Transcription factor Twist1 drives fibroblast activation to promote kidney fibrosis via signaling proteins Prrx1/TNC

The transcription factor Twist1 plays a vital role in normal development in many tissue systems and continues to be important throughout life. However, inappropriate Twist1 activity has been associated with kidney injury and fibrosis, though the underlying mechanisms involved remain incomplete. Here, we explored the role of Twist1 in regulating fibroblast behaviors and the development kidney fibrosis. Initially Twist1 protein and activity was found to be markedly increased within interstitial myofibroblasts in fibrotic kidneys in both humans and rodents. Treatment of rat kidney interstitial fibroblasts with transforming growth factor-ß1 (a profibrotic factor) also induced Twist1 expression in vitro. Gain- and loss-of-function experiments supported that Twist1 signaling was responsible for transforming growth factor-ß1-induced fibroblast activation and fetal bovine serum-induced fibroblast proliferation. Mechanistically, Twist1 protein promoted kidney fibroblast activation by driving the expression of downstream signaling proteins, Prrx1 and Tnc. Twist1 directly enhanced binding to the promoter of Prrx1 but not TNC, whereas the promoter of TNC was directly bound by Prrx1. Finally, mice with fibroblast-specific deletion of Twist1 exhibited less Prrx1 and TNC protein abundance, interstitial extracellular matrix deposition and kidney inflammation in both the unilateral ureteral obstruction and ischemic-reperfusion injury-induced-kidney fibrotic models. Inhibition of Twist1 signaling with Harmine, a ß-carboline alkaloid, improved extracellular matrix deposition in both injury models. Thus, our results suggest that Twist1 signaling promotes the activation and proliferation of kidney fibroblasts, contributing to the development of interstitial fibrosis, offering a potential therapeutic target for chronic kidney disease.

Long-Acting Nanohybrid Hydrogel Induces Persistent Immunogenic Chemotherapy for Suppressing Postoperative Tumor Recurrence and Metastasis.

Despite the continuous advancement of surgical resection techniques, postoperative tumor recurrence and metastasis remain a huge challenge. Here, we constructed an injectable curcumin/doxorubicin-loaded nanoparticle (NanoCD) hydrogel, which could effectively inhibit tumor regrowth and metastasis via reshaping the tumor immune microenvironment (TIME) for highly effective postsurgical cancer treatment. NanoCD was prepared by the controlled assembly of curcumin (CUR) and doxorubicin (DOX) via π-π stacking and hydrogen bonding in the presence of human serum albumin. To facilitate prolonged treatment of postsurgical tumors, NanoCD was further incorporated into the temperature-sensitive Poloxamer 407 gel (NanoCD@Gel) for intracavity administration. Mechanistically, DOX induced the generation of intracellular reactive oxygen species (ROS) and CUR reduced the ROS metabolism by inhibiting thioredoxin reductase (TrxR). The synergy of DOX and CUR amplified intracellular ROS levels and thus resulted in enhanced immunogenic cell death (ICD) of tumor cells. Upon being injected into the tumor cavity after resection, the in situ-generated NanoCD@Gel allowed the local release of CUR and DOX in a controlled manner to induce local chemotherapy and persistently activate the antitumor immune response, thereby achieving enhanced immunogenic chemotherapy with reduced systemic toxicity. Our work provides an elegant strategy for persistently stimulating effective antitumor immunity to prevent postsurgical tumor recurrence and metastasis.

Effects of oregano essential oil on in vitro ruminal fermentation, methane production, and ruminal microbial community.

Different inclusion rates of oregano essential oil (OEO) were investigated for their effects on ruminal in vitro fermentation parameters, total gas, methane production, and bacterial communities. Treatments were (1) control, 0 mg/L of OEO (CON); 13 mg/L (OEO1); 52 mg/L (OEO2); 91 mg/L (OEO3); and 130 mg/L (OEO4), each incubated with 150 mL of buffered rumen fluid and 1,200 mg of substrate for 24 h using the Ankom in vitro gas production system (Ankom Technology Corp., Fairport, NY). Treatment responses were statistically analyzed using polynomial contrasts. Digestibility of DM, NDF, and ADF increased quadratically with increasing OEO inclusion rates. Digestibility of DM and NDF were highest for OEO2, whereas ADF digestibility was highest for OEO3, compared with CON, with the remaining treatments being intermediate and similar. Ammonia nitrogen concentrations decreased from CON at a quadratic rate with increasing OEO inclusion rates, and OEO2 had the lowest concentration compared with the other groups. Total VFA, acetate, propionate, butyrate, valerate, and isovalerate concentrations linearly decreased with increasing OEO inclusion rates. Total gas production levels by CON and OEO4 were greater than those of OEO1, OEO2, and OEO3 in a quadratic response, and methane production linearly decreased from CON, compared with OEO4, at a decreasing rate with OEO inclusion rates. As determined by 16S rRNA sequencing, the α biodiversity of ruminal bacteria was similar among OEO inclusion rates. Increasing OEO inclusion rates linearly increased the relative abundance of Prevotella and Dialister bacteria. Several bacteria demonstrated different polynomial responses, whereas several bacteria were similar among increasing OEO inclusion rates. These results suggested that OEO supplementation can modify ruminal fermentation to alter VFA concentrations and reduce methane emissions by extensively altering the ruminal bacterial community, suggesting an optimal feeding rate for future animal studies of approximately 52 mg/L for mature ruminants.

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

High Sensitivity X-Ray Detectors with Low Degradation Based on Deuterated Halide Perovskite Single Crystals.

Methylammonium lead single crystal (MAPbI3 SC) possesses superior optoelectronic properties and low manufacturing cost, making it an ideal candidate for X-ray detection. However, the ionic migration of the perovskites usually leads to instability, dark current drift, and hysteresis of the detector, limiting their applications in well-established technologies. Here, a series of X-ray detectors of MAPbI3 SCs are reported with different degrees of deuteration (DxMAPbI3, x = 0, 0.15, 0.75, 0.99). By controlling the content of deuterium (D) in organic cations, the sensitivity, detection limits, ion migration, and resistivity of the detector can be controlled, thereby improving its performance. Due to stronger hydrogen bonds (N─D···I), the ion activation energy significantly increases to 886 meV. Consequently, the D0.99MAPbI3 SC detector shows more than five-fold enhancement, achieving a record-high mobility-lifetime (µτ) product of 5.39 × 10-2 cm2 V-1, with an ultrahigh sensitivity of 2.18 × 106 µC Gy-1 cm-2 under 120 keV hard X-ray and a low detection limit of 4.8 nGyair s-1, as well as long-term stability. The study provides a straightforward strategy for constructing ultrasensitive X-ray detection and imaging systems based on perovskite SCs.

Causal Effects of Oxidative Stress on Diabetes Mellitus and Microvascular Complications: Insights Integrating Genome-Wide Mendelian Randomization, DNA Methylation, and Proteome.

BACKGROUND: Oxidative stress (OS) is involved in the development of diabetes, but the genetic mechanisms are not completely understood. We integrated multi-omics data in order to explore the genetic relations between OS-related genes, diabetes mellitus, and microvascular complications using Mendelian randomization and colocalization analysis. METHODS: Summary-level data related to OS were acquired from respective studies of methylation, expression, and protein abundance quantitative trait loci. Genetic associations concerning diabetes, diabetic nephropathy (DN), and diabetic retinopathy (DR) were derived from the FinnGen study. Summary-data-based Mendelian randomization (SMR) analysis was conducted to evaluate the correlations between molecular features concerned with OS-related genes and diabetes mellitus, along with its microvascular complications. Additionally, we performed colocalization analysis to determine if the detected signal pairs shared a causal genetic variant. RESULTS: At the genetic level, we identified ten potential causal associations of oxidative stress genes with diabetes, along with microvascular complications, through SMR and colocalization analysis. After integrating the DNA methylation quantitative trait loci (mQTL) and expression QTL (eQTL) data, our analyses revealed a correlation between the methylation site cg26343298 and reduced expression of TP53INP1, supporting the protective role of cg26343298 methylation on type 2 diabetes (T2D) and diabetic nephropathy. Similarly, an inverse association was observed between gene methylation and expression in CHEK1 (cg07110182), confirming the beneficial effect of modification of CHEK1 by cg07110182 in diabetic retinopathy. In addition, upregulation of SUOX expression by cg22580629 was linked to a reduced risk of diabetic retinopathy. At circulating protein levels, genetically predicted a higher level of ICAM1 (OR 1.05, 95%CI 1.03-1.08) was positively connected with the risk of diabetic retinopathy. CONCLUSIONS: This SMR study elucidated that the TP53INP1 gene was putatively associated with T2D and DN risk, while the SUOX and CHEK1 genes were associated with DR risk through oxidative stress mechanisms. Additionally, our study showed a positive correlation between the ICAM-1 protein and DR. These findings may enhance our understanding of their pathogenesis and suggest new therapeutic targets for clinical practice.

Global and multi-partition local network analysis of scalp EEG in West syndrome before and after treatment.

West syndrome is an epileptic disease that seriously affects the normal growth and development of infants in early childhood. Based on the methods of brain topological network and graph theory, this article focuses on three clinical states of patients before and after treatment. In addition to discussing bidirectional and unidirectional global networks from the perspective of computational principles, a more in-depth analysis of local intra-network and inter-network characteristics of multi-partitioned networks is also performed. The spatial feature distribution based on feature path length is introduced for the first time. The results show that the bidirectional network has better significant differentiation. The rhythmic feature change trend and spatial characteristic distribution of this network can be used as a measure of the impact on global information processing in the brain after treatment. And localized brain regions variability in features and differences in the ability to interact with information between brain regions have potential as biomarkers for medication assessment in WEST syndrome. The above shows specific conclusions on the interaction relationship and consistency of macro-network and micro-network, which may have a positive effect on patients' treatment and prognosis management.

Value of turbo spin echo-based diffusion-weighted imaging in the differential diagnosis of benign and malignant solitary pulmonary lesions.

To quantitatively assess the diagnostic efficacy of multiple parameters derived from multi-b-value diffusion-weighted imaging (DWI) using turbo spin echo (TSE)-based acquisition techniques in patients with solitary pulmonary lesions (SPLs). A total of 105 patients with SPLs underwent lung DWI using single-shot TSE-based acquisition techniques and multiple b values. The apparent diffusion coefficient (ADC), intravoxel incoherent motion (IVIM) parameters, and lesion-to-spinal cord signal intensity ratio (LSR), were analyzed to compare the benign and malignant groups using the Mann-Whitney U test and receiver operating characteristic analysis. The Dstar values observed in lung cancer were slightly lower than those observed in pulmonary benign lesions (28.164 ± 31.950 versus 32.917 ± 34.184; Z = -2.239, p = 0.025). The LSR values were significantly higher in lung cancer than in benign lesions (1.137 ± 0.581 versus 0.614 ± 0.442; Z = - 4.522, p < 0.001). Additionally, the ADC800, ADCtotal, and D values were all significantly lower in lung cancer than in the benign lesions (Z = - 5.054, -5.370, and -6.047, respectively, all p < 0.001), whereas the f values did not exhibit any statistically significant difference between the two groups. D had the highest area under the curve (AUC = 0.887), followed by ADCtotal (AUC = 0.844), ADC800 (AUC = 0.824), and LSR (AUC = 0.789). The LSR, ADC800, ADCtotal, and D values did not differ statistically significantly in diagnostic effectiveness. Lung DWI using TSE is feasible for differentiating SPLs. The LSR method, conventional DWI, and IVIM have comparable diagnostic efficacy for assessing SPLs.

Primary Structural Units "D+A-" Ion Pairs Dominating Near-Infrared Photothermal Conversion of Organic Ionic Cocrystals.

The specific stacking mode of D/A blocks is often considered to largely determine the physicochemical properties of cocrystals. However, this rule may fail when encountering a large degree of (integer or near-integer) charge transfer situations. Herein, we explore the extensive correlations between the possible smallest structural units, stacking modes, and near-infrared photothermal conversion (NIR-PTC) properties of F4TCNQ-based cocrystals with typical features of integer-charge-transfer. Surprisingly, these cocrystals with distinct stacking modes display analogous D-A interactions, broad red-shift absorption, ultrafast (1-3 ps) relaxation dynamics of excited states, and excellent NIR-PTC properties. This supports that the resulting "D+A-" ion pairs from integer-charge-transfer may serve as the primary structural units beneath the secondary stacking modes to dominate the property of cocrystals. The stacking modes play an important but only secondary role. This work provides new insights into the structure-dynamics-property correlations and modular design of organic cocrystals for PTC and other applications.

Dietary oregano essential oil supplementation alters meat quality, oxidative stability, and fatty acid profiles of beef cattle.

This study was conducted to elucidate the effects of oregano essential oil (OEO) supplementation on the meat quality, antioxidant capacity, and nutritional value of the longissimus thoracis muscle in steers. Steers were divided into three groups (n = 9) and fed either a basal diet, or a basal diet supplemented with 130 mg/d OEO, or 230 mg/d OEO for 390 days. The results demonstrated that dietary OEO supplementation increased the total antioxidant capacity and activity of catalase, glutathione peroxidase, and superoxide dismutase, and decreased pH30min, pH24h, cooking loss, and malondialdehyde content. OEO increased the concentrations of polyunsaturated fatty acids and conjugated linoleic acid. In contrast, saturated fatty acids decreased, accompanied by increased essential amino acids, flavor amino acids, and total amino acids in the longissimus thoracis muscle. In summary, dietary OEO supplementation promotes the nutritional and meat quality of beef by maintaining its water-holding capacity and meat color, enhancing its antioxidative capacity, and preventing lipid oxidation.

Determining α-helical and ß-sheet secondary structures via pulsed electron spin resonance spectroscopy.

A new method has been developed to determine α-helical and ß-sheet secondary structural components of aqueous and membrane-bound proteins using pulsed electron paramagnetic resonance (EPR) spectroscopy. The three-pulse electron spin echo envelope modulation (ESEEM) technique was used to detect weakly coupled (2)H-labeled nuclei on side chains in the proximity of a strategically placed nitroxide spin-label up to 8 Å away. Changes in the ESEEM spectra for different samples correlate directly to periodic structural differences between α-helical and ß-sheet motifs. These distinct trends were demonstrated with α-helical (M2δ subunit of the acetylcholine receptor) and ß-sheet (ubiquitin) peptides in biologically relevant sample environments.

Convergence or divergence? The effects of economic openness on low-carbon innovation in Chinese manufacturing industry.

Low-carbon innovation can address both economic and environmental concerns; patterns of low-carbon innovation convergence can determine the effectiveness of mitigating the adverse consequences of climate change. Considering that economic openness has a huge impact on the development of innovation capability, this paper uses a conditional ß convergence model to examine the convergence of low-carbon innovation in Chinese manufacturing industry and its relationship with economic openness. We incorporate the spatial spillover effect into the convergence function by constructing spatial error model, spatial lag model, and spatial Durbin model. Based on a panel data set of 30 Chinese provinces over the period 2004-2016, the results show that low-carbon innovation in Chinese manufacturing industry has a strong feature of conditional ß convergence. The convergence rate of low-carbon innovation is slightly slowed down by economic openness, and the main reason is that the spillover effect is weak and the convergence rate is slow in lower open areas, so the convergence rate of the whole country is slowed down by that of the lower open areas. Although the economic openness in adjacent areas can contribute to the development of local innovation ability, but generally speaking, economic openness in local areas takes a stronger effect in promoting the convergence of low-carbon innovation than that in adjacent areas. The findings have important policy implications as they suggest the need for a more equal degree of economic openness among Chinese provinces to speed up the convergence of low-carbon innovation.

1,2-Dicarbofunctionalization of Trifluoromethyl Alkenes with Pyridinium Salts via a Cycloaddition/Visible-Light-Enabled Fragmentation Cascade.

Although trifluoromethyl alkenes have great synthetic potential, their 1,2-difunctionalization has been a challenge. In this Letter, we disclose the first 1,2-dicarbofunctionalization of trifluoromethyl alkenes with pyridinium salts via a cascade process involving a base-promoted [3 + 2] cycloaddition followed by a visible-light-mediated Norrish-type-II fragmentation. This protocol allows for the formation of pyridines bearing a trifluoromethyl-substituted quaternary center in moderate to excellent yields under mild conditions.

Values of Apparent Diffusion Coefficient and Lesion-to-Spinal Cord Signal Intensity in Diagnosing Solitary Pulmonary Lesions: Turbo Spin-Echo versus Echo-Planar Imaging Diffusion-Weighted Imaging.

OBJECTIVE: This study is aimed at comparing the image quality and diagnostic performance of mean apparent diffusion coefficient (ADC) and lesion-to-spinal cord signal intensity ratio (LSR) derived from turbo spin-echo diffusion-weighted imaging (TSE-DWI) and echo-planar imaging- (EPI-) DWI in patients with a solitary pulmonary lesion (SPL). METHODS: 33 patients with SPL underwent chest imaging using EPI-DWI and TSE-DWI with b = 600 s/mm2 in free breathing. A comparison of the distortion ratio (DR), signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) was drawn between the two techniques using a Wilcoxon signed-rank test. The interprotocol reproducibility between quantitative parameters of EPI-DWI and TSE-DWI was evaluated using a Bland-Altman plot. ADCs and LSRs derived from EPI-DWI and TSE-DWI were calculated and compared between malignant and benign groups using the Mann-Whitney test. RESULTS: TSE-DWI had similar SNR and CNR compared with EPI-DWI. DR was significantly lower on TSE-DWI than EPI-DWI. ADC and LSR showed slightly higher values with TSE-DWI, while the Bland-Altman analysis showed unacceptable limits of agreement between the two sequences. ADC and LSR of both DWI techniques differed significantly between lung cancer and benign lesions (P < 0.05). The LSR(EPI-DWI) showed the highest area under the curve (AUC = 0.818), followed by ADC(EPI-DWI) (AUC = 0.789), ADC(TSE-DWI) (AUC = 0.781), and LSR(TSE-DWI) (AUC = 0.748), respectively. Among these parameters, the difference in diagnostic accuracy was not statistically significant. CONCLUSIONS: TSE-DWI provides significantly improved image quality in patients with SPL as compared with EPI-DWI. However, there was no difference in diagnostic efficacy between these two techniques, according to ADC and LSR.

Comparative transcriptome and histological analyses provide insights into the skin pigmentation in Minxian black fur sheep (Ovis aries).

BACKGROUND: Minxian black fur (MBF) sheep are found in the northwestern parts of China. These sheep have developed several special traits. Skin color is a phenotype subject to strong natural selection and diverse skin colors are likely a consequence of differences in gene regulation. METHODS: Skin structure, color differences, and gene expression (determined by RNA sequencing) were evaluated the Minxian black fur and Small-tail Han sheep (n = 3 each group), which are both native Chinese sheep breeds. RESULTS: Small-tail Han sheep have a thicker skin and dermis than the Minxian black fur sheep (P < 0.01); however, the quantity of melanin granules is greater (P < 0.01) in Minxian black fur sheep with a more extensive distribution in skin tissue and hair follicles. One hundred thirty-three differentially expressed genes were significantly associated with 37 ontological terms and two critical KEGG pathways for pigmentation ("tyrosine metabolism" and "melanogenesis" pathways). Important genes from those pathways with known involvement in pigmentation included OCA2 melanosomal transmembrane protein (OCA2), dopachrome tautomerase (DCT), tyrosinase (TYR) and tyrosinase related protein (TYRP1), melanocortin 1 receptor (MC1R), and premelanosome protein (PMEL). The results from our histological and transcriptome analyses will form a foundation for additional investigation into the genetic basis and regulation of pigmentation in these sheep breeds.

Effects of oregano essential oil on the ruminal pH and microbial population of sheep.

Oregano essential oil (OEO), which has antimicrobial properties, may be used for altering the ruminal pH and microbial populations of sheep, as observed by the altered volatile fatty acid patterns. To further elucidate the effects of OEO on ruminal pH and microbial populations of sheep, 3 German merino sheep × local sheep crossbred rams with permanent ruminal fistulas were randomly assigned to a 3 × 3 Latin square design with 12-d periods. The treatments were as follows: control (CON); OEO4: OEO supplied at 4 g•d-1; and OEO7: OEO supplied at 7 g•d-1. Starting on day 11, rumen fluid was collected at 0 h, and at 4, 8, 12, 24 and 48 h after supplying OEO, and then pH values of rumen fluid were immediately measured. The abundance of microbial populations was determined by using qPCR. The ruminal pH values were similar among the sheep from all treatments. The abundance of ruminal fungi was higher for the sheep supplied OEO7 compared with the sheep supplied CON and OEO4, especially at 4 and 12 h. The abundance of ruminal protozoa decreased with supplied OEO, indicating that OEO could inhibit the protozoa. The abundance of the total ruminal bacteria was similar for the sheep from all treatments, but R. flavefaciens, R. albus and F. succinogenes increased in the sheep supplied OEO4 compared with those in the sheep supplied CON, however, the sheep supplied OEO7 had higher abundances of R. flavefaciens than the sheep supplied CON. These results demonstrated that supplying OEO to sheep did not affect the ruminal pH but could shift the rumen microbial population to one with less protozoa. Supplying OEO can preferentially enhance the growth of certain rumen microbial populations, but the shifts were influenced by the supply rate. Therefore, supplying low amount (i.e. 4 g•d-1) of OEO could have positive effects on ruminal microbial populations, whereas supplying elevated doses of OEO could be detrimental to those same ruminal microbial populations.

Investigating the Secondary Structure of Membrane Peptides Utilizing Multiple 2H-Labeled Hydrophobic Amino Acids via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

An electron spin echo envelope modulation (ESEEM) approach was used to probe local secondary structures of membrane proteins and peptides. This ESEEM method detects dipolar couplings between 2H-labeled nuclei on the side chains of an amino acid (Leu or Val) and a strategically placed nitroxide spin-label in the proximity up to 8 Å. ESEEM spectra patterns for different samples correlate directly to the periodic structural feature of different secondary structures. Since this pattern can be affected by the side chain length and flexibility of the 2H-labeled amino acid used in the experiment, it is important to examine several different hydrophobic amino acids (d3 Ala, d8 Val, d8 Phe) utilizing this ESEEM approach. In this work, a series of ESEEM data were collected on the AChR M2δ membrane peptide to build a reference for the future application of this approach for various biological systems. The results indicate that, despite the relative intensity and signal-to-noise level, all amino acids share a similar ESEEM modulation pattern for α-helical structures. Thus, all commercially available 2H-labeled hydrophobic amino acids can be utilized as probes for the further application of this ESEEM approach. Also, the ESEEM signal intensities increase as the side chain length gets longer or less rigid. In addition, longer side chain amino acids had a larger 2H ESEEM FT peak centered at the 2H Larmor frequency for the i ± 4 sample when compared to the corresponding i ± 3 sample. For shorter side chain amino acids, the 2H ESEEM FT peak intensity ratio between i ± 4 and i ± 3 was not well-defined.

The effect of periodic energy restriction on growth performance, serum biochemical indices, and meat quality in sheep.

Energy is a key factor regulated by the neuroendocrine system. The objective of this study was to compare growth performance, serum biochemical indices, carcass traits, meat quality, and nutrient composition of lambs fed restricted metabolizable energy (ME) intakes to mimic the seasonal changes of the natural grasslands of northwest China. Nineteen male Dorper × Small Tailed Han lambs were assigned to treatments, control (CON) fed at 1.0 MJ/W0.75 × d-1 throughout the 180 d study, and restricted ME (RES) fed by sequentially restricting ME every 30 d (0.56, 0.84, 1.0, 0.84, 0.56, and 0.28 MJ/W0.75 × d-1). Lambs were harvested at the end of the study. Lambs fed the CON diet demonstrated higher (P < 0.05) average daily gain (ADG) compared with RES-fed lambs (158.79 and 57.01 g/d, for CON and RES, respectively), although ADG were lower in each period with the last period being a negative ADG for RES-fed lambs resulting in a loss of body weight. Both treatments demonstrated lower ADG in the last 3 study periods compared with the first 2 periods, which may be explained by cold stress (<-3 °C) increasing the lamb's energy requirement. Feeding lambs a restricted ME diet resulted in lower (P < 0.05) blood serum glucose, triglycerides, total protein, calcium, phosphorus, immunoglobulin G, immunoglobulin M, and immunoglobulin A concentrations compared with CON-fed lambs, but both treatments were similar (P > 0.05) for cholesterol and high-density lipoprotein-cholesterol. Feeding lambs a restricted ME diet increased (P < 0.05) meat pH, while reducing the cooked meat rate compared with CON-fed lambs. Thus, the carcass yield, rib eye muscle area, and shear force were lower (P < 0.05) for RES-fed lambs compared with CON-fed lambs. The meat water concentration was higher (P < 0.05), whereas the concentrations of dry matter, crude fat, crude protein, and carbohydrate were lower (P < 0.05) for lambs fed a RES ME diet compared to CON-fed lambs. These results demonstrate that sequentially restricting ME intake, as natural grasslands might experience during seasonal forage quality and quantity changes, resulted in a reduction of body weight because lambs were mobilizing body reserves as a source of ME. The reduction in concentrations of key serum nutrients, as well as carcass traits and meat composition, demonstrate the body is responding to the meet the lambs ME requirements, which has implications for both meat quality and the animal's physiological functions.