A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems

Formation Mechanisms of Odor Substances in Mutton and Deodorization Methods: A Review

Mutton is a high-quality meat with high nutritional value. However, the typical species-specific flavor (odor) of mutton affects its quality and limits its consumer acceptance. Studies have shown that the major substances that cause mutton odor are 4-alkyl branched fatty acids, 3-methylindole, 4-methylphenol, stearic acids, and amino acids. The composition and formation pathway of mutton odor substances are complex and influenced by various factors such as nutrition, feeding system, variety, sex, age, and tissue position, so it is difficult to reduce mutton odor. Therefore, this article reviews the sources and formation mechanisms of mutton odor substances, the factors affecting the formation of mutton odor substances, and deodorization methods, which will provide a basis for scientifically regulating the flavor of mutton and its products

Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in Sulfolobus islandicus REY15A

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from Sulfolobus islandicus. It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc in vitro. These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. S. islandicus strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation

Infrared Imaging of Magnetic Octupole Domains in Non-collinear Antiferromagnets

Magnetic structure plays a pivotal role in the functionality of antiferromagnets (AFMs), which not only can be employed to encode digital data but also yields novel phenomena. Despite its growing significance, visualizing the antiferromagnetic domain structure remains a challenge, particularly for non-collinear AFMs. Currently, the observation of magnetic domains in non-collinear antiferromagnetic materials is feasible only in Mn$_{3}$Sn, underscoring the limitations of existing techniques that necessitate distinct methods for in-plane and out-of-plane magnetic domain imaging. In this study, we present a versatile method for imaging the antiferromagnetic domain structure in a series of non-collinear antiferromagnetic materials by utilizing the anomalous Ettingshausen effect (AEE), which resolves both the magnetic octupole moments parallel and perpendicular to the sample surface. Temperature modulation due to the AEE originating from different magnetic domains is measured by the lock-in thermography, revealing distinct behaviors of octupole domains in different antiferromagnets. This work delivers an efficient technique for the visualization of magnetic domains in non-collinear AFMs, which enables comprehensive study of the magnetization process at the microscopic level and paves the way for potential advancements in applications.Comment: National Science Review in pres

Effects of L-proline on the Growth Performance, and Blood Parameters in Weaned Lipopolysaccharide (LPS)-challenged Pigs

This trail was conducted to study the effect of L-proline on the growth performance, and blood parameter in the weaned lipopolysaccharide (LPS)-challenged pigs. Thirty six pigs (9.13±0.85 kg) were assigned randomly to dietary treatments in a 2×3 factorial arrangement in a 20-d growth assay. Factors were intraperitoneal injection with saline or LPS, and three dietary L-proline supplement levels (0%, 0.5%, or 1.0%). On d 10, blood samples were collected at 3 h after LPS (100 μg LPS/kg body weight [BW]) or saline injection. On d 20 of the trial, all pigs were orally administrated D-xylose (0.1 g/kg BW) at 2 h, and blood samples were collected at 3 h after LPS or saline injection. As a result, dietary supplementation with 0.5% proline had a tendency to increase average daily gain (ADG) in piglets during d 10 to 20 (p = 0.088). Without LPS challenge, dietary supplementation with 1.0% proline had no effect on growth hormone (GH) concentrations on d 10 (p>0.05), but decreased it after LPS challenge (p<0.05). There was LPS challenge×proline interaction for GH concentrations on d 10 (p<0.05). Dietary supplementation with 1.0% proline decreased glucagon concentration on d 10 after LPS challenge (p<0.05). In addition, dietary supplementation with proline increased superoxide dismutase (SOD) activity significantly on d 10 and 20 (p<0.05), and 1.0% proline increased heat shock proteins-70 concentration on d 10 (p<0.05). Moreover, proline supplementation increased diamine oxidase (DAO) concentrations after LPS challenge (p<0.05). There was LPS challenge×proline interaction for DAO (p<0.05). Furthermore, dietary supplementation with 1.0% proline increased the D-xylose level when no LPS challenge (p<0.05). These results indicate that proline supplementation could improve growth performance, increase SOD activities, and has a positive effect on the gastrointestinal tract digestibility in early weaned pigs

Chlorogenic Acid Decreases Intestinal Permeability and Increases Expression of Intestinal Tight Junction Proteins in Weaned Rats Challenged with LPS

Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21 ± 1 d of age; 62.26 ± 2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS

An underground positioning algorithm of offset error correction based on RSSI

In view of problems that the weighted centroid algorithm based on RSSI would appear error of three-side measuring area selection, only use distances between nodes to measure influence of beacon nodes, but neglect standard deviations of errors in every signal transmission path are different, an underground positioning algorithm of offset error correction based on RSSI was proposed. The algorithm combines with the idea of weighted centroid, and uses RSSI distance model to measure distance between nodes. After obtained the distance information, it can measure the distance error between nodes through probability distribution analysis of standard deviation of the random variables in the ranging error, and then corrects the offset error of the random variables, finally obtains the positioning results with higher precision. The simulation results show that the positioning accuracy improves 19% compared with the weighted centroid algorithm, and the stability of positioning results also has great improvement