Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume.

Mice are able to navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. We developed a protocol to monitor behavior and record Ca 2+ transients in dorsal CA1 stratum pyramidale neurons at the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. Ca 2+ transients were imaged by an epifluorescence miniscope focused through a GRIN lens on dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. We describe the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. We provide the step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. We provide information on real time tracking of the mouse position to automate the start of the trials and delivery of a sugar water reward. In addition, we provide information on the use of an Intan board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a FLIR camera tracking mouse position. Moreover, we delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using NorMCorre followed by identification of regions of interest (ROIs) with EXTRACT. Finally, we describe use of artificial neural network (ANN) machine learning to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume. SUMMARY: This protocol describes how to investigate the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol including the surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain and behavioral data to decode the mouse position from ROI neural activity.

Formulation development and characterization of plant-based alternatives to pâté using forest ingredients.

Due to the expanding global population and environmental concerns about meat production from livestock, there is a great demand for alternative ingredients. Beech achene (BA) and sessile oak acorn (SOA) were recently proposed as protein- and carbohydrate-rich novel food ingredients. This study used their roasted kernels to develop and characterize four formulations of spreadable vegetable paste (with 10% BAK, 10% SOAK, 5% SOAK + 5% BAK, and control-just with roasted sunflower kernel). The substitution of sunflower kernel with 10% BAK caused a decrease in the energy value of vegetable paste, while 10% SOAK and 5% SOAK + 5% BAK, an increase. Syneresis was higher in formulations with forest ingredients, most notably in those containing BAK. The SOAK also caused a decrease in the pH of vegetable pastes that included it. All forest formulations had a large total colour difference compared to the control sample, driven by its intensity decrease (less in that with BAK than in the other two). The acceptance rate was reasonable for all formulations, although the overall score was significantly lower (slightly liked) in the vegetable paste formulated only with BAK than in the others (moderately liked); thus, the consumer's purchase intention too (only 4.9% for that with 10% SOAK). Formulation with 10% BAK had a higher hardness, adhesiveness, gumminess, and chewiness than the others, while that with the 5% SOAK and 5% BAK mixture showed the most robust network structure. In conclusion, BA and SOA kernels are suitable for manufacturing plant-based alternatives to pâté if used in proper concentrations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05852-7.

Characterization of some Fagaceae kernels nutritional composition for potential use as novel food ingredients.

The present study attempts to characterise Fagaceae kernels as a promising source of nutritional compounds for potential use as novel food ingredients. Thus, the proximate and mineral composition of some kernels (beech achene-BA, sessile oak acorn-SOA, turkey oak acorn-TOA, and red oak acorn-ROA), total phenolic content, individual polyphenols, and cytotoxicity of their aqueous extracts, respectively, the fatty acid composition of kernel oils were investigated using physicochemical and analytical techniques. Results revealed that BAK is rich in lipid and protein, OAKs in carbohydrates. All tested kernels contain high oleic-linoleic acid oils. BAK is abundant in phenolic acids, OAKs in hydrolysable tannins. Only BA and SOA kernels exert cytotoxicity against human fibroblasts. In all kernels, macroelements are dominated by K and microelements by Cu, Mn, and Fe. In conclusion, BA and OA kernels could be alternatively used as protein-rich, respectively, starch-rich ingredients in food.

Fibre type-dependent response of broiler muscles to dietary antioxidant supplementation for oxidative stability enhancement.

The influence of dietary antioxidants and quality of oil on the oxidative and physico-chemical properties of chicken broiler breast and thigh meat stored was studied in either an oxygen-enriched (HiOx: 80% O2/20% CO2) or an air-permeable polyvinylchloride (PVC) packaging system during retail display at 2-4°C for up to 14 and 7 d, respectively. Broilers were fed on a diet with either a low-oxidised (peroxide value (POV) 23 meq O2/kg) or a high-oxidised (POV 121 meq O2/kg) oil, supplemented with or without an algae/selenium-based antioxidant with organic minerals, for 42 d. Lipid and protein oxidation, myofibrillar protein profile and purge loss were analysed. In both packaging systems, lipid oxidation (thiobarbituric acid-reactive substances [TBARS]) was inhibited by up to 65% and 57% in chicken breast and thigh, respectively, with an antioxidant-supplemented diet compared to those without. In both breast and thigh samples, protein sulfhydryls and water-holding capacity (purge loss) were better protected by the antioxidant dietary treatment, regardless of oil quality. Thigh muscles had up to sevenfold greater TBARS formation and more myosin heavy chain losses compared to breast samples. Antioxidant supplementation was more protective against lipid oxidation and water-holding capacity in the group fed on high-oxidised oil compared to those fed on low-oxidised oil. The results suggest that dietary antioxidants can minimise the negative impact of oxidised oil on broiler meat quality, and this protection was more pronounced for thigh than breast muscle, indicating inherent variations between muscle fibre types.

Augmentation of water-holding and textural properties of breast meat from oxidatively stressed broilers by dietary antioxidant regimens.

1. The impact of dietary antioxidants and degree of oil oxidation on textural attributes of chicken broiler breast meat stored in oxygen-enriched, air-permeable polyvinylchloride and skin packaging systems during retail display at 2-4°C for up to 21 d was assessed. 2. Broilers were fed on diets either with a low-oxidised oil (peroxide 23 mEq O2/kg) or with a high-oxidised oil (peroxide 121 mEq O2/kg), with or without an algae-based antioxidant and organic mineral antioxidant supplement for 42 d. 3. Fatty acids and radical scavenging activities of the diets were estimated. Meat colour, pH, myofibrillar protein profile and textural traits were measured. 4. Diets with high-oxidised oil reduced stearic, linoleic and linolenic acid content compared to low-oxidised oil samples, regardless of antioxidant supplementation. Meat colour and pH varied among dietary treatments throughout storage. Meat samples from the antioxidant dietary group, irrespective of oil oxidation level, had lower amounts of purge and cooking losses compared to the unsupplemented diets. For all packaging systems, meat shear force was significantly higher for broilers fed on high-oxidised diets. 5. The results demonstrate that dietary antioxidant supplementation can minimise the negative impact of oxidised oil on the quality of broiler meat packaged in different atmospheric environments.

Niobium direct detectors for fast and sensitive terahertz spectroscopy.

We report the performance of a niobium hot-electron bolometer designed for laboratory terahertz spectroscopy. The antenna-coupled detector can operate above 4.2 K and has fast (subnanosecond) response. Detailed microwave measurements of performance over a wide range of operating conditions were correlated with quantitative terahertz measurements. The maximum responsivity is 4 x 10(4) VW with a noise equivalent power at the detector of 2 x 10(-14) W/Hz(12), approaching the intrinsic thermal fluctuation limit for the device. This detector enables a variety of novel laboratory spectroscopy measurements.

Evaluation of low-ash poultry meal as a protein source in canine foods.

Eight mature female dogs (18.0 +/- 0.2 kg) were used in a replicated 4 x 4 Latin square experiment to determine the feeding value of low-ash poultry meal (PM) in a complete food fed to dogs. All foods contained graded concentrations of PM (10.4 to 32.5% DM), resulting in foods that were 10, 15, 20, and 25% CP. Daily DMI averaged 284 +/- 14 g/d. An increase in PM resulted in an increase in fecal moisture from 44.7 to 55.1% (linear; P < 0.01), and fecal DM output increased from 24.8 to 31.6 g/d (linear; P < 0.05). Ileal DM flow increased from 27.1 to 40.7 g/d (linear; P < 0.01). Small intestinal DM digestibility decreased from 90.4 to 86.1% (linear; P < 0.01) and total-tract DM digestibility decreased from 91.2 to 89.4% (linear; P < 0.01) as PM increased. Large intestinal DM digestibility increased from 8.4 to 21.1% with increasing PM (linear; P < 0.05). Fecal excretion of CP increased from 5.6 to 10.0 g/d (linear; P < 0.01) and ileal flow of CP increased from 6.9 to 15.6 g/d (linear; P < 0.01) as PM increased. Small intestinal CP digestibility was unaffected with treatment (P > 0.05). Large intestinal CP digestibility increased from 21.6 to 37.1% (linear; P < 0.05) with increasing PM. Total-tract CP digestibility increased from 81.0 to 86.6% (linear; P < 0.01) as PM increased. Arginine had the highest overall digestibility ranging from 88.5 to 91.3%, whereas cysteine had the lowest digestibility, ranging from 67.1 to 71.4%. These data indicate that PM is a highly digestible protein source for canine foods with inclusions of 10.4 to 32.5% of DM.

Heme oxygenase-1 protects against vascular constriction and proliferation.

Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.

Activation of NF-kappaB induced by H(2)O(2) and TNF-alpha and its effects on ICAM-1 expression in endothelial cells.

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB.

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.

X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin.

The FeZn derivative of purple acid phosphatase from porcine uterus (FeZnUf) and its phosphate complex (FeZnUf.PO4) have been characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges, to gain insight into the nature of the FeZn active site as well as the phosphate binding mode. Pre-edge data show that both FeZnUf and FeZnUf.PO4 have a 6-coordinate iron site. The iron site has an average Fe-O/N bond length of 2.01-2.02 A, which can be resolved into subshells of 1.92 and 2.11 A for FeZnUf.PO4. On the other hand, the zinc site has a shell of scatterers at 2.02-2.03 A plus one scatterer at ca. 2.4 A. These metal-ligand bond lengths are consistent with the nature of the ligands deduced from spectroscopic studies or identified in the crystal structure of the related kidney bean purple acid phosphatase (KBPAP). The outer-sphere analysis indicates an Fe-Zn separation of approximately 3.3 A in both FeZnUf and FeZnUf.PO4, consistent with the presence of an M2(mu-OR)2 diamond core as found in the crystal structures of KBPAP, calcineurin, and protein phosphatase 1. The Fe-P and Zn-P bond distances in FeZnUf.PO4 are determined to be 3.23 and 3.18 A, respectively, indicating that phosphate binds to the dinuclear center in a bidentate mode; such a mode has been observed in oxoanion complexes of KBPAP, calcineurin, and alkaline phosphatase, as well as in a number of synthetic FeFe and FeZn complexes. The implications of these structural results on the mechanism of phosphatase action are discussed.

Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.

Primary lung fibroblasts were isolated from patients with idiopathic pulmonary fibrosis (HIPF), from normal human lung tissue (NH), from rats treated with 75% oxygen and paraquat (PA), and from normal adult rats (NR). Serum-free media conditioned by each fibroblast strain were tested on the human A549 cell line (HIPF and NH media) or on primary alveolar epithelial cells (AEC) isolated from normal adult rats (PA or NR media). Over 20-h incubation, HIPF- or PA-conditioned media induced DNA fragmentation and significant decreases in total recoverable DNA and cell number of A549 or AEC, respectively; NH or NR media had no significant effect relative to serum-free unconditioned media. Apoptosis of A549 and AEC was detected by altered nuclear morphology and was confirmed by terminal deoxynucleotidyl transferase-mediated bio-dUTP nick end labeling. The endonuclease inhibitors 10 microM aurintricarboxylic acid and 50 microM zinc inhibited HIPF-induced apoptosis of A549 cells by 68 and 71%, respectively. Both apoptosis and necrosis were induced by HIPF and PA media in a concentration-dependent manner. These results suggest that altered fibroblasts emerging during fibrotic lung injury release a soluble factor(s) capable of inducing cell death and net loss of AEC.

X-ray absorption studies of the ferrous active site of isopenicillin N synthase and related model complexes.

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium (M(r) 38,400) is an iron-containing enzyme that aerobically catalyzes the four-electron oxidative ring closure reactions of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV), forming the beta-lactam and thiazolidine rings of isopenicillin N. Here, we report Fe K-edge X-ray absorption studies that provide insight into the iron coordination environment and the effect of substrate and nitric oxide binding. Our analysis reveals an iron(II) coordination environment consisting of two N/O-containing ligands at 2.01 +/- 0.02 A, three N/O ligands at 2.15 +/- 0.02 A, and one C/O scatterer at approximately 2.6-2.7 A. Three His ligands are associated with the 2.15-A shell, while an unsymmetrically chelated carboxylate is associated with a scatterer at 2.01 and at 2.6-2.7 A, a combination which is consistent with the ligand environment deduced from 1H NMR studies [Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. The remaining scatterer at 2.01 A is assigned to a coordinated solvent molecule, most likely hydroxide, which can act as the proton acceptor for the incoming substrate. ACV binding to Fe(II)IPNS evinces an Fe-S interaction at 2.35 +/- 0.02 A, indicative of the coordination of substrate cysteine thiolate to the metal center. Analysis of the Fe(II)IPNS-ACV-NO data reveals one Fe-N at 1.71 +/- 0.02 A, three Fe-(N,O) at 2.04 +/- 0.02 A, one Fe-S at 2.32 +/- 0.02 A, and one Fe-(C,O) at 2.61 +/- 0.02 A, the short Fe-N bond being derived from the binding of NO. Our EXAFS conclusions, supported by corresponding analysis of relevant model complexes, corroborate and refine the working model for the Fe(II) coordination environment developed from previous spectroscopic studies.

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase.

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters.

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.