Lipidomics for Determining Giant Panda Responses in Serum and Feces Following Exposure to Different Amount of Bamboo Shoot Consumption: A First Step towards Lipidomic Atlas of Bamboo, Giant Panda Serum and Feces by Means of GC-MS and UHPLC-HRMS/MS

Lipidic metabolites play essential roles in host physiological health and growth performance, serving as the major structural and signaling components of membranes, energy storage molecules, and steroid hormones. Bamboo, as wild giant pandas' exclusive diet, is the main determinant of giant pandas' lipidome, both as a direct source and through microbiota activity. Interestingly, the consumption of bamboo has attracted little attention from a lipidomic perspective. In the current study, we outline the lipidomic atlas of different parts of bamboo. By gas chromatography-mass spectrometry (GC-MS), we have been able to obtain the absolute quantification of 35 fatty acids pertaining to short chain fatty acids (8), medium chain fatty acids (6), long chain fatty acids (17), and very long chain fatty acids (4), while liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) allowed us to obtain the relative quantification of another 1638 lipids. Among the fatty acids quantified in absolute terms, eight showed significantly distinct concentrations among different bamboo parts. Subsequently, we investigated how the giant panda's serum and fecal lipidome adapt to the most important annual change in their diet, represented by the consumption of high amounts of bamboo shoots, typical of spring, the weight-gaining season. Five fatty acids were significantly altered in feces and two in serum, respectively, due to the different levels of bamboo shoot consumption. Furthermore, significant differences of the main bacteria strains were observed in feces between the two groups at the genus level, pertaining to Streptococcus, Leuconostoc, and Vagococcus. Correlations between giant panda fecal microbiome and lipidome were evaluated by Pearson correlation analysis. These findings suggest that a balanced diet, important for the overall lipidomic function and giant panda health, could be reached even in this remarkable case of a single food-based diet, by administering to the giant panda's combinations of different parts of bamboo, with specific lipidome profiles

Comparative transcriptome analyses of immune responses to LPS in peripheral blood mononuclear cells from the giant panda, human, mouse, and monkey

Gram-negative bacteria are major pathogens that can cause illnesses in giant pandas. Lipopolysaccharides (LPS), components of Gram-negative bacteria, can activate immune responses in mammals (i.e., humans and mice) through recognition by toll-like receptors (TLRs). However, the giant pandas’ immune response to LPS stimulation and the differences between the giant panda and other mammals are not fully known. In this study, we administrated peripheral blood mononuclear cells (PBMCs) from giant pandas, humans, C57BL/6 mice, and rhesus monkeys by LPS treatment at 6 h followed by RNA sequencing (RNA-seq), respectively, with control of non-stimulation. KEGG analyses of differentially expressed genes (DEGs) pathways indicated that LPS could activate the classic signaling pathway of NF-κB in PBMCs from those four tested species. Thus, similar to the other three species, NF-κB is an LPS-responsive regulator of innate immune responses in giant pandas. Furthermore, the expression patterns of adapter genes, inflammatory cytokine genes, chemokines, interferon genes, cytokine genes related to cell growth and development, costimulatory molecules, Th1/Th2 cytokine genes, Th17 cytokine genes, Th9, and Th22 cytokine genes were compared among giant pandas and three other species. Our data indicated that in addition to the similar expression patterns of certain genes among giant pandas and other species, the unique expression pattern response to LPS in giant pandas was also discovered. Furthermore, Th9, Th17, and Th22 cells might be involved in the response to LPS in giant pandas at this tested time point. This study reveals that LPS-induced immune responses have different sensitivities and response timelines in giant pandas compared with other mammals. This study facilitates further understanding of the role of the TLR signaling pathway and the immune system in giant pandas, which might be helpful for disease prevention and protection

Metagenomic Analysis of Bacteria, Fungi, Bacteriophages, and Helminths in the Gut of Giant Pandas

To obtain full details of gut microbiota, including bacteria, fungi, bacteriophages, and helminths, in giant pandas (GPs), we created a comprehensive microbial genome database and used metagenomic sequences to align against the database. We delineated a detailed and different gut microbiota structures of GPs. A total of 680 species of bacteria, 198 fungi, 185 bacteriophages, and 45 helminths were found. Compared with 16S rRNA sequencing, the dominant bacterium phyla not only included Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria but also Cyanobacteria and other eight phyla. Aside from Ascomycota, Basidiomycota, and Glomeromycota, Mucoromycota, and Microsporidia were the dominant fungi phyla. The bacteriophages were predominantly dsDNA Myoviridae, Siphoviridae, Podoviridae, ssDNA Inoviridae, and Microviridae. For helminths, phylum Nematoda was the dominant. In addition to previously described parasites, another 44 species of helminths were found in GPs. Also, differences in abundance of microbiota were found between the captive, semiwild, and wild GPs. A total of 1,739 genes encoding cellulase, β-glucosidase, and cellulose β-1,4-cellobiosidase were responsible for the metabolism of cellulose, and 128,707 putative glycoside hydrolase genes were found in bacteria/fungi. Taken together, the results indicated not only bacteria but also fungi, bacteriophages, and helminths were diverse in gut of giant pandas, which provided basis for the further identification of role of gut microbiota. Besides, metagenomics revealed that the bacteria/fungi in gut of GPs harbor the ability of cellulose and hemicellulose degradation

Laboratory-Based Investigation into Stress Corrosion Cracking of Cable Bolts

Cable-bolt failures due to stress corrosion cracking (SCC) could significantly compromise the sustainability and long-term stability of underground constructions. To fully understand the SCC of cable bolts, a two-step methodology was implemented: (i) long-term cable-bolt coupon tests using mineralogical materials collected from underground mines; and (ii) accelerated full-scale cable-bolt tests using an acidified solution. In the long-term tests, a novel three-point bending coupon was designed. The effects of mineralogical materials on SCC were evaluated under the simulated underground bolting conditions through the application of “corrosion cells”. For accelerated tests, SCC resistance of different type of cable bolts was examined using the new designed tensile-loading apparatus under the periodically increasing strain-rate loading mechanism. It was identified that mineralogical materials and applied stress intensity accelerated the corrosion process of the cable bolts. The number of wires and wire surface conditions in different types of cable bolt directly affected SCC susceptibility. The cable bolts with a greater number of wires provided higher resistance to SCC. The developed experimental methodologies can be applied to study SCC in other reinforcement materials and the results can be used to design optimal support systems in different environmental and geotechnical conditions

First Steps toward the Giant Panda Metabolome Database: Untargeted Metabolomics of Feces, Urine, Serum, and Saliva by 1H NMR

Differences in the concentration of metabolites in the biofluids of animals closely reflect their physiological diversities. In order to set the basis for a metabolomic atlas for giant panda (Ailuropoda melanoleuca), we characterized the metabolome of healthy giant panda feces (23), urine (16), serum (6), and saliva (4) samples by means of 1H NMR. A total of 107 metabolites and a core metabolome of 12 metabolites was quantified across the four biological matrices. Through univariate analysis followed by robust principal component analysis, we were able to describe how the molecular profile observed in giant panda urine and feces was affected by gender and age. Among the molecules modified by age in feces, fucose plays a peculiar role because it is related to the digestion of bamboo's hemicellulose, which is considered as the main source of energy for giant panda. A metagenomic investigation directed toward this molecule showed that its concentration was indeed positively related to the two-component system pathway and negatively related to the amino sugar and nucleotide sugar metabolism pathway. Such work is meant to provide a robust framework for further -omics research studies on giant panda to accelerate our understanding of the interaction of giant panda with its natural environment

Population genetics of Enterocytozoon bieneusi in captive giant pandas of China

Abstract Background Most studies on Enterocytozoon bieneusi are conducted based on the internal transcribed spacer (ITS) region of the rRNA gene, whereas some have examined E. bieneusi population structures. Currently, the population genetics of this pathogen in giant panda remains unknown. The objective of this study was to determine the E. bieneusi population in captive giant pandas in China. Results We examined 69 E. bieneusi-positive specimens from captive giant pandas in China using five loci (ITS, MS1, MS3, MS4 and MS7) to infer E. bieneusi population genetics. For multilocus genotype (MLG) analysis of E. bieneusi-positive isolates, the MS1, MS3, MS4, and MS7 microsatellite and minisatellite loci were amplified and sequenced in 48, 45, 50 and 47 specimens, respectively, generating ten, eight, nine and five types. We successfully amplified 36 specimens and sequenced all five loci, forming 24 MLGs. Multilocus sequence analysis revealed a strong and significant linkage disequilibrium (LD), indicating a clonal population. This result was further supported by measurements of pairwise intergenic LD and a standardized index of association (I S A) from allelic profile data. The analysis in STRUCTURE suggested three subpopulations in E. bieneusi, further confirmed using right’s fixation index (F ST). Subpopulations 1 and 2 exhibited an epidemic structure, whereas subpopulation 3 had a clonal structure. Conclusions Our results describe E. bieneusi population genetics in giant pandas for the first time, improving the current understanding E. bieneusi epidemiology in the studied region. These data also benefit future studies exploring potential transmission risks from pandas to other animals, including humans

Salt-Sensitive Signaling Networks in the Mediation of K+/Na+ Homeostasis Gene Expression in Glycyrrhiza uralensis Roots

We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K+/Na+ homeostasis in a perennial herb, Glycyrrhiza uralensis, during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na+ accumulation in root cells than in leaf cells. Na+ ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca2+, H2O2, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na+ efflux, due to the activated Na+/H+ antiport system in the plasma membrane (PM). Moreover, salt-elicited K+ efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca2+, H2O2, NO, and eATP. The salt-adaptive effects of these molecules (Na+ extrusion and K+ maintenance) were reduced by pharmacological agents, including LaCl3 (a PM Ca2+ channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na+/H+ antiport system in salinized roots most likely resulted from the upregulation of two genes, GuSOS1 and GuAHA, which encoded the PM Na+/H+ antiporter, salt overly sensitive 1 (SOS1), and H+-ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in G. uralensis roots. For example, Ca2+, H2O2, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca2+-SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of G. uralensis, increased the transcription of GuSOS3, GuCIPK, GuRbohD (respiratory burst oxidase homolog protein D), GuNIR (nitrate reductase), GuMAPK3, and GuMAPK6 (the mitogen-activated protein kinases 3 and 6). Moreover, GuMAPK3 and GuMAPK6 expression levels were enhanced by H2O2 in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca2+, H2O2, and NO signaling to maintain K+/Na+ homeostasis. We propose that a multiple signaling network regulated K+/Na+ homeostasis in NaCl-stressed G. uralensis roots

Fatty-binding protein and galectin of Baylisascaris schroederi: Prokaryotic expression and preliminary evaluation of serodiagnostic potential.

Baylisascaris schroederi is a common parasite of captive giant pandas. The diagnosis of this ascariasis is normally carried out by a sedimentation-floatation method or PCR to detect eggs in feces, but neither method is suitable for early diagnosis. Fatty acid-binding protein (FABP) and galectin (GAL) exist in various animals and participate in important biology of parasites. Because of their good immunogenicity, they are seen as potential antigens for the diagnosis of parasitic diseases. In this study, we cloned and expressed recombinant FABP and GAL from B. schroederi (rBs-FABP and rBs-GAL) and developed indirect enzyme-linked immunosorbent assays (ELISAs) to evaluate their potential for diagnosing ascariasis in giant pandas. Immunolocalization showed that Bs-FABP and Bs-GAL were widely distributed in adult worms. The ELISA based on rBs-FABP showed sensitivity of 95.8% (23/24) and specificity of 100% (12/12), and that based on rBs-GAL had sensitivity of 91.7% (22/24) and specificity of 100% (12/12)

Comparative analysis of gut microbiota among the male, female and pregnant giant pandas (Ailuropoda Melanoleuca)

The giant panda (GP) was the most endangered species in China, and gut microbiota plays a vital role in host health. To determine the differences of the gut microbiota among the male, female and pregnant GPs, a comparative analysis of gut microbiota in GPs was carried out by 16S rRNA and ITS high-throughput sequencing. In 16S rRNA sequencing, 435 OTUs, 17 phyla and 182 genera were totally detected. Firmicutes (53.6%) was the predominant phylum followed by Proteobacteria (37.8%) and Fusobacteria (7.1%). Escherichia/Shigella (35.9%) was the most prevalent genus followed by Streptococcus (25.9%) and Clostridium (11.1%). In ITS sequencing, 920 OTUs, 6 phyla and 322 genera were also detected. Ascomycota (71.3%) was the predominant phylum followed by Basidiomycota (28.4%) and Zygomycota (0.15%). Purpureocillium (4.4%) was the most prevalent genus followed by Cladosporium (2.5%) and Pezicula (2.4%). Comparative analysis indicated that the male GPs harbor a higher abundance of phylum Firmicutes than female GPs with the contribution from genus Streptococcus. Meanwhile, the female GPs harbor a higher abundance of phylum Proteobacteria than male GPs with the contribution from genus Escherichia/ Shigella. In addition, the shift in bacteria from female to pregnant GPs indicated that phylum Firmicutes increased significantly with the contribution from Clostridium in the gut, which may provide an opportunity to study possible associations with low reproduction of the GPs