had identical deduced amino acid sequences and shared 96% sequence identity at the nucleic acid level. In normal anthers, AGP expression was biphasic, indicating that AGP expression is required for starch biosynthesis both during meiosis and later during pollen maturation. AGP expression in stressed anthers was not affected during the first phase of starch accumulation, but was strongly inhibited during the second phase. We conclude from these results that the reduced starch deposition later in the development of stressed pollen could be the result of a lower expression of AGP. However, this inhibition of AGP expression is unlikely to be the primary cause of male sterility because anatomical symptoms of pollen abortion are observed prior to the time when AGP expression is inhibited.

Induction of Male Sterility in Wheat by Meiotic-Stage Water Deficit Is Preceded by a Decline in Invertase Activity and Changes in Carbohydrate Metabolism in Anthers.

Water deficit during meiosis in pollen mother cells of wheat (Triticum aestivum L.) induces male sterility, which can reduce grain set by 40 to 50%. In plants stressed during meiosis and then rewatered, division of pollen mother cells proceeds normally but subsequent pollen development is arrested 3 or 4 d later. An inhibition of starch accumulation within the pollen grain suggested that an alteration in carbohydrate metabolism or assimilate supply may be involved in pollen abortion. We measured levels of various carbohydrates and activities of key enzymes of Suc metabolism and starch synthesis at different stages of pollen development in anthers collected from well-watered and water-stressed plants. Compared to controls, soluble sugars increased in anthers stressed during meiosis, then decreased at later poststress stages. Sucrose and myoinositol accounted for part of the sugar accumulation. The activity of soluble acid invertase declined 4-fold during the stress period and never recovered thereafter. Sucrose synthase activity during starch accumulation in pollen was also lower in the anthers of plants stressed at meiosis. Stress had little negative effect on the activities of ADP-glucose pyrophosphorylase or soluble and granule-bound starch synthase during starch accumulation in pollen, although at the earlier stages, ADP-glucose pyrophosphorylase activity in stressed anthers was slightly lower compared to controls. The results suggest that carbohydrate starvation per se and inhibition of the enzymes of starch synthesis probably were not responsible for the stress-induced pollen abortion. Instead, an inability to metabolize incoming sucrose to hexoses may be involved in this developmental lesion.

Evidence against the Regulation of Grain Set by Spikelet Abscisic Acid Levels in Water-Stressed Wheat.

A possible role of abscisic acid (ABA) in the regulation of grain set in water-stressed wheat (Triticum aestivum L.) was investigated using a split root system to dry half the roots while the remainder were kept watered. Water uptake by the wet roots maintained the leaf water potential at the normal level, whereas the ABA produced in the dry roots was transported to the spike. This caused the spikelet ABA level to increase to the same extent as when the entire root system was stressed to permit a drop in the leaf water potential. In spite of this, the former treatment did not induce a reduction in grain set, whereas the latter did. Thus, contrary to previous reports, water stress-induced changes in spikelet ABA level alone do not appear to regulate grain set.

Water-stress-induced ethylene production in wheat : a fact or artifact?

Effects of water stress on ethylene evolution from excised leaf segments and intact plants of wheat (Triticum aestivum L. cv Katepwa) were studied. Excised leaf segments of 8 day or 6 week old plants were dried until they lost 8% of their fresh weight (water potential about -2.3 megapascals). These and nondried control leaf segments (water potential about -1.0 megapascal) were sealed in glass tubes, and their ethylene production rates were compared by head space analysis via gas-chromatography. The dried leaves of both ages produced significantly more ethylene than the corresponding controls. However, when 6 week old intact plants were water-stressed by withholding water supply, and their ethylene production measured using a continuousflow system, no increase in ethylene was deteceted despite a drop in water potential to -2.9 megapascals over 6 days. Even the leaf segments excised from plants that had been subjected to water stress for 2, 4, or 6 days produced no more ethylene (in sealed tubes) than the leaves from well-watered plants. In fact, the ethylene production by these segments decreased with the increase in the severity of stress experienced by the plants. The results show that the commonly reported overproduction of ethylene by excised leaves subjected to rapid drying represents an artifact, which has little relevance to the water stress responses of intact wheat plants.