Predicting enteric methane emission in lactating Holsteins based on reference methane data collected by the GreenFeed system

Methane emission is not included in the current breeding goals for dairy cattle mainly due to the expense and difficulty in obtaining sufficient data to generate accurate estimates of the relevant traits. While several models have been developed to predict methane emission from milk spectra using reference methane data obtained by the respiration chamber, SF6 and sniffer methods, the prediction of methane emission from milk mid-infrared (MIR) spectra using reference methane data collected by the GreenFeed system has not yet been explored. Methane emission was monitored for 151 cows using the GreenFeed system. Prediction models were developed for daily and average (for the trial period of 12 or 14 days) methane production (g/d), yield (g/kg DM intake (DMI)) and intensity (g/kg of fat- and protein-corrected milk) using partial least squares regression. The predictions were evaluated in 100 repeated validation cycles, where animals were randomly partitioned into training (80%) and testing (20%) populations for each cycle. The best performing model was observed for average methane intensity using MIR, parity and DMI with validation coefficient of determination (R²_val) and RMSE of prediction of 0.66 and 4.7 g/kg of fat- and protein-corrected milk, respectively. The accuracy of the best models for average methane production and average methane yield were poor (R²_val = 0.28 and 0.12, respectively). A lower accuracy of prediction was observed for methane intensity and production (R²_val = 0.42 and 0.17) when daily records were used while prediction for methane yield was comparable to that for average methane yield (R²_val = 0.16). Our results suggest the potential to predict methane intensity with moderate accuracy. In this case, prediction models for average methane values were generally better than for daily measures when using the GreenFeed system to obtain reference methane emission measurements.     

Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants

Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Ya?ez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.     

Positive Feedbacks in Seagrass Ecosystems: Implications for Success in Conservation and Restoration

Abstract Seagrasses are threatened by human activity in many locations around the world. Their decline is often characterized by sudden ecosystem collapse from a vegetated to a bare state. In the 1930s, such a dramatic event happened in the Dutch Wadden Sea. Before the shift, large seagrass beds (Zostera marina) were present in this area. After the construction of a large dam and an incidence of the “wasting disease” in the early 1930s, these meadows became virtually extinct and never recovered despite restoration attempts. We investigated whether this shift could be explained as a critical transition between alternative stable states, and whether the lack of recovery could be due to the high resilience of the new turbid state. We analyzed the depth distribution of the historical meadows, a long-term dataset of key factors determining turbidity and a minimal model based on these data. Results demonstrate that recovery was impossible because turbidity related to suspended sediment was too high, probably because turbidity was no longer reduced by seagrass itself. Model simulations on the positive feedback suggest indeed the robust occurrence of alternative stable states and a high resilience of the current turbid state. As positive feedbacks are common in seagrasses, our findings may explain both the worldwide observed collapses and the low success rate of restoration attempts of seagrass habitats. Therefore, appreciation of ecosystem resilience may be crucial in seagrass ecosystem management.     

Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues

Differentiation and maintenance of recirculating effector memory CD8 T cells (T(EM)) depends on prolonged cognate Ag stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (T(RM)), which contribute to rapid local protection upon pathogen re-exposure, is unknown. Memory CD8αβ(+) T cells within small intestine epithelium are well-characterized examples of T(RM), and they maintain a long-lived effector-like phenotype that is highly suggestive of persistent Ag stimulation. This study sought to define the sources and requirements for prolonged Ag stimulation in programming this differentiation state, including local stimulation via cognate or cross-reactive Ags derived from pathogens, microbial flora, or dietary proteins. Contrary to expectations, we found that prolonged cognate Ag stimulation was dispensable for intestinal T(RM) ontogeny. In fact, chronic antigenic stimulation skewed differentiation away from the canonical intestinal T cell phenotype. Resident memory signatures, CD69 and CD103, were expressed in many nonlymphoid tissues including intestine, stomach, kidney, reproductive tract, pancreas, brain, heart, and salivary gland and could be driven by cytokines. Moreover, TGF-β-driven CD103 expression was required for T(RM) maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal T(RM) differed from classic models of T(EM) ontogeny and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.     

Genetic, biochemical, and molecular characterization of the polypeptide transport-associated domain of Escherichia coli BamA

The BamA protein of Escherichia coli plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The C-terminal domain of BamA folds into an integral outer membrane β-barrel, and the N terminus forms a periplasmic polypeptide transport-associated (POTRA) domain for OMP reception and assembly. We show here that BamA misfolding, caused by the deletion of the R44 residue from the α2 helix of the POTRA 1 domain (ΔR44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ΔR44 site. This highlights the importance of the side chain orientation of the α2 helix residues for normal POTRA 1 activity. The ΔR44-mediated POTRA folding defect and its correction by the insertion of alanine were further demonstrated by using a construct expressing just the soluble POTRA domain. Besides misfolding, the expression of BamA(ΔR44) from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA(ΔR44) was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamA(ΔR44, A18S) background, OMP biogenesis improved dramatically, and this correlated with improved BamA folding, BamA-SurA interactions, and LptD (lipopolysaccharide transporter) biogenesis. The presence of the A18S substitution in the wild-type BamA protein did not affect the activity of BamA. The discovery of the A18S substitution in the α1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ΔR44 underscores the importance of the helix 1 and 2 regions in BamA folding.     

The PDZ scaffold NHERF-2 interacts with mGluR5 and regulates receptor activity

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to G(q) to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1 and 5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na(+)/H(+) exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.     

Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways

To define better the subcellular mechanism of heat shock (HS)-induced cardioprotection, we examined the effect of HS, as well as selective expression of individual HS proteins (HSPs), on cell injury in neonatal rat ventricular myocytes (NRVM). HS was induced in NRVM by a rapid elevation of temperature to 42 degrees C for 20 min followed by 20-24 h of recovery at 37 degrees C. Other NRVM were infected with a replication-deficient adenovirus encoding HSP27 or HSP70. On the same day, all groups were subjected to metabolic inhibition (MI). Cell injury was assayed by measurement of the percentage of total lactate dehydrogenase released, the percentage of cells staining with trypan blue, or TdT-mediated dUTP nick-end labeling, whereas cell signaling was assayed by immunoblot analysis and coimmunoprecipitation. Before MI, the viability of all treated groups did not differ significantly from control NRVM. HS resulted in a significant increase in HSP70 and HSP27 expression. Infection with either virus caused a significant increase in selective HSP content compared with control NRVM. HS protected NRVM from injury. Selective expression of HSP27 or HSP70 alone was not protective in NRVM, but dual infection with both viral vectors (HSP27 + HSP70) was protective. HS and HSP27 + HSP70 expression caused increased paxillin localization in the membrane fraction, which persisted in response to MI, compared with control NRVM. HS increased the integrin-paxillin-focal adhesion kinase interaction, whereas targeted inhibition of focal adhesion kinase activity abolished the integrin-paxillin association and resulted in an increase in cell death. HS and HSP27 + HSP70 expression increased the association of members of the focal adhesion complex and protected NRVM against irreversible injury. Cytoskeletal-based signaling pathways at focal adhesion junctions may represent a unique pathway of cardioprotection.     

DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells

Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.