Chemical composition and microbiota changes across musk secretion stages of forest musk deer.

Forest musk deer is the most important animal for natural musk production, and the musk composition changes periodically during musk secretion, accompanied by variation in the com-position of deer-symbiotic bacteria. GC-MS and 16S rRNA sequencing were conducted in this study, the dynamic changes to correlated chemical composition and the microbiota across musk secretion periods (prime musk secretion period, vigorous musk secretion period and late musk secretion period) were investigated by integrating its serum testosterone level in different mating states. Results showed that the testosterone level, musk composition and microbiota changed with annual cycle of musk secretion and affected by its mating state. Muscone and the testosterone level peaked at vigorous musk secretion period, and the microbiota of this stage was distinct from the other 2 periods. Actinobacteria, Firmicutes and Proteobacteria were dominant bacteria across musk secretion period. PICRUSt analysis demonstrated that bacteria were ubiquitous in musk pod and involved in the metabolism of antibiotics and terpenoids in musk. "Carbohydrates and amino acids," "fatty acids and CoA" and "secretion of metabolites" were enriched at 3 periods, respectively. Pseudomonas, Corynebacterium, Clostridium, Sulfuricurvum were potential biomarkers across musk secretion. This study provides a more comprehensive understanding of genetic mechanism during musk secretion, emphasizing the importance of Actinobacteria and Corynebacterium in the synthesis of muscone and etiocholanone during musk secretion, which required further validation.

[Systematic review and Meta-analysis of Gusongbao preparation in treatment of primary osteoporosis].

This study aims to provide evidence for clinical practice by systematically reviewing the efficacy and safety of Gusongbao preparation in the treatment of primary osteoporosis(POP). The relevant papers were retrieved from four Chinese academic journal databases and four English academic journal databases(from inception to May 31, 2022). The randomized controlled trial(RCT) of Gusongbao preparation in the treatment of POP was included after screening according to the inclusion and exclusion criteria. The quality of articles was evaluated using risk assessment tools, and the extracted data were subjected to Meta-analysis in RevMan 5.3. A total of 657 articles were retrieved, in which 15 articles were included in this study, which involved 16 RCTs. A total of 3 292 patients(1 071 in the observation group and 2 221 in the control group) were included in this study. In the treatment of POP, Gusongbao preparation+conventional treatment was superior to conventional treatment alone in terms of increasing lumbar spine(L2-L4) bone mineral density(MD=0.03, 95%CI[0.02, 0.04], P<0.000 01) and femoral neck bone mineral density, reducing low back pain(MD=-1.69, 95%CI[-2.46,-0.92], P<0.000 1) and improving clinical efficacy(RR=1.36, 95%CI[1.21, 1.53], P<0.000 01). Gusongbao preparation was comparable to similar Chinese patent medicines in terms of improving clinical efficacy(RR=0.95, 95%CI[0.86, 1.04], P=0.23). Gusongbao preparation was inferior to similar Chinese patent medicines in reducing traditional Chinese medicine syndrome scores(MD=1.08, 95%CI[0.44, 1.71], P=0.000 9) and improving Chinese medicine syndrome efficacy(RR=0.89, 95%CI[0.83, 0.95], P=0.000 4). The incidence of adverse reactions of Gusongbao preparation alone or combined with conventio-nal treatment was comparable to that of similar Chinese patent medicines(RR=0.98, 95%CI[0.57, 1.69], P=0.94) or conventio-nal treatment(RR=0.73, 95%CI[0.38, 1.42], P=0.35), and the adverse reactions were mainly gastrointestinal discomforts. According to the available data, Gusongbao preparation combined with conventional treatment is more effective than conventional treatment alone in increasing lumbar spine(L2-L4) bone mineral density and femoral neck bone mineral density, reducing low back pain, and improving clinical efficacy. The adverse reactions of Gusongbao preparation were mainly gastrointestinal discomforts, which were mild.

Correlation Analysis between Muskrat (Ondatra zibethicus) Musk and Traditional Musk.

Muskrat musk is considered to be a potential substitute for traditional musk. However, little is known about the similarity between muskrat musk and musk, and whether it is related to muskrat age. In this study, muskrat musk (MR1, MR2, and MR3) were from 1, 2, and 3-year-old muskrats, respectively, and white musk (WM) and brown musk (BM) were picked from male forest musk deer. The results indicated that muskrat musk had higher similarity to WM than BM. Further research showed that RM3 had the highest matched degree with WM. By significantly different metabolite analysis, we found that 52 metabolites continue to increase from 1- to 3-year-old muskrats. In total, 7 and 15 metabolites were significantly decreased in RM1 vs. RM2 and RM2 vs. RM3, respectively. Meanwhile, 30 and 17 signaling pathways were observed from increased and decreased metabolites, respectively. The increased metabolites mainly entailed enrichment in amino acid biosynthesis and metabolism, steroid hormone biosynthesis, and fatty acid biosynthesis. In conclusion, muskrat musk from three-year-old muskrat is a relatively good substitute for white musk, and the result also implies that these biological processes of amino acid biosynthesis and metabolism, steroid hormone biosynthesis, and fatty acid biosynthesis are beneficial to the secretion of muskrat musk.

Study on Anti-inflammatory Mechanism of Blueberry based on Network Pharmacology and Molecular Docking Technology.

The Batman-TCM research platform based on network pharmacology was used to predict the reverse targets of 11 active components of blueberry. The anti-inflammatory target genes of these components were extracted by comparing them with the anti-inflammatory drug target genes in the GeneCards database. GO enrichment and KEGG pathway, as well as protein interaction analysis of these anti-inflammatory target genes, were carried out using the String database. The antiinflammatory component-target-action pathway map of blueberry was constructed using the Cytoscape software. The molecular docking between seven components and two targets was validated using the Autodock-vina program. The results showed that 7 components had anti-inflammatory activity and acted on 84 anti-inflammatory targets. KEGG and GO analysis showed that the main active components of blueberry could inhibit inflammation by inhibiting the production of inflammatory factors and enhancing immunity. Network analysis revealed that the main anti-inflammatory targets of blueberry active components were TNF, ESR1, AGTR1, and IGF1. Based on molecular docking analysis, the main components of blueberry integrate with 2 important targets in inflammatory networks. Collectively, we characterized the anti-inflammatory effect of blueberry by multi-component, multi-target, and multi-pathway. The molecular mechanism of the multi-target effect of blueberry was preliminarily expounded, thereby providing a scientific basis for exploring the material basis and mechanism of the anti- inflammatory action of blueberry. BACKGROUND: Non-steroidal anti-inflammatory drugs, such as aspirin, have beneficial effects in the treatment of inflammation but they often have undesired side effects. In contrast, various natural remedies, with their unique natural, safe and effective ingredients, have achieved good effects in the treatment of inflammation and become widely used for anti-inflammatory medication. OBJECTIVE: To provide scientific basis for exploring the material basis and mechanism of antiinflammatory action of blueberry. METHODS: The anti-inflammatory target genes of these components were extracted by comparing them with the anti-inflammatory drug target genes in the GeneCards database. GO enrichment and KEGG pathway, as well as protein interaction analysis of these anti-inflammatory target genes, were carried out by using the String database. The anti-inflammatory component-target-action pathway map of blueberry was constructed using the Cytoscape software. The molecular docking between seven components and two targets was validated using the Autodock-vina program. The results showed that 7 components had anti-inflammatory activity and acted on 84 anti-inflammatory targets. RESULTS: 7 components had anti-inflammatory activity and acted on 84 anti-inflammatory targets. KEGG and GO analysis showed that the main active components of blueberry could inhibit inflammation by inhibiting the production of inflammatory factors and enhancing immunity. Network analysis revealed that the main anti-inflammatory targets of blueberry active components were TNF, ESR1, AGTR1 and IGF1. Based on molecular docking analysis, the main components of blueberry integrate with 2 important targets in inflammatory networks. CONCLUSION: The molecular mechanism of the multi-target effect of blueberry was preliminarily expounded, thereby providing a scientific basis for exploring the material basis and mechanism of antiinflammatory action of blueberry.

Systematic review and Meta-analysis of Gusongbao preparation in treatment of primary osteoporosis / 中国中药杂志

This study aims to provide evidence for clinical practice by systematically reviewing the efficacy and safety of Gusongbao preparation in the treatment of primary osteoporosis(POP). The relevant papers were retrieved from four Chinese academic journal databases and four English academic journal databases(from inception to May 31, 2022). The randomized controlled trial(RCT) of Gusongbao preparation in the treatment of POP was included after screening according to the inclusion and exclusion criteria. The quality of articles was evaluated using risk assessment tools, and the extracted data were subjected to Meta-analysis in RevMan 5.3. A total of 657 articles were retrieved, in which 15 articles were included in this study, which involved 16 RCTs. A total of 3 292 patients(1 071 in the observation group and 2 221 in the control group) were included in this study. In the treatment of POP, Gusongbao preparation+conventional treatment was superior to conventional treatment alone in terms of increasing lumbar spine(L2-L4) bone mineral density(MD=0.03, 95%CI[0.02, 0.04], P<0.000 01) and femoral neck bone mineral density, reducing low back pain(MD=-1.69, 95%CI[-2.46,-0.92], P<0.000 1) and improving clinical efficacy(RR=1.36, 95%CI[1.21, 1.53], P<0.000 01). Gusongbao preparation was comparable to similar Chinese patent medicines in terms of improving clinical efficacy(RR=0.95, 95%CI[0.86, 1.04], P=0.23). Gusongbao preparation was inferior to similar Chinese patent medicines in reducing traditional Chinese medicine syndrome scores(MD=1.08, 95%CI[0.44, 1.71], P=0.000 9) and improving Chinese medicine syndrome efficacy(RR=0.89, 95%CI[0.83, 0.95], P=0.000 4). The incidence of adverse reactions of Gusongbao preparation alone or combined with conventio-nal treatment was comparable to that of similar Chinese patent medicines(RR=0.98, 95%CI[0.57, 1.69], P=0.94) or conventio-nal treatment(RR=0.73, 95%CI[0.38, 1.42], P=0.35), and the adverse reactions were mainly gastrointestinal discomforts. According to the available data, Gusongbao preparation combined with conventional treatment is more effective than conventional treatment alone in increasing lumbar spine(L2-L4) bone mineral density and femoral neck bone mineral density, reducing low back pain, and improving clinical efficacy. The adverse reactions of Gusongbao preparation were mainly gastrointestinal discomforts, which were mild.

Dynamic Changes in the Gut Microbial Community and Function during Broiler Growth.

During the entire growth process, gut microbiota continues to change and has a certain impact on the performance of broilers. Here, we used 16S rRNA gene sequencing to explore the dynamic changes in the fecal bacterial communities and functions in 120 broilers from 4 to 16 weeks of age. We found that the main phyla (Firmicutes, Fusobacteria, Proteobacteria, and Bacteroides) accounted for more than 93.5% of the total bacteria in the feces. The alpha diversity of the fecal microbiota showed a downward trend with time, and the beta diversity showed significant differences at various time points. Then, the study on the differences of microbiota between high-weight (HW) and low-weight (LW) broilers showed that there were differences in the diversity and composition of microbiota between high- and low-weight broilers. Furthermore, we identified 22 genera that may be related to the weight change of broilers. The analysis of flora function reveals their changes in metabolism, genetic information processing, and environmental information processing. Finally, combined with microbial function and cecal transcriptome results, we speculated that microorganisms may affect the immune level and energy metabolism level of broilers through their own carbohydrate metabolism and lipid metabolism and then affect body weight (BW). Our results will help to expand our understanding of intestinal microbiota and provide guidance for the production of high-quality broilers. IMPORTANCE The intestinal microbiota has a certain impact on the performance of broilers. However, the change of intestinal microbiota after 4 weeks of age is not clear, and the mechanism of the effect of microorganisms on the weight change of broilers needs more exploration. After 4 weeks of age, the alpha diversity of microorganisms in broiler feces decreased, and the dominant bacteria were Firmicutes, Fusobacteria, Proteobacteria, and Bacteroides. There were differences in microbiota diversity and composition between high- and low-weight broilers. Intestinal microorganisms may affect the immune level and energy metabolism level of broilers through their own carbohydrate metabolism and lipid metabolism and then affect the body weight. The results are helpful to increase the understanding of intestinal microbiota and provide reference for the production of high-quality broilers.

Improvements of Flexural Properties and Thermal Performance in Thin Geopolymer Based on Fly Ash and Ladle Furnace Slag Using Borax Decahydrates.

This paper elucidates the influence of borax decahydrate addition on the flexural and thermal properties of 10 mm thin fly ash/ladle furnace slag (FAS) geopolymers. The borax decahydrate (2, 4, 6, and 8 wt.%) was incorporated to produce FAB geopolymers. Heat treatment was applied with temperature ranges of 300 °C, 600 °C, 900 °C, 1000 °C and 1100 °C. Unexposed FAB geopolymers experienced a drop in strength due to a looser matrix with higher porosity. However, borax decahydrate inclusion significantly enhanced the flexural performance of thin geopolymers after heating. FAB2 and FAB8 geopolymers reported higher flexural strength of 26.5 MPa and 47.8 MPa, respectively, at 1000 °C as compared to FAS geopolymers (24.1 MPa at 1100 °C). The molten B2O3 provided an adhesive medium to assemble the aluminosilicates, improving the interparticle connectivity which led to a drastic strength increment. Moreover, the borax addition reduced the glass transition temperature, forming more refractory crystalline phases at lower temperatures. This induced a significant strength increment in FAB geopolymers with a factor of 3.6 for FAB8 at 900 °C, and 4.0 factor for FAB2 at 1000 °C, respectively. Comparatively, FAS geopolymers only achieved 3.1 factor in strength increment at 1100 °C. This proved that borax decahydrate could be utilized in the high strength development of thin geopolymers.

The Maternal Milk Microbiome in Mammals of Different Types and Its Potential Role in the Neonatal Gut Microbiota Composition.

Maternal milk, a main source of nutrition for neonates in early life, has attracted attention. An increasing number of studies have found that maternal milk has a high microbial diversity, as well as factors that might influence this diversity. However, there is a lack of knowledge regarding the effects of host diet and phylogeny on maternal milk microbes and the contribution of the maternal milk microbiota to the neonatal gut microbiota. Here, we analyzed the maternal milk and fecal microbiota of nine species (lion, dog, panda, human, mouse, rhesus macaque, cow, goat, and rabbit) of mammals of three type groups (herbivore, omnivore, and carnivore) using 16S rRNA amplicon sequencing. Our study provided evidence of host diet and phylogeny on the maternal milk microbiota. Moreover, functional prediction revealed that the carnivores had a significantly higher percentage of base excision repair, glycerolipid metabolism, taurine and hypotaurine metabolism, inorganic ion transport and metabolism, and nucleotide metabolism; while arginine and proline metabolism showed enrichment in the herbivore group. Source-tracking analysis showed that the contributions of bacteria from maternal milk to the microbiota of neonates of different mammals were different at day 3 after neonatal birth. Overall, our findings provided a theoretical basis for the maternal milk microbiota to affect neonatal fecal microbiota at day 3 after neonatal birth.

Differential expression profiles of microRNAs in musk gland of unmated and mated forest musk deer (Moschus berezovskii).

BACKGROUND: The formation of musk is a complex biophysical and biochemical process that change with the rut of male forest musk deer. We have reported that the mating status of male forest musk deer might result to the variations of chemical composition and microbiota of musk and its yields. Critical roles for microRNAs (miRNAs) of multi-tissues were profiled in our previous study; however, the role for miRNAs of the musk gland remains unclear in this species. METHODS: In this study, we used Illumina deep sequencing technology to sequence the small RNA transcriptome of unmated male (UM) and mated male (UM) of Chinese forest musk deer. RESULTS: We identified 1,652 known miRNAs and 45 novel miRNAs, of which there were 174 differentially expressed miRNAs between UM and MM. chi-miR-21-5p, ipu-miR-99b and bta-miR-26a were up-regulated in UM among the 10 most differentially expressed miRNAs. Functional enrichment of the target genes showed that monosaccharide biosynthetic process, protein targeting, cellular protein catabolic process enriched higher in MM. Meanwhile, structural molecule activity, secretion by cell, regulated exocytosis and circulatory system process enriched more in UM, hinting that the formation of musk in UM was mediated by target genes related to exocytosis. The miRNA-mRNA pairs such as miR-21: CHD7, miR143: HSD17B7, miR-141/200a: Noc2 might involve in musk gland development and musk secretion, which need to be verified in future study.

Distribution patterns of microsatellites and development of its marker in different genomic regions of forest musk deer genome based on high throughput sequencing.

Forest musk deer (Moschus berezovskii, FMD) is an endangered artiodactyl species, male FMD produce musk. We have sequenced the whole genome of FMD, completed the genomic assembly and annotation, and performed bioinformatic analyses. Our results showed that microsatellites (SSRs) displayed nonrandomly distribution in genomic regions, and SSR abundances were much higher in the intronic and intergenic regions compared to other genomic regions. Tri- and hexanucleotide perfect (P) SSRs predominated in coding regions (CDSs), whereas, tetra- and pentanucleotide P-SSRs were less abundant. Trifold P-SSRs had more GC-contents in the 5'-untranslated regions (5'UTRs) and CDSs than other genomic regions, whereas mononucleotide P-SSRs had the least GC-contents. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs had different distributions in different genomic regions. The RCN of trinucleotide P-SSRs had increased significantly in the CDSs compared to the transposable elements (TEs), intronic and intergenic regions. The analysis of coefficient of variability (CV) of P-SSRs showed that the RCN of mononucleotide P-SSRs had relative higher variation in different genomic regions, followed by the CV pattern of RCN: dinucleotide P-SSRs > trinucleotide P-SSRs > tetranucleotide P-SSRs > pentanucleotide P-SSRs > hexanucleotide P-SSRs. The CV variations of RCN of the same mono- to hexanucleotide P-SSRs were relative higher in the intron and intergenic regions, followed by that in the TEs, and the relative lower was in the 5'UTR, CDSs and 3'UTRs. 58 novel polymorphic SSR loci were detected based on genotyping DNA from 36 captive FMD and 22 SSR markers finally showed polymorphism, stability, and repetition.

Physiological Implications of Orexins/Hypocretins on Energy Metabolism and Adipose Tissue Development.

Orexins/hypocretins and their receptors (OXRs) are ubiquitously distributed throughout the nervous system and peripheral tissues. Recently, various reports have indicated that orexins play regulatory roles in numerous physiological processes involved in obesity, energy homeostasis, sleep-wake cycle, analgesia, alcoholism, learning, and memory. This review aims to outline recent progress in the research and development of orexins used in biochemical signaling pathways, secretion pathways, and the regulation of energy metabolism/adipose tissue development. Orexins regulate a variety of physiological functions in the body by activating phospholipase C/protein kinase C and AC/cAMP/PKA pathways, through receptors coupled to Gq and Gi/Gs, respectively. The secretion of orexins is modulated by blood glucose, blood lipids, hormones, and neuropeptides. Orexins have critical functions in energy metabolism, regulating both feeding behavior and energy expenditure. Increasing the sensitivity of orexin-coupled hypothalamic neurons concurrently enhances spontaneous physical activity, non-exercise activity thermogenesis, white adipose tissue lipolysis, and brown adipose tissue thermogenesis. With this comprehensive review of the current literature on the subject, we hope to provide an integrated perspective for the prevention/treatment of obesity.

[Microbial diversity of musk across its maturation process in forest musk deer].

Musk,with unique and intense perfume,was a kind of deep brown precious medicinal material in traditional Chinese medicine. However,the immature musk in musk pot was white and stench. Given the fact that bacterial diversity generated odorous metabolites in animal hosts,in this study,musk samples at three different mature stages,including MJ( the end of June),MA( the end of August) and MO( the end of October) were harvested from three male forest musk deer,and then next-generation sequencing was used to intensively survey the bacterial communities in musk harvested at different mature stages. RESULTS: indicated that the average OTUs per sample at the end of June,August and October were 47 116. 00 ± 1 567. 24( SE),52 009. 00 ± 8 958. 75( SE) and50 004. 67±4 135. 57( SE),respectively. Feature of the musk 16 S rRNA gene showed a total of 418 genera belonging to 52 phyla were observed in all samples. The main microbiota was bacteria,which accounted for 98. 82%,99. 95% and 99. 58% in MJ,MA and MO,respectively. At phylum level,Firmicutes was the most abundant bacterial of MA( 32. 75%) and MO( 39. 19%). While,the major bacterial in MJ was Proteobacteria( 49. 14%). PICRUSt analysis revealed the functions of bacterial in MJ were mainly involved in secretion,while bacterial functions of MA and MO were mainly involved in amino acid or other substance metabolism,which was in accord with the musk secretion physiological process of forest musk deer. This is the first study involved in the bacterial diversity in musk of forest musk deer across the maturation process,while may provide a new insight into the musk generation mechanism.

Gut Microbiome of Chinese Forest Musk Deer Examined across Gender and Age.

Animal gut microbiota begins to colonize after birth and is functionally indispensable for maintaining the health of the host. It has been reported that gender and age influence the composition of the intestinal microbiome. However, the effects of gender and age on the intestinal microorganism of forest musk deer (FMD) remain unclear. The aim of this study was to establish the relationship between the structure and composition of fecal microbiota of male and female forest musk deer with age. Here, Illumina Miseq 300PE sequencing platform targeting 16S rRNA V3-V4 hypervariable region applied to define the fecal microbiota of male and female FMD with two age groups, juvenile (age 1-2 years) and adult (age 4-10 years). Alpha diversity index did not show significant difference in bacterial diversity between the males and females or among age groups. The intestinal microbiota of FMD was dominated by three phyla, the Firmicutes, Proteobacteria and Bacteroidetes regardless of gender and different ages. Higher proportions of Proteobacteria were found in adult male and juvenile female individuals. The composition of Bacteroidetes was stable with the gender and age of FMD. Interestingly, the relative abundance of genera Clostridiales and Bacteroidales were higher in the juvenile FMD. Conversely, proportions of Pseudomonas and Lachnospiraceae were abundant in the adult FMD. Higher proportions of Ruminococcaceae, Dore, and 5-7N15 were found in the juvenile male groups. They may reflect the different immune resistance of male and female individuals at different stages of development. This study explored the fecal microbiota composition of forest musk deer in relation to gender and age, which may provide an effective strategy for developing intestinal microecological preparations and potential musk deer breeding.

Effect of Bitter Compounds on the Expression of Bitter Taste Receptor T2R7 Downstream Signaling Effectors in cT2R7/pDisplay-Gα16/gust44/pcDNA3.1 (+) Cells.

Bitterness is an important taste sensation for chickens, which provides useful sensory information for acquisition and selection of diet, and warns them against ingestion of potentially harmful and noxious substances in nature. Bitter taste receptors (T2Rs) mediate the recognition of bitter compounds belonging to a family of proteins known as G-protein coupled receptors. The aim of this study was to identify and evaluate the expression of T2R7 in chicken tongue tissue and construct cT2R7-1 and cT2R7-2-expressing HEK-293T cells to access the expression of PLCß2 and ITPR3 after exposure with different concentrations of the bitter compounds. Using real-time PCR, we show that the relative expression level of T2R7 mRNA in 5, 1, 0.1, and 10-3 mM of camphor and erythromycin solutions and 5 mM of chlorpheniramine maleate solutions was significantly higher than that in 50 mM KCL solutions. We confirmed that the bitter taste receptor T2R7 and downstream signaling effectors are sensitive to different concentrations of bitter compounds. Moreover, T2R7-1 (corresponding to the unique haplotype of the Tibetan chicken) had higher sensitivity to bitter compounds compared with that of T2R7-2 (corresponding to the unique haplotype of the Jiuyuan black-chicken). These results provide great significance of taste response on dietary intake to improve chicken feeding efficiency in poultry production and have certain reference value for future taste research in other bird species.

Genomic Identification and Expression Analysis of the Cathelicidin Gene Family of the Forest Musk Deer.

The forest musk deer (Moschus berezovskii) is a small-sized artiodactyl species famous for the musk secreted by adult males. In the captive population, this species is under the threat of infection diseases, which greatly limits the increase of individual numbers. In the present study, we computationally analyzed the repertoire of the cathelicidin (CATHL) family from the genome of forest musk deer and investigated their expression pattern by real-time PCR. Our results showed that the entire genome of forest musk deer encodes eight cathelicidins, including six functional genes and two pseudogenes. Phylogenetic analyses further revealed that all forest musk deer cathelicidin members have emerged before the split of the forest musk deer and cattle and that forest musk deer CATHL3L2 and CATHL9 are orthologous with two cattle pseudogenes. In addition, the gene expression results showed that the six functional genes are not only abundantly expressed in the spleen and lung, but are also differently expressed in response to abscesses, which suggests that forest musk deer cathelicidins may be involved in infections. Taken together, identification and characterization of the forest musk deer cathelicidins provide fundamental data for further investigating their evolutionary process and biological functions.

microRNA and Other Small RNA Sequence Profiling across Six Tissues of Chinese Forest Musk Deer (Moschus berezovskii).

The Chinese forest musk deer (Moschus berezovskii) is an economically important species distributed throughout southwest China and northern Vietnam. Occurrence and development of disease are aggravated by inbreeding and genetic diversity declines in captive musk deer populations. Deep transcriptomics investigation may provide a promising way to improve genetic health of captive and wild FMD population. MicroRNAs (miRNAs), which regulate gene expression by targeting and suppressing of mRNAs, play an important role in physiology and organism development control. In this study, RNA-seq technology was adopted to characterize the miRNA transcriptome signature among six tissues (heart, liver, spleen, lung, kidney, and muscle) in Chinese forest musk deer at two years of age. Deep sequencing generated a total of 103,261,451 (~87.87%) good quality small RNA reads; of them 6,622,520 were unique across all six tissues. A total of 2890 miRNAs were identified, among them 1129 were found to be expressed in all tissues. Moreover, coexpression of 20 miRNAs (>2000RPM) in all six tissues and top five highly expressed miRNAs in each tissue implied the crucial and particular function of them in FMD physiological processes. Our findings of forest musk deer miRNAs supplement the database of transcriptome information for this species and conduce to our understanding of forest musk deer biology.

[Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells].

OBJECTIVE: To evaluate the effect of zoledronate acid (ZA) on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (BMSCs). METHODS: The BMSCs isolated from the SD rats were cultured with different concentrations of ZA (1, 5, 10, and 20 µmol·L), and the contro1 group received the same volume of culture medium but without ZA. Cell counting kit-8 was used to detect proliferation activity in each group. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability in each group. The gene expression levels of ALP, bone morphogenetic protein-2 (BMP-2), typeⅠcollagenase (COL-Ⅰ), runt-related transcription factor-2 (Runx-2), zinc finger structure transcription factor (Osx), osteocalcin (OCN), and osteopontin (OPN) were evaluated by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Zoledronate at 1 µmol·L⁻¹ concentration had no effect on the proliferation and osteogenic differentiation of BMSCs. No significant difference was observed between this group and the control group (P>0.05). When the ZA concentration was more than 1 µmol·L⁻¹, ZA inhibited the proliferation and osteogenic differentiation of BMSCs, and the effect was concentration dependent. The difference between each group and the control group was statistically significant (P<0.05). At ZA concentration of 5 µmol·L⁻¹, ZA enhanced the expression of ALP, BMP-2, COL-Ⅰ, Runx-2, Osx, OCN, and OPN (P<0.05). However, at ZA concentration of more than 5 µmol·L⁻¹, the expression levels of osteogenicrelated genes in each group was lower than those of the control group (P<0.05). CONCLUSIONS: Low ZA concentration has no effect on the proliferation and osteogenic differentiation of BMSCs. ZA at 5 µmol·L⁻¹ concentration inhibits the proliferation but promotes the osteogenic differentiation of BMSCs. High ZA concentration inhibits the proliferation and osteogenic differentiation of BMSCs.

Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells / 华西口腔医学杂志

OBJECTIVE@#To evaluate the effect of zoledronate acid (ZA) on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (BMSCs).@*METHODS@#The BMSCs isolated from the SD rats were cultured with different concentrations of ZA (1, 5, 10, and 20 μmol·L), and the contro1 group received the same volume of culture medium but without ZA. Cell counting kit-8 was used to detect proliferation activity in each group. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability in each group. The gene expression levels of ALP, bone morphogenetic protein-2 (BMP-2), typeⅠcollagenase (COL-Ⅰ), runt-related transcription factor-2 (Runx-2), zinc finger structure transcription factor (Osx), osteocalcin (OCN), and osteopontin (OPN) were evaluated by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Zoledronate at 1 μmol·L⁻¹ concentration had no effect on the proliferation and osteogenic differentiation of BMSCs. No significant difference was observed between this group and the control group (P>0.05). When the ZA concentration was more than 1 μmol·L⁻¹, ZA inhibited the proliferation and osteogenic differentiation of BMSCs, and the effect was concentration dependent. The difference between each group and the control group was statistically significant (P<0.05). At ZA concentration of 5 μmol·L⁻¹, ZA enhanced the expression of ALP, BMP-2, COL-Ⅰ, Runx-2, Osx, OCN, and OPN (P<0.05). However, at ZA concentration of more than 5 μmol·L⁻¹, the expression levels of osteogenicrelated genes in each group was lower than those of the control group (P<0.05).@*CONCLUSIONS@#Low ZA concentration has no effect on the proliferation and osteogenic differentiation of BMSCs. ZA at 5 μmol·L⁻¹ concentration inhibits the proliferation but promotes the osteogenic differentiation of BMSCs. High ZA concentration inhibits the proliferation and osteogenic differentiation of BMSCs.

Isolation, primary culture, and morphological characterization of gland epithelium from forest musk deer (Moschus berezovskii).

Research of epithelial cells in musk gland is lacking. There are no good characterized epithelial cell lines that can provide complementary in vitro models for in vivo research. We successfully cultivated epithelial cells of musk gland for the first time. The protocol described here produces epithelial cell lines from the mature secreting musk gland. Based on morphological observation, epithelial cells of musk gland were isolated and cultured in vitro. After the third passage, the musk gland-derived cells were filled with many lipid droplets and proliferated well. We used gas chromatography and mass spectrometry to explore the chemical composition of lipid droplets in the musk gland-derived cells. The main components of secreted lipid droplet were alkanes, esters, amines, alcohols, ketones, organic acids, and aldehydes. Muscone, which is the main active compound of musk, was not found. This is a new attempt in the field of animal musk to obtain naturally secreted animal musk in vitro by cloning specialized cells. In conclusion, this study provides a reference at the cellular level to further analyze the biology and physiology of the musk gland epithelium and secretion mechanism of musk deer.

Illumina-based de novo transcriptome sequencing and analysis of Chinese forest musk deer.

The Chinese forest musk deer (Moschus berezovskii Flerov) is an endangered artiodactyl mammal. The musk secreted by sexually mature males is highly valued for alleged pharmaceutical properties and perfume manufacturing. However, the genomic and transcriptomic resources of musk deer remain deficiently represented and poorly understood. Next-generation sequencing technique is an efficient method for generating an enormous amount of sequence data that can represent a large number of genes and their expression levels. In the present study, we used Illumina HiSeq technology to perform de novo assembly of heart and musk gland transcriptomes from the Chinese forest musk deer. A total of 239,383 transcripts and 176,450 unigenes were obtained, of which 37,329 unigenes were matched to known sequences in the NCBI nonredundant protein (Nr) database; 31,039 unigenes were assigned to 61 GO terms, and 11,782 to 332 KEGG pathways. Additionally, 592 and 2282 differentially expressed genes were found to be specifically expressed in the heart and musk gland, respectively. The abundant transcriptomic data generated in the present report will provide a comprehensive sequence resource for Chinese forest musk deer as well as lay down a foundation which will help in accelerating genetic and functional genomics research in this species.