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Delta-tocotrienol as a vitamin E isomer has received much attention because of its diverse biomedical applications. Microbial biosynthesis of delta-tocotrienol is a promising strategy for its economic and environmental advantages. Here, we accomplished complete biosynthesis of delta-tocotrienol in Saccharomyces cerevisiae from glucose. We first constructed and incorporated a heterologous pathway into the genome of S. cerevisiae by incorporating the genes hpd (from Pseudomonas putida KT2440), hpt (from Synechocystis sp. PCC 6803), and vte1 (from Arabidopsis thaliana) for the biosynthesis of delta-tocotrienol. We further enhanced the biosynthesis of the precursor geranylgeranyl diphosphate by overexpressing the thmg1 and ggppssa (from Sulfolobus acidocaldarius) genes, leading to a production titer of delta-tocotrienol of 1.39 ± 0.01 mg/L. Finally, we optimized the fermentation medium using the response surface methodology, enabling a high-titer production of delta-tocotrienol (3.56 ± 0.25 mg/L), ∼2.6-fold of that of the initial culture medium. Fed-batch fermentation in a 2 L fermenter was further used to enhance the production titer of delta-tocotrienol (4.10 ± 0.10 mg/L). To the best of our knowledge, this is the first report on the de novo biosynthesis of delta-tocotrienol in S. cerevisiae, and the highest titer obtained for microbial production of delta-tocotrienol.

Additional file 4. Indirectness of the network meta-analysis.

Additional file 1: Table S1. Final search strategy for PubMed. Table 2. Final search strategy for Clinical Trial gov of Controlled Trials. Table 3. Final search strategy for Cochrane Central Register of Controlled Trials

Additional file 6. Overal evaluattion of the network meta-analysis.

Additional file 5. Incoherence of the network meta-analysis.

Additional file 3. Heterogeneity of the network meta-analysis.

Additional file 2: Figure 1. Treatment strategy for all-cause mortality. Figure 2. Risk of bias in all trials. Figure 3. Assessment of risk of bias of the included studies (I²=0%)

Self-sustained populations of captive red panda were challenged by low reproductive success rate. Defining the different types of testicular cell and increasing the knowledge of male fertility was critical for the improvement of reproductive efficiency in red panda captive populations. Here, we firstly report the histological structure in red panda testes at various ages. Besides, our data also showed that markers for red panda testicular cells including ITGA6, PLZF, THY1, VASA, DAZL, GATA4, VASA, and αSMA were expressed in adult testis tissue. To further examine the localizations of ITGA6, VASA, VIM, and αSMA, the immunohistochemical analysis was performed and the result showed the typical staining patterns of undifferentiated spermatogonia, multiple types of germ cells, Sertoli cells, and peritubular myoid cells (PMCs), respectively. For the testicular tissue digestion, two-step enzyme digestion was used to obtain the cell suspension. The red panda Sertoli cells, expressing Sertoli cell marker VIM, were cultured and conserved in this study, which can be used for red panda Sertoli cell establishment and as the feeder cells for red panda spermatogonial stem cells (SSCs) growing.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) constitute a class of cells with significant self-renewal and multilineage differentiation properties and have great potential for therapeutic applications and the genetic conservation of endangered animals. In this study, we successfully isolated and cultured UC-MSCs from the blood vessels of giant panda umbilical cord (UC). The cells were arranged in a vortex or cluster pattern and exhibited a normal karyotype, showing the morphological characteristics of fibroblasts. In addition, we found that basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promoted cell proliferation, whereas stem cell factor (SCF) did not promote cell proliferation. Cultured UC-MSCs were negative for CD34 (hematopoietic stem cell marker) and CD31 (endothelial cell marker), but positive for MSC markers (CD44, CD49f, CD105, and CD73) and stem cell markers (KLF4, SOX2, and THY1). Similar to other MSCs, giant panda UC-MSCs have multiple differentiation ability and can differentiate into adipocytes, osteoblasts and chondrocytes. Giant panda UC-MSCs are new resources for basic research as cell models following their differentiation into different cell types and for future clinical treatments of giant panda diseases.

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.

Menaquinone-7 (MK-7), a valuable vitamin K2, plays an important role in the prevention of osteoporosis and cardiovascular calcification. We chose B. subtilis 168 as the chassis for the modular metabolic engineering design to promote the biosynthesis of MK-7. The biosynthetic pathway of MK-7 was categorized into four modules, namely, the MK-7 pathway (Module I), the shikimate (SA) pathway (Module II), the methylerythritol phosphate (MEP) pathway (Module III), and the glycerol metabolism pathway (Module IV). Overexpression of menA (Module I) resulted in 6.6 ± 0.1 mg/L of MK-7 after 120 h fermentation, which was 2.1-fold that of the starting strain BS168NU (3.1 ± 0.2 mg/L). Overexpression of aroA, aroD, and aroE (Module II) had a negative effect on the synthesis of MK-7. Simultaneous overexpression of dxs, dxr, yacM, and yacN (Module III) enabled the yield of MK-7 to 12.0 ± 0.1 mg/L. Moreover, overexpression of glpD (Module IV) resulted in an increase of the yield of MK-7 to 13.7 ± 0.2 mg/L. Furthermore, del...

Background/Aims: Giant pandas, an endangered species, are a powerful symbol of species conservation. Giant pandas may suffer from a variety of diseases. Owing to their highly specialized diet of bamboo, giant pandas are thought to have a relatively weak ability to resist diseases. The spleen is the largest organ in the lymphatic system. However, there is little known about giant panda spleen at a molecular level. Thus, clarifying the regulatory mechanisms of spleen could help us further understand the immune system of the giant panda as well as its conservation. Methods: The two giant panda spleens were from two male individuals, one newborn and one an adult, in a non-pathological condition. The whole transcriptomes of mRNA, lncRNA, miRNA, and circRNA in the two spleens were sequenced using the Illumina HiSeq platform. EBseq and IDEG6 were used to observe the differentially expressed genes (DEGs) between these two spleens. Gene Ontology and KEGG analyses were used to annotate the function of DEGs. Furthermore, networks between non-coding RNAs and protein-coding genes were constructed to investigate the relationship between non-coding RNAs and immune-associated genes. Results: By comparative analysis of the whole transcriptomes of these two spleens, we found that one of the major roles of lncRNAs could be involved in the regulation of immune responses of giant panda spleens. In addition, our results also revealed that microRNAs and circRNAs may have evolved to regulate a large set of biological processes of giant panda spleens, and circRNAs may function as miRNA sponges. Conclusion: To our knowledge, this is the first report of lncRNAs and circRNAs in giant panda, which could be a useful resource for further giant panda research. Our study reveals the potential functional roles of miRNAs, lncRNAs, and circRNAs in giant panda spleen.

Through the implementation of giant panda ex situ conservation breeding programs, the giant panda has successfully been protected and has attained many conservation achievements. As of 2019, there were nearly 600 captive giant pandas worldwide, and the number of captive populations and the richness of genetic diversity have basically reached a self-sustainable state. However, to date, most captive adult male giant pandas do not mate naturally, and the reason is unclear, which has serious implications for implementation of the reintroduction training program of captive giant pandas. We used a combination of methods evaluating life history traits, behavioral observation, and bioinformatics statistical analysis and conducted comprehensive research on the factors that affected the natural mating success rates of captive adult giant pandas. The results showed that the management mode of singleton and parent rearing during cub development and mixed male and female group housing during the stage of subadult learning skills and experience could effectively improve the natural reproduction success rate of captive adult male giant pandas. Using odor stimulation during the mating period could change the courtship behavior of adult giant pandas in captivity, as males, when courting estrous females, behaved in a way that did not lead to successful mating. Applying these results to the breeding management and mating strategies of captive male giant pandas could not only help improve appropriate reproductive behavior and subsequently increase reproductive performance but also may provide more candidates for China's giant panda reintroduction programs.

Mesenchymal stem cells (MSC) have strong therapeutic potential due to their capacity for self-renewal and multilineage differentiation. MSCs can also be useful in preserving the current genetic diversity of endangered wildlife. To date, MSCs from various species have been studied, but only a few species of endangered wild animals have been reported. Adult bone marrow (BM) is a rich source of mesenchymal stem cells. The aim of this study was to isolate and characterize MSCs derived from the BM of red pandas. Red panda BM-MSCs isolated from five individuals were fibroblast-like cells, similar to other species. Cultured BM-MSCs with normal karyotype were negative for the hematopoietic line marker CD34 and the endothelial cell marker CD31 but were positive for MSC markers, including CD44, CD105 and CD90. RT-PCR and western blot analysis showed self-renewal and pluripotency genes, including Oct4, Sox2 and Klf4, were also expressed in red panda BM-MSCs. Finally, red panda BM-MSCs had the potential for differentiation into osteogenic, adipogenic and neuron-like cells by using a combination of previously reported protocols for other species. We have therefore demonstrated that cells harvested from red panda bone marrow are capable of extensive in vitro multiplication and multilineage differentiation, which is an essential step toward their use in the preservation of red pandas biological diversity and future studies on MSC applications in endangered species.

The aim of this study was to determine the metabolic response in giant pandas (Ailuropoda melanoleuca) to the consumption of certain parts of bamboo above ground growth. Giant pandas were provisioned with three species of bamboo: Phyllostachys bissetii, of which they only consume the culm (culm group); Bashania fargesii, of which they only consume the leaves (leaf group); and Qiongzhuea opienensis, of which they only consume the shoots (shoot group). The “culm” group absorbed the highest amount of calories and fiber, but was in short energy supply (depressed tricarboxylic acid cycle activity), and high fiber level diet might reduce the digestibility of protein. The “culm” and “leaf” groups absorbed less protein, and had a lower rate of body mass growth than the “shoot” group. Digestion of fiber requires energy input and yields low caloric extraction from the culm and leaf, and protein intake is important for increasing body mass. However, long-term consumption of shoots may have a potentially negative effect on the health because of high protein composition. Therefore, a balanced diet consisting of diverse plant parts of bamboo is important for the overall metabolic function and health of captive giant pandas.

In present study, we report on bone marrow (BM) mesenchymal stem cells (MSCs) that are isolated from giant pandas. Cells were collected from the BM of two stillborn giant pandas. The cells were cultured and expanded in 10% fetal bovine serum medium. Cell morphology was observed under an inverted microscopy, and the proliferation potential of the cells was evaluated by counting cell numbers for eight consecutive days. Differentiation potentials of the cells were determined by using a variety of differentiation protocols for osteocytes, adipocytes, neuron cells, and cardiomyocytes. Meanwhile, the specific gene expressions for MSCs or differentiated cells were analyzed by RT-PCR. The isolated cells exhibited a fibroblast-like morphology; expressed mesenchymal specific markers such as cluster of differentiation 73 (CD73), SRY (sex determining region Y)-box 2 (SOX-2), guanine nucleotide-binding protein-like 3 (GNL3), and stem cell factor receptor (SCFR); and could be differentiated into osteocytes and adipocytes that were characterized by Alizarin Red and Oil Red O staining. Under appropriate induction conditions, these cells were also able to differentiate into neuroglial-like or myocardial-like cells that expressed specific myocardial markers such as GATA transcription factors 4 (GATA-4), cardiac troponin T (cTnT), and myosin heavy chain 7B (MYH7B), or neural specific markers such as Nestin and glial fibrillary acidic protein (GFAP). This study demonstrated stem cells recovery and growth from giant pandas. The findings suggest that cells isolated from the BM of giant pandas have a high proliferative capacity and multiple differentiation potential in vitro which might aid conservation efforts.

Luteinizing hormone (LH) is one of the main pituitary hormones that regulate ovulation, however its role has not been studied in giant panda. In this study, we developed an ELISA method for the detection of panda urinary LH. We analyzed urinary hormones of 24 female pandas during 36 breeding periods, we found females could easily be impregnated if the first mating occurred within 10 hours after LH peak. We also found the patterns of the ratios of urinary LH and progestagen in pandas that bred and successfully gave birth were significantly different from those that bred but failed to give birth. These data was the first to provide the urinary LH profiles during the estrous and gestational periods in pandas, and demonstrated that the appearance of the urinary LH peak indicated the timing of ovulation. The LH detection together with estrogen analysis makes the window for successful mating narrower than previously reported. Moreover, detection of urinary LH and progestagen can be used to discriminate between pregnancies and pseudopregnancies/miscarriages in the species. Thus, our findings suggest that LH not only plays a critical role in regulating ovulation but also plays an important role in maintaining pregnancy in the giant panda.

The functional adaptive changes in cyanide detoxification in giant panda appear to be response to dietary transition from typical carnivore to herbivorous bear. We tested the absorption of cyanide contained in bamboo/bamboo shoots with a feeding trial in 20 adult giant pandas. We determined total cyanide content in bamboo shoots and giant panda’s feces, levels of urinary thiocyanate and tissue rhodanese activity using color reactions with a spectrophotometer. Rhodanese expression in liver and kidney at transcription and translation levels were measured using real-time RT-PCR and immunohistochemistry, respectively. We compared differences of rhodanese activity and gene expressions among giant panda, rabbit (herbivore) and cat (carnivore), and between newborn and adult giant pandas. Bamboo shoots contained 3.2 mg/kg of cyanide and giant pandas absorbed more than 65% of cyanide. However, approximately 80% of absorbed cyanide was metabolized to less toxic thiocyanate that was discharged in urine. Rhodanese expression and activity in liver and kidney of giant panda were significantly higher than in cat, but lower than in rabbit (all P