A time-resolved multi-omics atlas of transcriptional regulation in response to high-altitude hypoxia across whole-body tissues.

High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we use sheep as an animal model for low-to-high altitude translocation. We generate multi-omics data including whole-genome sequences, time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterize transcriptional changes of all genes in each tissue, and examine multi-tissue temporal dynamics and transcriptional interactions among genes. Particularly, we identify critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identify TAD-constrained cis-regulatory elements, which suppress the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicate that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provide time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals.

Rational Design and Hierarchical Assembly of a Genetically Engineered Resilin-Silk Copolymer Results in Stiff Hydrogels.

Genetically engineered protein polymers, which can combine different unique peptide sequences from natural protein materials, offer great opportunities for making advanced materials with well-defined structures and properties. Here we report for the first time biosynthesis and self-assembly of a recombinant resilin-silk (RS) copolymer consisting of repeating units of silk and resilin blocks. The copolymer in aqueous solution self-assembled into nanoparticles, and the assembled nanoparticles further form nano- to microscale fibers in a time-dependent manner at body temperature, whereas such fibers were not formed upon incubation of the copolymer at either low or high temperatures. In contrast, a resilin-like polypeptide without the silk blocks exhibited a typical thermoresponsive dual-phase transition behavior and was incapable of self-assembling into fibers. More interestingly, the microscale fibers self-assembled from a moderately concentrated RS solution (20 wt %) could interact to give a self-supporting, semitransparent hydrogel with elastic modulus at approximately 195 Pa. Furthermore, photo-cross-linking of either freshly prepared or annealed RS copolymer led to the formation of stiff hydrogels and the material mechanical property was superior upon annealing of the RS solution for a longer time up to 4 h, with elastic modulus ranging from 2.9 to 7.0 kPa. These results not only shed light on the fundamental hierarchical assembly mechanism of a new family of genetically engineered RS copolymer but also suggest future opportunities for these thermoresponsive polymers in fabrication of hydrogel materials with tunable mechanical properties for diverse applications.

Fabrication of Protein Films from Genetically Engineered Silk-Elastin-Like Proteins by Controlled Cross-Linking.

Protein films are an important class of materials for applications in biomedicine and biotechnology. The rational design of protein polymer sequence and selection of customized cross-linking offers unique opportunities to engineer desirable functionalities into these materials. Here we report the fabrication of a series of films with tunable physiochemical properties from genetically engineered silk-elastin-like proteins (SELPs). The SELPs were recombinantly biosynthesized with different ratios of silk-to-elastin blocks and periodic cysteine residues incorporated in the elastin blocks. A disulfide cross-linking method was developed for the preparation of the SELP films under mild oxidative conditions with a low concentration of hydrogen peroxide, in comparison with the physical cross-linking method used with the organic solvent methanol. Film properties were characterized for solubility, water absorption, hydrophilicity, surface roughness, and cyto-compatibility. The results indicated that customized cross-linking supported the fabrication of films from the SELP proteins with tunable features, including smooth, water stable film materials with cyto-compatibility. Interestingly, hydrogen peroxide oxidation was a preferred cross-linking method for the cysteine-containing SELPs with a low ratio of the silk-to-elastin blocks, whereas methanol treatment was suitable for fabricating films from the SELPs with a high ratio of silk-to-elastin blocks into stable films with rougher surfaces. We anticipate that an appropriate combination of polymer design and cross-linking might be a useful strategy for the preparation of protein films for diverse applications.

Rationally Designed Redox-Sensitive Protein Hydrogels with Tunable Mechanical Properties.

Protein hydrogels are an important class of materials for applications in biotechnology and medicine. The fine-tuning of their sequence, molecular weight, and stereochemistry offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here we report a new family of redox-sensitive protein hydrogels with controllable mechanical properties composed of recombinant silk-elastin-like protein polymers (SELPs). The SELPs were designed and synthesized with different ratios of silk-to-elastin blocks that incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally responsive in solution and rapidly formed hydrogels at body temperature under physiologically relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative gelation was performed at other physiologically relevant temperatures, and this resulted in hydrogels with tunable mechanical properties, thus, providing a secondary level of control over hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive features and tunable mechanical properties may be potentially useful as biomaterials with broad applications in controlled drug delivery and tissue engineering.

Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

[Effect of Electroacupuncture at "Yanglingquan" (GB 34) Acupoint on White Blood Cell Count and Gallbladder Wall Thickness in Rabbits with Acute Cholecystitis].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Yanglingquan"(GB 34) acupoint on white blood cell (WBC) count and gallbladder wall thickness in rabbits with acute cholecystitis, so as to explore the inherent correlation between the gallbladder and GB 34 acupoint according to the theory of Chinese medicine. METHODS: Thirty-two male rabbits were randomly and equally divided into control group, model group, EA "Yanglingquan"(GB 34) group (EA-GB 34) and EA "Yinlingquan"(SP 9) group (EA-SP 9). The acute cholecystitis model was induced by intra-gallbladder injection of escherichia coli liquid. EA stimulation was delivered once per day for consecutive 7 days. WBC count and gallbladder wall thickness were measured to assess the effects of EA treatment. HE staining was carried out to examine the pathological changes of the gallbladder tissue. RESULTS: Compared with the control group,WBC count and gallbladder wall thickness of the model group were significantly increased (P<0. 05), and those changes were reversed by EA intervention at either GB 34 or SP 9 acupoints (P<0. 05). As to WBC count, there was no significant difference between EA-GB 34 and EA-SP 9 groups (P>0. 05), whereas for reducing gallbladder wall thickness, EA stimulation at GB 34 acupoint resulted in a better effect than that of EA at SP 9 acupoint (P<0. 05). CONCLUSION: EA produces positive therapeutic benefits on acute cholecystitis in rabbits. It seems that stimulation at "Yan - glingquan" acupoint gives rise to a better effect than that of "Yinlingquan" acupoint in particular for the reduction of gallbladder wall thickhess.

[Effect of electroacupunctu-e intervention on serum IL-17 and IL-23 contents in rheumatoid arthritis rats].

OBJECTIVE: To observe the influence of electroacupuncture (EA) stimulation of "Zusanli" (ST 36) and "Kunlun" (BL 60) on serum and knee-joint IL-17 and IL-23 contents in rheumatoid arthritis (RA) rats so as to study its underlying mechanism in improving RA. METHODS: Male Wistar rats were randomly allocated to normal control, RA model, EA and Prednisolone groups (n = 8 in each group). The RA model was established by intra-planta injection of incomplete Freund's adjuvant and type II collagen. EA (2 Hz/100 Hz,1-2 mA)was applied to bilateral "Zusanli"(ST 36) and "Kunlun"(BL 60) for 30 min, once daily for 10 days. The rats' ankle diameter was measured, and IL-17 and IL-23 contents in the serum and the knee-joint cavity were assayed by ELISA and Western blot, respectively. RESULTS: In comparison with the normal control group, the rats' ankle diameter, serum IL-17 and IL-23 contents and knee-joint IL-17 and IL-23 protein expression levels were significantly increased in the model group (P < 0.05). After EA and Prednisolone treatment, compared with the model group, all the rats' ankle diameter, serum IL-17 and IL-23 contents and knee-joint IL-17 and IL-23 protein expression levels were decreased remarkably (P < 0.05). No obvious differences were found between the EA and Prednisolone groups in the aforementioned indexes (P > 0.05), except IL-17 protein expression level (being markedly lower in the Prednisolone group than in the EA group). CONCLUSION: EA intervention can reduce inflammatory reaction of the ankle-joint in RA rats, which may be related to its effects in down-regulating serum and knee-joint IL-17 and IL-23 levels.

Semaphorin 4D and hypoxia-inducible factor-1α overexpression is related to prognosis in colorectal carcinoma.

AIM: To investigate semaphorin 4D (Sema4D) and hypoxia-inducible factor-1α (HIF-1α) expression in colorectal carcinoma and evaluate their clinicopathological and prognostic significance. METHODS: Eighty-six curatively resected colorectal carcinoma patients at different stages of disease were randomly selected from the group of patients who underwent surgery, and none of them received preoperative radiochemotherapy. Normal proximal adjacent bowel tissue, which served as an internal control, was obtained from 52 randomly selected patients. Immunohistochemistry was performed to analyze the expression of Sema4D and the tumor angiogenesis-related protein HIF-1α in normal colorectal tissues and colorectal carcinoma tissues. The relationships between the expression and clinical characters and prognosis were analyzed. RESULTS: HIF-1α and Sema4D were positively expressed in 58% and 60% of colorectal carcinoma tissues, respectively. Significantly lower expression levels were observed in normal mucosa (8% and 12%, respectively). HIF-1α and Sema4D expression was closely correlated with histological tumor type, tumor-node-metastasis (TNM) stage, and lymphatic metastasis (P<0.05), but not with age or tumor size (P>0.05). HIF-1α and Sema4D protein expression was significantly correlated with prognosis of colorectal carcinoma, as determined by Spearman rank correlation analysis (r=0.567; P<0.01). Multivariate Cox analysis revealed that only Sema4D expression played a significant role in predicting patient prognosis (P<0.05). CONCLUSION: These findings suggest that HIF-1α and Sema4D expression correlates with histological tumor type, TNM stage, and lymphatic metastasis in colorectal carcinoma and that Sema4D is a prognostic indicator of colorectal carcinoma.

[Analysis of the fractal properties of the birth weight of Liangshan semi-fine wool sheep.].

Understanding of animal population genetic structure about quantitative traits is an important step in animal breeding. In the present study, the data of birth weight of the Liangshan semi-fine wool sheep was first studied by the fractal theory of nonlinear theory. The information dimension, correlation dimension, none-scale range, and the scale range were calculated. The results showed that (1) the information dimensions of the birth weight from 1996 to 2004 ranged from 0.66529 to 0.90675. The none-scale range and the range of the variation were very large. This indicates that the population had large dimensions of the BW (birth weight), large variation, and great potential in breeding; (2) the correlation dimensions ranged from 0.62438 to 0.86528, which indicates that the genetic structures of the population were highly correlated; (3) both of the two dimensions could reveal the fractal properties of the population genetic structure through two different aspects, and it was useful in studying the population genetic structure and animal breeding.

[Differentiation of Bos grunniens, Bos Taurus, and Bubalus from meat products mixture based on mitochondrion 12S rRNA gene].

Mitochondrial DNA sequence is highly conserved within species. Gene 12S rRNA is able to endure degeneration and high temperature, which allows identification of feedstuff, fresh meat, processed meat, and traceability. In the present study, three unique restriction sites were detected in the fragments of mitochondrial 12S rRNA gene regions amplified with universal primer, which were able to distinguish Bos grunniens, Bos. taurus, and Bubalus in fresh meat and processed meat mixture. The fragment of yak was digested to 134 bp and 318 bp, scalper 134 bp and 318 bp, and buffalo 86 bp and 367 bp. The specific locus and digestion were verified by sequencing analysis. There was no difference between PCR amplification products from various treatments at different temperatures (i.e., 100, 120, 140, 160, and 180). However, the sig-nal was weak at 120 and above. This method is simple, fast and cheap in identification of fresh meat and processed meat.

[Construction of genetic linkage map and QTL detection of chromosome 3 in sheep].

Based on the reference family of Sichuan liangshan semi-fine sheep, the genetic linkage map was constructed by Crimap 2.4 software, which was used to detect the QTLs for growth traits by QTLExpress with nine microsatellite markers located on chromosome 3 in sheep. The results indicated that: (1) the mean of the polymorphic information content was 0.606 (0.378-0.738), and the mean heterozygosity was 0.650 (0.404-0.766); (2) the length of the linkage map was 339.8 cM, which was longer than the length of map built by the international major drawing organization for sheep with the average distance of markers of 42.5 cM; (3) The QTLs for weaning weight, weaning daily gain, and 2.5 year weight were detected at 99 cM, 219 cM, 273 cM, respectively. The effect of QTLs on weaning daily gain trait and 2.5 year weight trait were significant, except for weaning weight trait.