Our study indicated that the LHZZ particle has preventive and treatment effect on colorectal adenoma through multi-component, multi-target and multi-pathway.The Luo-hua-zi-zhu (LHZZ) granule has been widely used for the treatment of colorectal adenoma (CRA), which is a precursor of colorectal cancer (CRC). However, the active components of LUZZ and its mechanism of action against CRA have not yet been elucidated. This study was designed to investigate the effect of LHZZ on CRA and explore its pharmacological mechanisms. First, a total of 24 chemical constituents were identified in the 50% aqueous methanol extract of LHZZ granule based on the mass fragment patterns and mass spectral library using the high resolution UPLC-Q-TOF MS/MS system. Subsequently, based on a network pharmacology study, 16 bioactive compounds and 28 targets of the LHZZ associated with CRA were obtained, forming a compound-target network. Molecular docking tests showed tight docking of these compounds with predicted targeted proteins. The protein–protein interaction (PPI) network identified AKT1, CASP3, TP53 and EGFR as hub targets. The Kyoto Encyclopedia of Genes and Genomes pathway network and pathway-target-compound network revealed that the apoptosis pathway was enriched by multiple signaling pathways and multiple targets, including the hub targets. Finally, the reliability of the core targets was evaluated using molecular docking technology and  Colorectal adenoma (CRA) is the precancerous lesion of colorectal cancer (CRC) . CRA is Callicarpa nudiflora Hook, belonging to the family Verbenacase, is a perennial evergreen shrub or low arbor and its dried leaves and twigs are used as traditional Chinese herbal medicine, Luo-hua-zi-zhu (LHZZ), for the treatment of inflammation and bleeding. LHZZ granule is a Chinese patent medicine, which is included in Chinese Pharmacopoeia 2015 edition. As a drug for anti-inflammation, detoxification, convergence and hemostasis, LHZZ has been widely used in clinical treatment of inflammation caused by bacterial infection, acute infectious hepatitis, respiratory tract and gastrointestinal bleeding , HPLC grade methyl tert-butyl ether from CNW, AR grade acetic acid and GR grade ethanol from Sinopharm Chemical Reagent , and Cremophor EL (BR grade) from Shanghai Yuanye . Purified water used throughout the study was prepared by a purified water system 611VP . LHZZ granule was provided by the Jiangxi Puzheng Pharmaceutical Co., Ltd. . and stored in the laboratory of the Shanghai University of TCM.LHZZ granule (25 mg) was accurately weighed and placed in a round bottom flask with 25 ml 50% methanol, mixed well, sealed and soaked for 0.5 h at room temperature, and ultrasonically treated for 30 min using an ultrasonic cleaning instrument . The extract solution was centrifuged at 14,000 rpm for 15 min at room temperature, then filtered through a 0.22-μm microporous membrane before qualitative analysis.XRpump, a SIL-20ACXRautosampler, a CTO-10Avp column oven and a Shiseido C18 column  equipped with a guard column  was used for the chromatographic separation of LHZZ granule. A linear gradient program with a mobile phase system including solvent A  and solvent B  as follow: 95–5% A at 0.01–18 min, 5% A at 18–19 min, 5–95% A at 19–20 min, and 95% A at 20–25 min. The temperature of the analytical column and autosampler were maintained at 30°C and 4°C respectively. The total run time for a LC–MS/MS analysis was 25 min.A Shimadzu HPLC, consisting of a CBM-20A system controller, a LC-20ADThe instrumental settings of Q-TOF-MS/MS were as follow: ion source gas 1 (GSI) and gas 2 (GS2) were both set to 50 psi, curtain gas (CUR) was set to 40 psi, ion spray voltage floating (ISVF) was set to 3500 V in the positive mode while 3200 V was set in the negative mode, ion source temperature (TEM) was 350°C, collision energy (CE) was 60 V, collision energy spread (CES) was 15 V, declustering potential (DP) was 100 V, and nitrogen was used as a nebulizer and auxiliary gas. Samples were analyzed in both positive and negative ionization modes with a scanning mas-to-charge (m/z) range from 120 to 1,500. The quantification was performed via peak area ratio of interest analytes to IS. The Applied Biosystems Analyst version 1.5.1 software was used to control the LC–ESI/MS/MS system as well as collect and process the data.https://pubchem.ncbi.nlm.nih.gov/). Then, we predicted the target of bioactive components using public databases, namely the Swiss Target Prediction and STITCH, with the species limited to ‘Homo sapiens’. Finally, we standardized the target names using the UniProtKB (https://www.uniprot.gov/).We obtained information on the structure of the bioactive components, including molecular structures, canonical smiles, and their ‘sdf’ files from the product databases of PubChem (http://www.cytoscape.org/) to construct a compound-target network, The CytoNCA plugin (v2.1.6) was used to measure the topology scores of the nodes, including the betweenness, closeness, and subgraph centrality. The option “without weight” was selected , the DrugBank database (http://www.drugbank.ca/), the DisGeNET database (https://www.disgenet.org/), and the GeneCards database (https://www.genecards.org/). The species “Homo sapiens” was selected and the target gene information related to CRA was collected and integrated.Colorectal adenomas, colon precancerous lesions and Colorectal polyp are search terms used to separately search in the OMIM database (http://bioinfogp.cnb.csic.es/tools/venny/index.html) online tool. The Venn diagram was draw. A common target network was constructed using the Cytoscape.The target prediction results of the active ingredients of the LHZZ granule are matched with the search results of the CRA-related targets, and the overlapping target  is selecThe GeneMANIA tool can not only construct a PPI network, but also find a series of genes related to the input gene based on a large amount of function-related data, and analyze the interaction between them, such as co-expression and co-localization . In the https://david.ncifcrf.gov/). The species was set to be "Homo Sapiens", and GO function enrichment analysis and KEGG pathway enrichment was performed. GO functional analysis includes biological process , cellular component , molecular function . It is visualized as histogram and bubble chart by the OmicShare cloud platform (http://www.omicshare.com).The common target of LHZZ and CRA obtained by the above screening was imported into the DAVID6.8 database . The water molecules and excess inactive ligands were removed by PyMOL software. The proteins were hydrogenated and charged into AutoDockTools1.5.6 software and exported to pdbqt format. According to the published methods, the molecular docking simulation of potential targets and their corresponding components is carried out by using AutoDock vina software. The global optimal binding conformation is obtained, and the docking results are visualized by the DiscoveryStudioVisualizer.The core compounds were screened under the condition that the "Degree" parameter of the node in the "active ingredient-target-disease" network was greater than the mean. The mol2 file of the core compound was downloaded from the TCMSP database, imported into the AutoDockTools1.5.6 software, and saved in pdbqt format. The 3D structure of the key target proteins was downloaded from PDB database (2.IH-CRA cells were obtained from the China Center for Type Culture Collection (CCTCC), whose storage number is C2019307. We have applied for the national invention patent, patent number: 201911261397.9 . Cells were cultured in Ham‘s F 12 nutrient medium , supplemented with 10% fetal bovine serum . Cells were cultured at 37°C and 5% CO2. The absorbance at 450 nm was measured by a microplate reader . Cell survival was calculated as: absorbance/absorbance of control × 100%. The Graphpad prism 7.0 software was used to analyze and plot the data.IH-CRA cells in the logarithmic phase were seeded at 1 × 104cells/well in 96-well plates. After incubation for 24 h, IH-CRA cells were exposed to LHZZ . After treatment for 48 h and 72h, 20 ml of Cell Counting Kit (CCK-8) assay solution  was added to each well, and cells were incubated for 4 h at 37°C and 5% COg at 4°C for 15 min. Proteins were separated via sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . After blocking in 5% bovine serum albumin  for 1.5 h at room temperature, the membranes were incubated with primary antibodies against AKT1  at 4°C overnight. Next, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (proteintech) at room temperature for 1 h. The Enhanced Chemiluminescence Detection Kit  was used to detect and visualize protein bands.Total protein was obtained from treated cells in radioimmunoprecipitation assay buffer  and centrifuged at 12,000 × p-values of <0.05 or <0.01 were regarded as statistically significant. The statistical analysis was performed by SPSS 20.0 .All data are expressed as the mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) followed by a least-significant difference test. A total of 24 chemical constituents were identified in the 50% aqueous methanol extract of LHZZ granule based on the mass fragment patterns and mass spectral library using the high resolution UPLC-Q-TOF MS/MS system. These chemical constituents include Apigenin, luteolin, and Prostaglandin B1 et al. . The maiThe 24 components isolated from the LHZZ were analyzed by the Swiss Target Prediction database. A total of 359 potential targets were screened after removing duplicates in the target prediction. Subsequently, we used Cytoscape to construct a visual "compound-target" network with 383 nodes and 770 edges . Nodes rThe intestinal adenoma-related targets were collected from the human genome database. In the OMIM, DisGeNET, DrugBank and GeneCard, with the number of these targets 173,3, 25 and 111respectively. A total of 271 LHZZ targets were obtained by removing duplicates in these four kinds of databases. These targets were intersected with component targets to obtain intersection targets 28, as shown in In order to supplement other related genes in the network and find the key signaling pathways, we imported 28 target genes into the GeneMANIA tool to obtain a PPI network. The percentage in the results stands for the weight of interaction relationship in the network. Our result revealed that in the interaction between targets of the network, 21.82% were co-expression and 32.68% had physical interactions. There were also relationships of co-localization and shared protein domains . The calp-value < 0.05 and gene number 6 as screening conditions, a total of 30 items related to biological process, molecular function and cellular composition were obtained. It was suggested that LHZZ may play a role in inhibiting CRA through the regulation of metabolic process, inflammatory factors, cell proliferation, protein transport, transcription factor activity and other biological processes. In the KEGG pathway enrichment analysis, a total of 71 enrichment results were obtained. Among them, cancer-related pathway, PI3K-Akt signaling pathway, p53 signaling pathway and cell apoptosis are closely associated with cell proliferation, metabolic disorder and protein transport, that are in consistent with the results of GO enrichment. KEGG pathways and gene pathways with p ≤ 0.05 are significantly abundant. The first 20 components are graphed using the OmicShare cloud platform , molecular function (MF) and cellular component (CC) , as welllatform . StatistBased on the network analysis of several representative signaling pathways, biological processes and molecular functions, the target pathway/functional network is constructed. There are 28 nodes and 73 edges in the network graph . Multipld-glucopyranoside and AKT1  is Isoacteoside and AKT1, and the second highest binding affinity (−11.8 kJ/mol) is Luteolin-3”-O-β-and AKT1 . Using Dand AKT1 . It can  to AKT1  mainly t to AKT1  is mainlCCK-8 assays were performed to determine the anti-CRA effect of treatment with LHZZ at different concentrations  for 48 and 72 h in IH-CRA cells. As shown in We further explored the molecular mechanism of LHZZ involved in inducing apoptosis in IH-CRA cells by Western blotting. As shown in There are approximately 1.4 million new CRC patients, and approximately 700,000 deaths worldwide each year . About 8For complex diseases such as CRA, network pharmacology has unique advantages in predictive analysis . We screin vitro validation experiments confirmed the results of network pharmacology.The top targets in PPI network analysis are TP53, CASP3, EGFR and AKT1. TP53 is a tumor suppressor protein that can induce cell cycle arrest and apoptosis, and its mutations are common in various cancers. Some previous meta analysis show that, TP53 could be used as a biomarker for screening inflammatory bowel disease-related colorectal cancer and dysplasia . Caspasein vitro experiments. P53 can prevent CRA by promoting DNA repair, cell cycle control, and programmed cell death. Studies (According to the GO analysis of LHZZ granule on CRA, 30 biological processes were screened. These processes include those that regulate metabolic processes, inflammatory factors, protein transport, transcription factor activity, regulation of cell proliferation, et al. LHZZ may adjust the microenvironment of intestinal adenomas. In the results of the enrichment analysis of the KEGG pathway of LHZZ on CRA, the top 20 signaling pathways were screened out. Among them, the most enriched targets are cancer pathways that involve in pro-inflammatory carcinogenesis, miRNA regulatory mechanisms, PI3K-Akt signaling pathways, p53 signaling pathways, etc. According to the previous research findings , PI3K/Ak Studies  have fou Studies , a varie Studies . The actIn summary, the Apigenin, Prostaglandin, Brefeldin A, Isoacteoside, Verbascoside and luteolin are the main active components LHZZ granule. Their targets of anti-colorectal adenoma are AKT1, TP53, CASP3 and EGFR. LHZZ participates in the regulation of CRA cell proliferation through cancer pathway, metabolic pathway, PI3K-Akt pathway, p53 pathway and microRNAs. This study provides a rationale for using LHZZ for the treatment of CRA.


Despite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons.+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system.To gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NADOur data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain. Axon degeneration leads to loss of connectivity and brain function upon trauma including head injury and stroke. Understanding the mechanisms of axon degeneration to either prevent or delay it, therefore, represents a promising avenue to develop effective therapies for these brain disorders .Axonal injury is followed by degeneration of the distal part of the axon that is detached from the soma. This tightly controlled process of axon self-elimination following a lesion is termed WD  and has + or the NAD+ synthesising enzymes, which when depleted from the axon trigger the process of fragmentation Terminauxsoftware , 44, 45.The resolution of our multiphoton microscope (0.25 × 0.25 × 2.5 in X-Y-Z) is sufficient to resolve gaps along the axons of few microns. Axons were excluded from analysis in cases where the visibility was compromised .The length of the distal portions from the edge of the lesion mask to the terminal ending was measured for all axons. ‘Ending’ and ‘close to the lesion’ were used to describe each end of the disconnected axon where ending refers to the terminal ending of the axon. Axons were grouped as either unbranched or branched, where the total length was less than or more than 600 μm.Figures were prepared using Microsoft Office Suite, Adobe Illustrator and BioRender.t tests or Mann Whitney U tests were performed, unless otherwise stated. Results were considered significant when p < 0.05.Statistical analyses were performed in Microsoft Excel, R or GraphPad Prism. Unless otherwise stated, all measurements are given as the mean ± standard error of the mean (SEM). Student’s Additional file 1. Canty et al. Additional file 1.pdf. Examples of fragmenting cortical axons.  Representative images and tracings of two short axon endings undergoing rapid onset WD (roWD) with time post-lesion and percentage of axon lost indicated. (c) Representative images and tracings of a branched, longer axon undergoing Wallerian degeneration (WD) with time post-lesion and percentage of axon lost indicated. Dotted boxes refer to the axon segments shown below. Scale bar is 20 μm in a, 25 μm in b and 32 μm in c.Additional file 2. Canty et al. Additional file 2.pdf. Probing the onset mechanism of lesioned axons. Schematic depiction of ‘protective factor’ and ‘degeneration trigger factor’ regulated axon degeneration paradigms following a single or double lesion. Lightning bolt represents lesion site. Purple arrows indicate movement of protective factor out of the lesioned axon via one (single lesion) or two routes (double lesion). Red arrows indicate movement of a degeneration trigger into the lesioned axon via one (single lesion) or two routes (double lesion). Additional file 4. AAD involves axoplasmic but not axolemmal fragmentation in spinal cord sensory axons after axotomy. (a) Axoplasmic fragmentation, AAD, occurs after laser-mediated lesion of ascending sensory neurons expressing a cytosolic form of GFP . (b) Axons remain intact for longer in ascending Thy1-L15 sensory axons expressing a membrane-bound form of GFP . Note beading of axons, but no fragments for up to ~ 2 h postlesion. (c) The mean onset of sensory axon fragmentation is significantly shorter for GFP-M (cytosolic GFP) compared to L15 (membrane-targeted GFP) axons. (d) Mean distance of severed proximal and distal axon tips to the lesion site in GFP-M  and L15 . Scale bar 50 μm for a and b. *** p < 0.001.Additional file 5. Axon degeneration modalities. Schematic depicts the characteristics of axon degeneration for acute axonal degeneration in the spinal cord , rapid onset Wallerian degeneration in the cortex  and Wallerian degeneration (WD) in the cortex (grey) and spinal cord (pink). From top to bottom, illustrated degeneration characteristics include the site of injury along the axon, the length of disconnected axon, the onset time at which fragmentation commences, and the fragmentation rate.


Recent landmark studies have shown a survival benefit from using genomic profiling to guide targeted personalised therapy in men with metastatic castrate refractory prostate cancer (mCRPC) . The poiPatients referred to a standard NHS diagnostic service between Jan 2019 -Dec 2021 were screened using the pre-biopsy MRI report and presenting PSA . Eligibl62 men were recruited of whom 52 (84%) had cancer samples sufficient and met quality for sequencing. Cohort details are in Focusing on the 37 men with CPG4-5/metastatic disease, all but two had combined androgen deprivation therapy (ADT) and radiotherapy or systemic therapy alone (ADT and chemotherapy). Follow up was available for a median of 26 months (range 15-35). In this CPG4-5/metastatic sub-group; overall there were 12/37 (32%) progression events; 9 new or worsening metastases and 4 who progressed and died of prostate cancer. Three others developed rapid biochemical relapse after first-line treatment. Of the 5 men with DDR pathway mutations, 2/5 progressed; one with a BRCA2 mutation died from disease and another developed new metastasis. Interestingly 1 of the men with ATM loss of uncertain significance, relapsed rapidly after surgery. Of the 6 men with any Pi3K-Akt mutation there was 1 death from prostate cancer and 1 progression despite treatment. Combining men with either a DDR or a Pi3K pathogenic pathway mutation 4/11 (36%) progressed and/or died of disease (1 man had both BRCA2 and PI3K mutation). In men without any reported DDR or PI3K mutations (pathogenic or uncertain), 5/22 (23%) progressed in the follow-up period.(i)Targeting men with high PSA and/or MRI defined high probability lesions(ii)Histology identification of tissue to be profiled at routine pathology assessment(iii)Assign prognostic/metastatic category at MDT and determine likely outcomes vs competing morbidity for case selection(iv)Sequencing of those men with a high likelihood of progression or cancer mortality –: CPG4-5/metastatic diseaseIt is now clear that specific genomic signatures add value in management decisions and impact disease trajectory and survival outcomes . FallingWe have shown that these steps are possible in a routine NHS diagnostic pathway and offers a rationale way to maximise the detection of potentially actionable mutations while avoiding over-investigation. Using this method DDR and PI3K mutations were detected in 11% and 19% in CPG4-5/metastatic disease and these men seemed to be over-represented in cases that progressed over the short term .a priori knowledge of BRCA status may allow modifications in earlier stages of management and future proof for evolutions in genomic testing . BRCA2 status for instance is already an independent predictor of poor survival in the Predict Prostate algorithm  are now approved by NICE (as well as the European Medicines Agency and US FDA) . It coullgorithm .In summary we report a pragmatic method to optimise selection of men for molecular profiling of prostate cancer at the point of diagnosis. Working within standard diagnostic pathways our method could be adopted by any NHS (or other) unit without much resourcing. The upfront information could be used not only for future drug selection, but also influence decision making and personalised follow-up.Supplementary File


The transcriptome profiles of hypothalamus, tail-fat and perirenal fat tissues from these four groups were determined using paired-end sequencing for RNA expression analysis. There are differences in cold tolerance between Hu and Altay sheep. Under cold exposure of the lambs: (1) UCP1-dependent thermogenesis and calcium- and cAMP-signaling pathways were activated; and (2) different fat tissues were activated in Hu and Altay lambs. Several candidate genes involved in thermogenesis including UCP1, ADRB3, ADORA2A, ATP2A1, RYR1 and IP6K1 were identified. Molecular mechanisms of thermogenesis in the sheep are discussed and new avenues for research are suggested.Thermogenesis plays an important role in the survival of sheep exposed to low temperatures; however, little is known about the genetic mechanisms underlying cold adaptation in sheep. We examined 6 Altay (A) and 6 Hu (H) six-month-old ewe lambs. Altay sheep are raised in northern China and are adapted to dry, cold climates, while Hu sheep are raised in southern China and are adapted to warm, humid climates. Each breed was divided into two groups: chronic cold sheep, exposed to −5 °C for 25 days (3 A As a consequence of the long, harsh winters in the high-altitude alpine meadows in Northern China, sheep often suffer from hypothermia. Consequently, the ability to withstand cold-exposure is vital for their survival. To maintain body temperature when exposed to low air temperature, sheep must increase their heat production (thermogenesis), which involves shivering thermogenesis (ST) and non-shivering thermogenesis (NST) . In new-When lambs, like other mammals, are exposed to cold, the hypothalamic–pituitary axis releases catecholamines to stimulate mitochondria in the adipocytes. These cells generate heat when the β-3 adrenergic receptor (ADRB3) enhances electron transfer chain uncoupling, via uncoupling protein 1 (UCP1). Mammals can also produce heat by triggering creatine cycling .Differences in the capability to withstand cold-exposure in sheep have been reported between breeds  and in nIn this study, two breeds of sheep were examined. The Altay sheep are indigenous to the Altay Prefecture of Northern Xinjiang Province in China, and are well-adapted to low temperatures. This Prefecture has an average January (winter) temperature of −16.3 °C (Climate-Data.org), with prolonged snow cover. Altay sheep have coarse wool and a fat tail, and are raised for high meat quality . TraditiBased on different selection backgrounds between breeds, we hypothesized that these breeds would display differences in resistance to cold as well as in genetic regulatory mechanisms when exposed to low temperature. To test this hypothesis, we used an RNA-seq approach to examine the transcriptome profiles of fat and hypothalamus tissues from Altay and Hu sheep when maintained under cold and thermo-neutral conditions.c, n = 3) and one group was kept under thermo-neutral conditions at 20 °C . The Hu lambs were divided similarly, one group was cold-exposed  and one group under thermo-neutral conditions at 20 °C . The lambs were acclimatized to the conditions for 7 days prior to the study.Six Altay and six Hu ewe-lambs, six-months of age and of similar body mass (mean 29.2 ± 2.47 kg), were purchased from Zhongmu Sheep Farm in the Altay region in September 2018, where they had been fed alfalfa pellets ad libitum and had free access to water. All lambs had been weaned at 2.5 months of age. Wool length did not differ between the breeds and averaged 8.9 ± 2.15 cm. Each breed was divided into two groups matched for body mass and were maintained in individual metabolic cages in temperature-controlled rooms. For the Altay lambs, one group was cold-exposed at −5 °C , and then maintained at −5 °C for 25 days. The humidity was 88% ± 6.5. The thermo-neutral lambs were kept at 20 °C for the 35-day duration of the study, and the humidity was 87.5% ± 9.9.On day 36, rectal temperature of the lambs was measured at 06:00, 14:00 and 22:00 using a mercury thermometer. They were then slaughtered and tissue samples were collected from the hypothalamus, perirenal fat (left kidney) and tail-fat (tail tip). The samples were snap-frozen in liquid nitrogen and stored at −80 °C.c (n = 3), Aw (n = 3), Hc (n = 3) and Hw (n = 3) for each tissue depot were not pooled.A total of 36 RNA samples were extracted from the three different tissues of each lamb in each of the four groups using Trizol reagent . The RNA was checked for quality to ensure that the RNA Integrity Number (RIN) was >7.0, and the 28S:18S rRNA ratio was >1.0 and was quantified using an Agilent 2100 bioanalyzer . The RNA samples from three separate lambs  was isolated from the total RNA, which was obtained from the samples, using an oligo dT extraction kit (NEB next poly (A) mRNA magnetic isolation module, NEB, USA). Purified mRNA was fragmented into small pieces with a fragment buffer at an appropriate temperature , and a first-strand cDNA was synthesized from the fragmented mRNA using random oligonucleotide primers and reverse transcriptase . The synthesis of the second-strand used DNA polymerase I and RNase H treatments. A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and the products were purified by Ampure XP Beads and then dissolved in EB solution. The product was validated on the Agilent Bioanalyzer  for quality control. The double stranded PCR products were heated, denatured and circularized by the splint oligo sequence to obtain the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with the ssCir DNA by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process to create DNA nanoball (DNB). The DNBs were loaded into the patterned Nano-array and single end 100 bases reads were generated on a BGIseq500 platform .Ovis aries  using the HISAT alignment tool . HISAT is based on the burrows-wheeler transform and Ferragina-Manzini (FM) index methods [p value was calculated by the permutation tests [The raw sequence reads, which included low quality , adaptor (reads that contained the adaptor sequences) and highly unknown base N content (reads which contained more than 5 percent base of undetermined base information), were filtered out using quality control software SOAPnuke (BGI) , and the methods . We mapp methods   for functional annotation. Based on the GO annotation results, GO enrichment analysis uses the phyper function in the R statistical software to calculate hypergeometric probabilities of DEGs .Gene ontology (GO) enrichment analysis provides a systematic method of defining the function of gene products ,18, and p ≤ 0.01 was used to accept whether DEG results from GO analyses were significant.The DEGs were enriched on three default GO terms, ‘molecular function’, ‘biological process’ and ‘cellular component’. A level of adjusted http://www.kegg.jp/kegg/pathway.html/ (accessed on 26 April 2019). The KEGG enrichment analysis method was consistent with the GO enrichment.The KEGG pathway enrichment was used to identify genes and metabolic pathways that involved the DEGs, according to methods described in the KEGG database  , ADIPOQ and LPL (lipoprotein lipase) were analyzed using an RT-qPCR approach. The primers for RT-qPCR validation were designed using Oligo7  (β-actin as a reference gene to verify the relative level of expression. The RT-qPCR amplification of cDNA pools used a PrimerScript RT reagent kit (Takara) with gDNA Erase (Takara), according to the manufacturer’s instructions. The RT-qPCR reactions used an Agilent Mx3000P system , and reactions contained 2 µL of cDNA, 0.8 µL forward and reverse primers (10 uM), 10 µL TB GreenTM Premix Ex Taq II, 6 µL RNAase free water, and 0.4 µL ROX Reference Dye II (50×) in a total volume of 20 µL. The thermal profile for amplification followed a two-step approach: 1) pre-denaturation for 15 s at 95 °C; then 2) 5 s at 95 °C and 34 s at the Tm listed in −△△ct method [To test selected gene expression differences which had been identified by the RNA-seq analysis, the expressions of four candidate genes, namely, ik, USA) , with β-https://string-db.org (accessed on 26 April 2019). The PPI pairs of proteins encoded by DEGs were retrieved using the tools of Cytoscape (version 3.6.1) [UCP1, ATP2A1, RYR1 and IP6K1) were chosen to construct a regulatory network.All the differentially expressed transcripts used a hierarchical clustering algorithm and prediction methods were based on information derived from string database (n 3.6.1) . The canp < 0.05 was accepted as significant. Results are presented as means ± SEM.All qPCR and rectal temperature data were analyzed by t-tests and one-way ANOVA. . A level of The datasets generated and analyzed during the current study are available confidentially to editors and reviewers, and all transcriptome data were submitted to the NCBI sequence read archive (SRA: accession PRJNA542078).The study design and all procedures on sheep were approved by the Academic Committee of Northwestern Institute of Eco-Environment Resources, Chinese Academy of Sciences (protocol number: CAS201810082).Rectal temperatures ranged between 38.4 °C and 39.3 °C. All lambs maintained normal rectal temperature  and appep = 0.001). The hypothalamus and adipose tissue were distinguished significantly in the CPCo 1 , and tail fat and perirenal fat were also distinguished in the CPCo 2 (7.7% of variance in the CPCo 1 of 48.3%). The samples of different tissues were divided into three groups. There was more organizational distance between adipose tissues than between breeds or temperature treatment. The distance between duplicates was relatively far, which meant dispersion of the sample was relatively large. In addition, the distance between three biological duplicates in the fat tissue of cold exposed Hu lambs was relatively far when compared to the other treatments. The PCoA indicated that the dispersion in the H-tailc and H-peric was caused by individual differences among the three cold-exposed Hu lambs, but this dispersion did not affect subsequent analyses.To examine the global difference between different tissues, breeds and treatments in the transcriptome sequence in 36 samples, constrained principal coordinate analysis (CPCoA) by Bray-Curtis distances was done for every biological repeat of different tissue and treatment . The 36 The average matching ratio of the clean reads to the genome was 87.8%, and the average matching ratio of the clean reads to the gene sequence was 78.2%. The sequencing and mapping data are summarized in A total of 23,736 genes were annotated according to the Oar reference genome Oar_v4.0, including 20,649 previously identified genes and 3087 potentially novel genes, that is, where genes mapped to unannotated regions of the genome. Venn diagrams compared the different tissues, temperatures and breeds according to the global genes which were annotated . In a coComparisons of the DEGs up- and down regulation between the two breeds and at the different temperatures for the three tissues are presented in p value < 0.01). For the MF GO category, DEGs in the hypothalamus tissue were enriched in transporter activity, heme and tetrapyrrole binding and ion transmembrane transporter channel activity related functions; the perirenal fat tissue was enriched in receptor activity and heme binding; and the tail-fat tissue was enriched in chemokine, cytokine activity, receptor binding and receptor activity. For the CC GO category, DEGs in the hypothalamus tissue were enriched in the plasma membrane parts and both intrinsic and integral components of the membrane; the perirenal fat tissue was enriched in membrane parts and extracellular regions; and the tail-fat tissue was enriched in intrinsic and integral components of the membrane, and membrane parts. For the top 10 up- and down-DEGs enriched CC and MF terms (q < 0.05), DEGs in the hypothalamus, perirenal fat and tail-fat tissues are presented in The GO (gene ontology) analyses revealed that the DEGs could be categorized into molecular function (MF), cellular component (CC) and biological process (BP). The selected significant GO term annotations are presented in A summary of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses of the DEGs in the different tissues and groups according to q-value (q < 0.01) is presented in In the hypothalamus, the calcium signaling pathway was significantly enriched in the breeds during cold exposure. The phosphatidylinositol signaling system and cyclic AMP (cAMP) signaling pathway were both enriched in the −5 °C Altay and Hu lambs, but more DEGs were enriched in the Hu lambs. Both the Rap1- and Ras signaling pathways were significantly enriched in the Hu lambs, but not in the Altay lambs. cAMP- and calcium signaling pathways were up-regulated in the −5 °C Altay lambs compared to the 20 °C Altay lambs and −5 °C Altay lambs compared to the −5 °C Hu lambs, but down-regulated in the −5 °C Hu lambs compared to the 20 °C Hu lambs. In the perirenal fat tissue, the NF-kappa B-, the calcium- and the rap1-signaling pathways were significantly enriched in the 20 °C Altay lambs compared to the 20 °C Hu lambs and the −5 °C Altay lambs compared to the −5 °C Hu lambs. With exposure to low temperature, the Ras-signaling pathway and steroid hormone biosynthesis, and cAMP signaling pathway were significantly enriched in the Altay and Hu lambs. Fat digestion and absorption pathway was up-regulated in the −5 °C Altay lambs compared to the 20 °C Altay lambs. The cAMP signaling pathway, ABC transporters, adrenergic signaling in cardiomyocytes, carbohydrate digestion and absorption and cardiac muscle contraction were all up-regulated in the −5 °C Altay lambs compared to the −5 °C Hu lambs. ABC transporters were down-regulated in the −5 °C Hu lambs compared to the 20 °C Hu lambs. In the tail-fat tissue, the two breeds were enriched in the calcium-, phosphatidylinositol-, cAMP-, Rap1- and Ras signaling pathways at low temperature, but fewer DEGs were enriched in the −5 °C Altay lambs in the calcium signaling pathway than in the Hu lambs. The PI3K-Akt signaling pathway and adrenergic signaling in cardiomyocytes were down-regulated in the −5 °C Altay lambs compared to the 20 °C Altay lambs. The NF-kappa B-, rap1- and MAPK signaling pathways and cytokine-cytokine receptor interaction were up-regulated in the −5 °C Hu lambs compared to the 20 °C Hu lambs, and calcium signaling pathway, insulin secretion, ABC transporters, ascorbate and aldarate metabolism and pentose and glucoronate interconversions were down-regulated in the −5 °C Hu lambs compared to the 20 °C Altay lambs. In contrast, pathways of ascorbate and aldarate metabolism and pentose and glucoronate interconversions were up-regulated in the −5 °C Altay lambs compared to the −5 °C Hu lambs, and NF-kappa B-, rap1-, ras-, MAPK- and PI3K-Akt signaling pathways, adrenergic signaling in cardiomyocytes pathway and cardiac muscle contraction pathways were all down-regulated in the −5 °C Altay lambs compared to the −5 °C Hu lambs.UCP1, RYR1, ADIPOQ and LPL, were selected for RT-qPCR analysis. The UCP1 and ADIPOQ genes were involved in the UCP1-dependent thermogenesis pathway [LPL gene was not only involved in the UCP1-dependent thermogenesis of brown adipose tissue (BAT) but also in the thermogenesis of hypothalamus [RYR1 gene participated in the calcium signaling pathway [To substantiate the RNA-seq results, four DEGs from different pathways, including  pathway , LPL genthalamus ,24, and  pathway . The qPCATP2A1 (SERCA1) and CKM (creatine kinase) had a direct protein-protein interaction with each other in the tail-fat tissue of the −5 °C Altay lambs compared to the 20 °C Altay lambs, but there was no relationship in the −5 °C tail-fat of Hu lambs. The UCP1 protein did not have a relationship in the tail-fat tissue of −5 °C Altay lambs compared to the 20 °C Altay lambs, which suggests there may be different strategies of thermogenesis in the two breeds.The PPI analysis, done on Cytoscape software, was based on transcriptome DEGs and String database, which evaluates and scores the relationship between two genes. Protein clustering analyses are illustrated in The heat map of several candidate genes between breeds and tissues are presented in Transcriptome profiling provides new insights into the mechanisms underlying the tolerance of sheep to cold-exposure. In this context, we undertook a comparative RNA-Seq study of three tissues  from cold-exposed Altay and Hu sheep. All lambs maintained normal rectal temperatures with no significant difference among groups. The highest number of DEGs occurred in the tail-fat of −5 °C Altay and Hu lambs  with 2787 down-regulated DEGs and 593 up-regulated DEGs, which suggests that some genes expressed in the tail-fat tissue have important roles in response to cold when compared to those in other tissues. The Altay sheep have a fat tail which stores energy and is important in overcoming periods of sparse winter pasture, and also in preventing hypothermia ,27. In cUCP1 were up-regulated in tail-fat of −5 °C Hu lambs compared to the 20 °C Hu lambs, in perirenal fat of −5 °C Altay lambs compared to the −5 °C Hu lambs and −5 °C Altay lambs compared to the 20 °C Altay lambs. The results indicated that adipose tissue was browning and thermogenic responses had occurred in tail-fat of Hu lambs and in perirenal fat of Altay lambs under cold exposure.Thermogenic genes like The GO analysis revealed that more up-regulated DEGs enriched in a larger number of metabolic process-related GO terms in Hu lambs than in Altay lambs after cold exposure, which suggests that Hu lambs had a greater response to low temperature. Up-regulated DEGs were significantly enriched in the GO term of GTP binding in tail fat of −5 °C compared to 20 °C Hu lambs, and up-regulated DEGs were significantly enriched in the GO term of ATP binding in perirenal fat of −5 °C Altay lambs compared to −5 °C Hu lambs. GTP binding capacity to BAT mitochondria or UCP1 determines the thermogenesis potential of BAT to respond to cold exposure . At ther2+ transport pathway [2+ concentration from a basal level of 0.05 uM to 0.2–0.7 uM, could trigger heat production in BAT cells in rabbits [According to KEGG pathway analyses, upon cold exposure, Hu lambs activated more signal transduction pathways in the hypothalamus and tail-fat tissue to regulate thermogenesis than Altay lambs. In contrast, the Altay lambs activated more pathways in the perirenal fat tissue at low temperatures. The results revealed that the cAMP- and calcium signaling pathways were the major pathways involved in the different tissues from the −5 °C Altay and Hu lambs. The cAMP signaling pathway was enriched in all cold-exposed lambs, which suggested that it plays an important role in the response to cold exposure for different sheep breeds. In addition, cAMP was also enriched in the tail-fat of the −5 °C compared to 20 °C Hu lambs and in the hypothalamus in all cold-exposed lambs. cAMP, known as the second messenger, is involved in triggering energy metabolic and thermogenic processes. Low temperature stimulates an increase in the intracellular concentration of cAMP ,35,36, w pathway , leading pathway . Althoug pathway ,42, as w pathway , with a  pathway ,42. Calc pathway ,45. In a rabbits . The resUCP1 thermogenesis pathway was identified. Some marker genes like UCP1, UCP2 (uncoupling protein 2) and ADRB3 gene were up-regulated to a greater extent in perirenal fat of the −5 °C Altay lambs compared to 20 °C Altay lambs, and in tail-fat of the −5 °C Hu lambs compared to 20 °C Hu lambs. These results indicated that the tail-fat and perirenal fat of Hu and Altay lambs were involved in UCP1 thermogenesis.There was evidence that a calcium signaling pathway was enriched in the present study, although no direct UCP1 in Hu lambs. The hypothalamus of Altay and Hu lambs participated in heat production through different pathways, and different fat tissues of Hu and Altay lambs were involved in UCP1 thermogenesis. The candidate genes heatmap analysis indicated that the pathways related candidate genes had opposite regulatory effects between breed and within the same breed after cold exposure in hypothalamus tissue, but not in fat tissues. The large difference in the hypothalamus tissue suggests that it may play an important role in the cold response in sheep.Given the different homeostatic pathways and genes identified in this study, we concluded that all three tissues were involved in producing heat; but under a cold challenge, the cAMP signaling pathway displayed significant differences among breeds. The calcium signaling pathway may be inhibited in producing heat, being dependent on We screened the DEGs related to thermogenesis from the significant enriched signal transduction pathways within the GO categories.ADORA2A) was an important gene in the cAMP signaling pathway, which was down-regulated in the perirenal fat and tail-fat, and had a down-regulated trend in the hypothalamus of −5 °C Altay lambs compared to Hu lambs. It was up-regulated in perirenal fat of −5 °C compared to 20 °C Altay lambs and tail-fat of −5 °C compared to 20 °C Hu lambs. ADORA2A, one of the receptors of adenosine in this study, is an important thermogenic marker expressed in adipose tissues [ADORA2A gene regulated lipolysis in perirenal fat of Altay lambs and tail-fat of Hu lambs.In the present study, adenosine A2A receptor  plays an important role in lipolysis in WAT by activating hormone sensitive lipase (LIPE), and it could alter Ca2+ influx and the rate of mitochondrial oxidative consumption [HTR2B and LIPE were up-regulated in the hypothalamus and perirenal fat tissue of the −5 °C Hu lambs. These genes promote fat mobilization and heat production in cold-exposed lambs, when the calcium- and cAMP signaling pathways enhance heat production in cold-exposed sheep.In the present study, a large number of calcium signaling related genes were correlated with cold tolerance. chondria . In BAT mulation . Studies muscles ,57,58. Rernating . We pressumption ,60,61. HUCP1, formerly known as “thermogenin”, is a key thermogenic gene in cold-induced NST [UCP1 gene in perirenal fat of −5 °C Altay lambs and in tail-fat of the −5 °C Hu lambs indicated that these adipose tissues underwent browning. Its transcription factor PPARGC1A (peroxisome proliferator-activated receptor γ coactivator-1 α), which regulates mitochondrial biogenesis and respiration, adaptive thermogenesis, gluconeogenesis and other metabolic processes [UCP1 thermogenic pathway verified that the traditional thermogenic pathway existed in this study. The up-regulation of these three key UCP1-dependent thermogenic genes (including ADRB3) in perirenal fat of −5 °C Altay lambs and tail-fat of the −5 °C Hu lambs verified that cold exposure induced UCP1-dependent thermogenesis in different adipose tissue of different sheep breeds. The PPARGC1A and UCP1 genes were inconsistently expressed in tail-fat of cold-exposed Altay lambs because PPARGC1A gene was an upstream gene, which might not involve transcriptional regulation of downstream genes—UCP1.uced NST  and one rocesses , was up-IP6K1) was enriched in the phosphatidylinositol signaling system and up-regulated in all tissues of −5 °C Altay lambs compared to Hu lambs and −5 °C compared to the 20 °C Altay lambs, and down-regulated in the fat tissues of 20 °C Altay lambs compared to Hu lambs. However, the regulatory trends were reversed in the −5 °C compared to the 20 °C Hu lambs in fat tissues. In recent studies, IP6K1 was reported to be related to adenosine monophosphate kinase (AMPK)-mediated thermogenesis [IP6K1 gene did not only differ between breeds, but also relieved the inhibition of AMPK-mediated UCP1-dependent thermogenesis by reducing gene expression. This provides further evidence that tail-fat of cold-exposed Hu lambs was involved in UCP1-dependent thermogenesis.The inositol hexakisphosphate kinase-1 gene  was down-regulated in the tail-fat of −5 °C Altay lambs compared to Hu lambs but there was has no difference in the tail-fat of −5 °C compared to 20 °C Altay lambs, which suggests that this gene may not be involved in thermogenesis of BAT, but may play an important role in fat deposition in −5 °C Altay lambs compared to Hu lambs.The enditure , which sntration ,24, and  in mice . The broPrevious studies on cold stress in sheep focused mainly on physiological responses ,68,69. W


Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, yet as of currently, there is no disease-modifying therapy that could delay its progression. Paeonia lactiflora Pall. is the most frequently used herb in formulas for PD in Traditional Chinese Medicine and also a potential neuroprotective agent for neurodegenerative diseases, while its mechanisms remain poorly understood. In this study, we aim to explore the underlying mechanism of P. lactiflora in treating PD utilizing a network pharmacology approach.Methods: The protein targets of P. lactiflora ingredients and PD were first obtained from several databases. To clarify the key targets, a Protein-Protein-Interaction (PPI) network was constructed and analyzed on the String database, and then enrichment analysis was performed by the Metascape platform to determine the main Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes pathways. Finally, the Ingredient-Target-Pathway (I-T-P) network was constructed and analyzed by Cytoscape software.Results: Six active ingredients of P. lactiflora -catechin) as well as six core targets strongly related to PD treatment  were identified. The main pathways were shown to involve neuroactive ligand-receptor interaction, Calcium signaling pathway, PI3-Akt signaling pathway, TNF signaling pathway, and apoptosis signaling pathway. The main biological process included the regulation of neurotransmitter levels.Conclusion:P. lactiflora may retard neurodegeneration by reducing neuroinflammation, inhibiting intrinsic and extrinsic apoptosis, and may improve motor and non-motor symptoms by regulating the levels of neurotransmitters. Our study has revealed the mechanism of P. lactiflora in the treatment of PD and may contribute to novel drug development for PD. Parkinson’s disease (PD) is becoming the fastest growing neurological disorder surpassing Alzheimer’s disease (AD), with the number of patients expected to double to 14.2 million by 2040 . The cliMucuna pruriens seeds and P. lactiflora roots. Many of these herbs were discovered in traditional medicine practice, and now verified by modern experiments to play a role in various pathological process of PD.For this reason, medicinal plants with multiple ingredients have received widespread attention for PD neuroprotection. A previous research has already summarized more than 50 types of plants against MPTP induced neurotoxicity , includiP. lactiflora is reported to be the most frequently used herb in the treatment of PD , indications of P. lactiflora involve excessive sweating, abdominal pain, contracture of limbs, headache and dizziness, irregular menstruation, et al.  accounts for an incredible 28.55% of its total pharmaceutical industry , P. lactnt of PD . The driAliments  and is ssymptoms . Literatnvulsant . Accordi, et al. . Diarrheextracts ; hence i pathway , and pae pathway . Further pathway , with an pathway . TherefoP. lactiflora in the treatment of PD based on a network pharmacology approach from a holistic view.Network pharmacology is a novel approach to analyze drug mechanisms, identify new targets, and expand new indications at a systematic level, which can comprehensively reflect the mechanism of drugs on disease networks, thus providing support for drug discovery for complex diseases and natural products . NetworkP. lactiflora, an initial screening based on the absorption, distribution, metabolism, and excretion (ADME) properties [oral availability (OB)≥ 30% and drug-likeness (DL) ≥ 0.18], was conducted by the TCM Systems Pharmacology  database (https://pubchem.ncbi.nlm.nih.gov/) and the concentration of active components in this study were retrieved from literature.To obtain the active ingredients of database . OB reprdatabase . Since pdatabase , it is shttp://www.swisstargetprediction.ch/) database  (https://www.disgenet.org/) (https://www.drugbank.ca)  , Disgeneet.org/) , and Drubank.ca) , followehttps://string-db.org)  network on the STRING11.0 database (-db.org) . Set theape3.7.2  to identhttp://metascape.org/gp/index.html). Metascape platform has integrated over 40 knowledgebases with monthly data updates and friendly interactive interface  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out on the Metascape platform  network of P. lactiflora by ADME screening , family and other genes .P. lactiflora in the treatment of PD, and BCL2 is also among the targets. AKT, also known as protein kinase B, plays a key role in a variety of signaling pathways with more than 100 substrates. AKT phosphorylation induces the binding of accessory proteins necessary for the anti-apoptosis gene BCL2 expression, which in turn inhibits apoptosis by regulating the permeability of the mitochondrial outer membrane -catechin may regulate PTGS2 to inhibit intrinsic and extrinsic apoptosis in the treatment of PD.We found that AKT1, CASP3 and PTGS2 are the key targets of membrane . Anothermembrane . Evidencmembrane . In addimembrane . AccordiP. lactiflora may regulate DRD1, ADORA2A, and SLC6A3 in the PD pathway and AKT1 downstream to relieve motor symptoms.According to the classical model of basal ganglion circuit, dyskinesia in PD is caused by unbalanced activity of direct and indirect pathways. The decline of dopamine transmitter released from the substantia nigra to the striatum leads to a decrease in the excitability of the direct pathway  and the over-activation of the indirect pathway , which collectively suppress the cortical excitability . A2A recP. lactiflora for PD, as well as serotonergic synaptic pathway, cholinergic synaptic pathway, and dopaminergic synaptic pathway also being observed, suggesting that P. lactiflora may relieve NMS through the regulation of a variety of neurotransmitter pathways.NMS are key determinants of quality of life. However, limited treatments are available . MainstrP. lactiflora components. For example, kaempferol inhibits the activation of NLRP3 inflammasome through the synergistic effect of ubiquitin and autophagy, thus promotes the survival of dopaminergic neurons in PD models induced by LPS and SNCA  inhibitor is a first-line treatment for AD dementia, and possibly useful for PD dementia  in a dose-dependent manner .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anthropogenic stressors can disrupt cetacean foraging ability and reduce energy available for reproduction. Predicting such ecological consequences is limited by our understanding of cetacean energy metabolism. We here show that cetaceans have undergone substantial evolutionary changes in nutrient signaling metabolic pathways, rendering mechanistic physiological frameworks derived from model organisms inappropriate. Foraging disruption caused by human activities is emerging as a key issue in cetacean conservation because it can affect nutrient levels and the amount of energy available to individuals to invest into reproduction. Our ability to predict how anthropogenic stressors affect these ecological processes and ultimately population trajectory depends crucially on our understanding of the complex physiological mechanisms that detect nutrient availability and regulate energy metabolism, foraging behavior and life-history decisions. These physiological mechanisms are likely to differ considerably from terrestrial mammalian model systems. Here, we examine nucleotide substitution rates in cetacean and other artiodactyl genomes to identify signatures of selection in genes associated with nutrient sensing pathways. We also estimated the likely physiological consequences of adaptive amino acid substitutions for pathway functions. Our results highlight that genes involved in the insulin, mTOR and NF-ĸB pathways are subject to significant positive selection in cetaceans compared to terrestrial artiodactyla. These genes may have been positively selected to enable cetaceans to adapt to a glucose-poor diet, to overcome deleterious effects caused by hypoxia during diving (e.g. oxidative stress and inflammation) and to modify fat-depot signaling functions in a manner different to terrestrial mammals. We thus show that adaptation in cetaceans to an aquatic lifestyle significantly affected functions in nutrient sensing pathways. The use of fat stores as a condition index in cetaceans may be confounded by the multiple and critical roles fat has in regulating cetacean metabolism, foraging behavior and diving physiology. Phocoena phocoena) by causing displacement from important habitat and feeding grounds  are becoming a pervasive and prevalent threat to many cetacean species and are a key priority in cetacean conservation policy e.g. . One impOne significant barrier to achieving this understanding is that these physiological mechanisms are likely to differ considerably from those of terrestrial mammal model systems. Cetaceans evolved from terrestrial mammals approximately 53–56 million years ago and drastically adapted their anatomy, physiology and metabolism during transition to an aquatic lifestyle. In particular, the ability to maintain body temperature and energy reserves required several critical morphological and physiological adaptations . BlubberBalaenoptera physalus) is impacted by lower but not larger than normal blubber thickness  with lower amounts of blubber angle their body in such a way to reduce the cost of transport when swimming against the force of buoyancy  as in terrestrial mammals such as humans and laboratory rodent models. In these models, both reduced and increased fat mass  lead to reduced reproductive success  signaling and NF-ĸB signaling. Leptin and insulin represent the classic model feedback system that can lead to changes in behavioral and metabolic responses in mammals . Howeverhttp://www.genome.jp/kegg/) and the ingenuity pathway analysis (IPA) program . We obtained reference genome assemblies for human, mouse, 16 cetacean species and 37 artiodactyl species from NCBI  was retained and the nucleotide sequence of the aligned genomic region was extracted.We identified 532 human reference genes involved in the leptin, insulin, p53, mTOR, SIRT or NF-ĸB signaling pathways using the Kyoto Encyclopedia of Genes and Genomes website , we identified the specific codons under positive selection in the cetacean lineage using the Bayes Empirical Bayes (BEB) method . We thenwww.ingenuity.com). This software allows for visualizing and analysing prebuilt biological pathways using measured values at particular genes. The links between the genes in IPA are knowledge based and therefore represent regulatory roles with either activator or inhibitor effects. This allows for predicting the regulatory impact a gene may have on the pathway. We used pre-build pathways for the p53 signaling pathway, the insulin signaling pathway, the mTOR signaling pathway, the leptin signaling pathway and the NF-ĸB signaling pathway. IPA did not have a prebuilt sirtuin signaling pathway, so we manually constructed this pathway based on the summarized data by  between the positive and neutral branch-site models was used as a measure for visualization. This allowed us to see how genes under positive selection may interact with each other and what the potential impact the gene may have on a pathway level.We then assessed the pathway-level impacts of those genes that were identified with a functionally relevant amino acid substitution (PROVEAN score ≤ −2.5), using the molecule activity predictor (MAP) function in IPA. This function can predict the upstream  or downstream  effect of a molecule in a pathway, allowing us to examine how these genes may impact energy metabolism by simulating directional consequences on downstream molecules and inferred activity upstream from the gene. The algorithm of the MAP function divides the molecules of the pathway into two sets: known or unknown expression based on the uploaded dataset. In our case, we used the PROVEAN score as a measure of ‘expression’. The algorithm identifies the unknown molecules that are connected to the known molecules by directional relationships (i.e. activated or inhibited) based on the underlying scientific knowledge (pre-existing content in IPA). For each molecule, it will then calculate a z-score using the findings between the unknown molecules and its known neighbors. Once the value of a molecule is known, it is moved to the ‘known set’ and the algorithm is repeated until all molecules have been evaluated. The result of the MAP function is the visualized predicted regulation of the pathway based on the genes affected by the functionally relevant amino acid substitution. Although this approach assumes that the identified beneficial amino-acid substitutions cause a concomitant change in protein expression, it provides a reasonable functional representation of those parts of the pathway that may be affected downstream due to functionally relevant amino acid substitution of any gene higher up in the signaling cascade.Hippopotamus amphibius (0) from the PAML null models were < 1 for all but one genes , consistent with an expected baseline of strong purifying selection on protein function across all taxa and all codons (f < 1) instead of positive selection (ωf > 1). Only 7 of 532 genes (1.3%) showed an indication of positive selection (ωf > 1), but none of these estimates were significant  in the cetacean group on a subset of codons in 133 of 532 genes (25%)  .Using the BEB method in PAML, a total of 1936 codons among 133 genes (mean 14.78 codons per gene) were under positive selection in the cetacean lineage. The vast majority (91%) of amino-acid substitutions in cetacea were predicted to have some functional effects (PROVEAN score < 0) and 56% of substitutions may have strong, biologically significant, effects . Only 8.5% of substitutions had a positive PROVEAN score, suggesting functional neutrality .Using IPA to visualize genes under positive selection in cetacea at the pathway level, we found evidence of selection in insulin signaling , mTOR side novo lipid synthesis.Using the MAP function in IPA to predict potential pathway-level regulatory effects of functionally relevant amino acid substitutions identified by PROVEAN, we found several instances of directional consequences on downstream molecules and inferred activity upstream of the gene in the pathway. Unsurprisingly, glucose metabolism is expected to be altered with changes in insulin signaling , SIRT3 sSublethal stressors caused by anthropogenic disturbances can trigger a stress response regulated via the hypothalamic–pituitary–adrenal axis and signals to release energy in the form of glucose from glycogen stores or amino acids (gluconeogenesis) and from fat stores to allow a rapid response to optimize survival (i.e. fight and flight response) . CetaceaThere are indications that cetaceans underwent evolutionary adaptation to key genes involved in energy metabolism and this may impact the ways in which cetaceans cope with varied environmental pressures (Tursiops truncatus) where glucose levels were significantly higher during starvation (Mirounga angustirostris), components of the insulin signaling pathway were reduced including PI3K and IRS1  . mTORC1 regulates processes related to growth and differentiation while mTORC2 plays a regulatory role in the insulin cascade . This miTaken together, our findings provide novel insights into the role of the insulin, mTOR and NF-ĸB signaling pathways in the adaptation of cetaceans to an aquatic life. Our focus on biological pathway-level expected changes as adaptations to an aquatic lifestyle across the whole cetacean group has opened up novel and exciting research avenues into phenotypic and molecular evolution of particular cetacean taxa and candidate genes. Population genomics studies will be required to determine spatio-temporal structure of adaptive genetic diversity at key genes in nutrient sensing pathways. Our work has also provided a framework for examining physiological trait evolution throughout cetartiodactyla.These results mean that condition measures based on adiposity must be used with caution. Blubber thickness alone provides little insight into the health of cetaceans  have led to changes in energy metabolism, which may also affect the way energy investment decisions are reached in cetaceans. Blubber is a complex tissue (D.L. and D.D. designed the study. D.D. and M.W. wrote the manuscript, and D.D. performed the pathway level analyses and interpretation with input from D.L. M.W. performed the gene-level analyses with input from A.D. and J.S. D.D., M.W. and D.L. interpreted the data. All authors read and commented on the manuscript.Data will become openly available after uploading on Dryad.suppl_data_coaa136Click here for additional data file.


Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast  All isoprenoids derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)2. In nature, two routes are leading to IPP and DMAPP; the mevalonic acid (MVA) pathway and the methylerythritol phosphate (MEP) pathway5 17, and non-LTR Long INterspersed Element (LINE)18 retrotransposons. Lastly, contrasting to the more well documented respiro-fermentative S. cerevisiae, Y. lipolytica stands an obligate aerobe. Yarrowia lipolytica metabolizes a varied type of carbon substrates, including lipids, paraffin, oils, acetate, and glycerol, and is capable of accumulating a high ratio of cell weight in lipid21.The oleaginous yeast Y. lipolytica could utilize the MEP pathway for isoprenoid biosynthesis. Here, we present a more in-depth investigation of the existence of the MEP pathway in Y. lipolytica using three independent approaches; the spiking of the authentic standard, the inhibition of the pathway-specific inhibitor, and analysis of the incorporation of the stable carbon isotope in the natural product. This study suggested that the MVA and MEP pathways co-exist in Y. lipolytica.This study raises an intriguing hypothesis of whether Y. lipolytica in which the nitrogen source was limited. Interestingly, the chromatographic feature was similar to the previous report on the MEP analysis in E. coli, where a larger peak with the same MRM was detected next to the MEP peak22. To confirm that the possible MEP chromatographic peak found in Y. lipolytica is comparable to the analytical standard; the spiking analysis of the authentic MEP standard was carried out. The results showed that the annotated MEP peak detected in Y. lipolytica was identical to the analytical MEP standard, thereby meeting the criteria for metabolite identification based on The Metabolomics Standards Initiative (MSI) for level 1 identification  was found only in the sample from the culture extract of Streptomyces lavendulae, has been shown to inhibit bacterial isoprenoid biosynthesis23. It was later demonstrated that fosmidomycin specifically inhibits 1-deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR)25, which catalyzes the conversion of the DXP to MEP in the specific step of the MEP pathway. In this study, Y. lipolytica cultivated in nitrogen limiting condition was treated with fosmidomycin and analyzed with LC/QqQ/MS. The data show that the MEP peak that was elucidated with the authentic standard spiking earlier was reduced in the presence of fosmidomycin. The result suggested that DXR-like activity was present in Y. lipolytica and inhibited by fosmidomycin to have reduced the MEP content  of 37928. When 1-13C-labeled glucose was added to the culture medium of Y. lipolytica, the incorporation of the 13C carbon isotope increased the m/z of the fragment to the maximum of 383. Thus, this fragment of m/z 383 was selected as the parent ion for the product ion scan  are included; these data are known to possess a high correlation to a physical phenomenon35. Upon our data analysis, we stumbled upon a very startling data where one of the chromatographic peaks was annotated as MEP. Although the LC/QqQ/MS annotated data are widely accepted in the field of metabolomics due to its selectivity and sensitivity36, we took extra steps to verify the results by further checked the annotation with the authentic standard spiking technique. The spiking of the authentic standard provided the analytical chemistry evidence of the existence of the MEP pathway enzyme(s) in Y. lipolytica. Note that in the nitrogen limiting condition, another peak around 7.40 min that also only occurs in the nitrogen limiting condition, this might be a compound that is important for adapting to the nitrogen limiting condition or related to this MEP-like pathway. It would be interesting for future studies. The next issue we address is the evidence from the biological perspective. One of the best ways to assess the MEP pathway activity is to evaluate the enzyme specific to the MEP pathway; the DXP reductoisomerase37. However, no DXR gene has been annotated in the genome of Y. lipolytica and the BLAST homology search42 returns no significant alignment. We further investigated this issue with an in-house bioinformatics pipeline , 2. the ergosterol molecule provide multiple fragments that could be analyzed for the pathway differences, and they provide more mass different; therefore, the effect of the MEP or MVA pathway can be clearly observed, 3. the extraction and purification method for ergosterol was well established. This analysis was grounded on the fact that these building blocks are biosynthesized by completely different routes, the MVA pathway, and the MEP pathway  involved in the MEP-like pathway. Firstly, the authentic standard confirmed that the MEP was detected in Y. lipolytica. Secondly, the MEP peak was affected by the specific MEP pathway inhibitor fosmidomycin. Finally, the 13C labeled glucose feeding showed that the ergosterol fragments were matched with the theoretical calculations of the mass increased from both the MVA pathway and the MEP pathway. The genes involved in the MEP synthesis in Y. lipolytica and the mechanism on how nitrogen limiting conditions trigger this phenomenon remain unclear. Our findings reported here may spark new investigations on the possibility of the MEP pathway-like existence in yeasts that may be activated in specific environmental conditions. Moreover, this finding might open a new avenue for oleaginous yeast control and modification for further industrial applications.Altogether, our results suggested that the MVA pathway and the MEP-like pathway co-exist in Yarrowia lipolytica PO1d obtained from the National University of Singapore were pre-cultivated for 17 h in synthetic complete (SC) medium  in 100 mL Erlenmeyer flasks at the constant temperature of 30 °C and the shaking speed of 200 rpm. The cells were then inoculated into 20 mL SC medium without ammonium sulfate (nitrogen limiting condition) to obtain the initial optical density at 600 nm of 0.1 in 100 mL Erlenmeyer flasks at the constant temperature of 30 °C and the shaking speed of 200 rpm. For the sample collection, 5 OD600 units of cells were collected at 36 h by means of fast filtration using the 0.45 µm pore size, 47 mm diameter nylon membrane (Millipore). The cells were then immersed in liquid nitrogen immediately for metabolism quenching and kept at -80 °C until extraction. For extraction, 1.8 mL of extraction solvent -10 camphorsulfonic acid as an internal standard) was added to each 2-mL sampling tube with filtered sample and incubated at -30 °C for 1 h. After incubation, 700 µL of the solution was transferred to a new tube containing 350 µL of water. The mixture was mixed using a vortex and centrifuged at 16,000 × g for 3 min at 4 °C to separate polar and non-polar phases. 700 µL of the upper polar phase was transferred to a new tube via syringe filtration . The sample was centrifugally concentrated for 2 h and freeze-dried overnight. After reconstituting in 100 µL ultra-pure water, the sample was centrifuged at 16,000 × g for 3 min at 4 °C and transferred to an LC glass vial. All experiment was done in triplicates.43 using a Nexera UHPLC system  coupled with LC/QqQ/MS 8030 Plus (Shimadzu). The column used was CERI ODS L-column 2 metal free column , the mobile phase was (A): 10 mM tributylamine and 15 mM acetate in ultra-pure water, (B): methanol. The flow rate was set to 0.2 mL/min, the column oven temperature was set to 45 °C. The mobile phase (B) was increased from 0 to 15%, 50%, and 100% from 1.0 to 1.5 min, 3.0 to 8.0 min, and 8.0 to 10.0 min, respectively; held until 11.5 min, decreased to 0% from 11.5 min and held at 0% until 20 min. The analysis mode was negative ion detection mode. The injection volume was 3 µL, probe position was + 1.5 mm, desolvation line temperature was 250 °C, heat block temperature was 400 °C, nebulizer gas flow was 2 L/min, and drying gas flow was 15 L/min. All analysis was done in triplicates.The MEP authentic standard was purchased from Sigma-Aldrich (product number 52131). The samples were analyzed based on the method adopted from Dempo et al.Y. lipolytica, DXP reductoisomerase inhibitor (Fosmidomycin) was used. Fosmidomycin sodium salt hydrate ≥ 95% (NMR) was purchased from Sigma-Aldrich (product number F8682). Yarrowia lipolytica the main culture, was cultivated and collected as mentioned above, using medium with limited nitrogen source with the addition of 100 µM inhibitor. The samples were analyzed using the LCMS method above. All analysis was done in triplicates.To assess the DXR activity, the enzyme specific to the MEP pathway in Y. lipolytica, we analyze ergosterol in Y. lipolytica, which is the natural end product derived from DMAPP. The 1-13C D-glucose (98–99%) was purchased from Cambridge Isotope Laboratories (Item Number CLM-420-PK). Yarrowia lipolytica PO1d were pre-cultivated overnight in synthetic complete (SC) medium  in 100 mL shake flasks at 30 °C and 200 rpm. The pre-cultured cells were inoculated into 20 mL 10% 1-13C D-glucose SC medium without ammonium sulfate to obtain the initial optical density at 600 nm of 0.1 in 100 mL shake flasks at 30 °C and 200 rpm. For sample collection, the cell pellet was collected at 36 h by centrifugation at 4 °C, 10,000 rpm, 10 min. The cell pellet was then submerged in liquid nitrogen and subjected to lyophilization overnight for further use. For extraction, 1 g of lyophilized cell pellet was extracted by 20 mL of extraction solvent  for 2 h with stirring. After incubation, the solution was filtered with filter paper with cut-off diameter of 3 µM (Advantech No.6), the extraction was repeated three times. The solution was evaporated until dryness using a rotary evaporator. The ergosterol was then separated from the crude extract by open column chromatography; the fraction containing ergosterol was confirmed by GCMS (Supplementary Fig. To further assess if the MEP pathway was working in Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.Supplementary Information 15.Supplementary Information 16.Supplementary Information 17.Supplementary Information 18.Supplementary Information 19.Supplementary Information 20.Supplementary Information 21.Supplementary Information 22.


Bioengineering has demonstrated the potential of utilising mesenchymal stem cells (MSCs), growth factors, and mechanical stimuli to treat cartilage defects. However, the underlying genes and pathways are largely unclear. This is the first study on screening and identifying the hub genes involved in mechanically enhanced chondrogenesis and their potential molecular mechanisms.The datasets were downloaded from the Gene Expression Omnibus (GEO) database and contain six transforming growth factor-beta-3 (TGF-β3) induced bovine bone marrow-derived MSCs specimens and six TGF-β3/dynamic-compression-induced specimens at day 42. Screening differentially expressed genes (DEGs) was performed and then analysed via bioinformatics methods. The Database for Annotation, Visualisation, and Integrated Discovery (DAVID) online analysis was utilised to obtain the Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment. The protein-protein interaction (PPI) network of the DEGs was constructed based on data from the STRING database and visualised through the Cytoscape software. The functional modules were extracted from the PPI network for further analysis.The top 10 hub genes ranked by their connection degrees were IL6, UBE2C, TOP2A, MCM4, PLK2, SMC2, BMP2, LMO7, TRIM36, and MAPK8. Multiple signalling pathways  may impact the sensation, transduction, and reaction of external mechanical stimuli.This study provides a theoretical finding showing that gene UBE2C, IL6, and MAPK8, and multiple signalling pathways may play pivotal roles in dynamic compression-enhanced chondrogenesis. Cartilage has little self-renewable ability due to its instinctive physiologies , 2, whicInspired by the physiology of native articular cartilage subjected to the dynamic joint environment, mainly under compression and shearing conditions , the sigMicroarray technology provides a powerful tool for exploring the gene regulation pattern and molecular mechanisms involved in mechanical-enhanced chondrogenesis. It enables to investigate thousands of gene expression patterns . The micFigure GO enrichment and KEGG pathway enrichment analyses were performed to identify the biological function of DEGs. In GO terms, a negative regulation of angiogenesis, in utero embryonic development, and inflammatory responses provided the most significant enrichment in the biological process. The most significant enrichment in the cellular component was created through the cytoplasm, transcription elongation factor complex, and cortical actin cytoskeleton. Haemoglobin binding and ATP binding represented the most significant enrichment in the molecular function. A full list of enriched GO terms is shown in Table The PPI network of all DEGs Fig. , construThe MCODE generated five sub-clusters, which reflect the high modularisation of a gene network. The top three amongst five modules contain nine of ten hub genes and are shown in Fig. Chondrocytes respond to mechanical stimuli through regulating gene expression, proliferation, and metabolic functions. However, little is known about the key genes, signalling pathways, and proteins. Chondrocytes have been considered a post-mitotic tissue with nearly no cellular turnover. They are surrounded by an extracellular matrix comprised of glycosaminoglycan (GAG) and collagen and are subjected to daily dynamic compression. During the in vitro culture, growth factors such as bone morphogenetic protein (BMP) and the TGF-β superfamily are indispensable for the chondrogenic differentiation of MSCs . HoweverFurthermore, in Huang’s  originalThe high-throughput microarray technology combined with bioinformatics analysis has been widely used in providing new insight into gene expression changes and molecular mechanisms. In the present study, the GEO database was utilised to obtain microarray raw data. A total of 236 DEGs were identified between TGF-β3-induced and TGF-β3/dynamic-compression-induced MSCs, including 178 up-regulated genes and 58 down-regulated genes. After that, the DEGs were analysed by GO functional enrichment analysis and classified into three groups, which were subsequently further clustered, based on functions and signalling pathways.The results of GO functional enrichment analysis showed that the DEGs were mainly enriched in the GO terms of inflammatory response, in utero embryonic development and negative regulation of angiogenesis. This conforms to previous studies showing that the inflammatory response was involved in chondrogenic regulation. Inflammatory factors have been recognised as an important driving force leading to cartilage breakdown, and their down-regulation is vital for constructing initial collagen networks. A previous animal study revealed that the three-day cyclic compression of 0.5 MPa at 0.5 Hz on bovine chondrocytes counteracted the cartilage degradation induced by IL-1 . TherefoMoreover, KEGG pathways enrichment analysis was performed. Because the KEGG database integrates data on genomes, chemical molecules and biochemical systems, including pathways, drug, disease, gene sequences, and genomes, some irrelevant disease clusters might be unexpectedly enriched. These disease-related clusters were screened and removed from the results and discussion. The KEGG pathway enrichment of DEGs and module analysis showed that the PI3K-Akt signalling pathway, toll-like receptor signalling pathway and TNF signalling pathway were highly enriched. Studies have demonstrated that the activation of the PI3K-Akt pathway promotes the terminal differentiation of chondrocytes and inhibits the hypertrophic differentiation of chondrocytes , 24. TheThe PPI network was constructed to predict the connections of proteins encoded by DEGs. The top 10 hub genes were screened according to connection degree as follows: IL6, UBE2C, TOP2A, MCM4, PLK2, SMC2, BMP2, LMO7, TRIM36, and MAPK8. Nine of them functioned in two of the top three most significant modules, suggesting that these genes play a more important role in chondrogenesis and are enhanced by dynamic compression. The Modules 1 and 3 were extracted from the PPI network. UBE2C, TOP2A, MCM4, PLK2, SMC2 LMO7, and TRIM36 were contained in Module 1, which were mainly enriched in GO terms related to the cellular metabolic process. These genes have closed relationships with the cell cycle and proliferation, and some of them were found overexpressed in various tumours. Moreover, UBE2C , TOP2A  and MCM4It was reported that the downregulation of PLK2 inhibited the degree of inflammation of knee joint synovial tissue and inhibited the cartilage collagen destruction in rats . In receThis study analysed the gene expression profiles between TGF-β3-induced and TGF-β3/dynamic-compression-induced MSCs using a bioinformatics approach. 236 DEGs were found and annotated into GO terms and KEGG pathways, followed by constructing a PPI network and module mining. To our knowledge, this is the first time that genes, including UBE2C, IL6, and MAPK8, are identified to play a pivotal role in dynamic compression enhanced chondrogenesis via regulating proliferation, apoptosis and inflammatory response. Multiple signalling pathways, including the PI3K-Akt signalling pathway, toll-like receptor signalling pathway, TNF signalling pathway, and MAPK pathway, may be involved in sensation, transduction, and reaction of external mechanical stimuli. Although this is the first study giving a comprehensive genetic perspective on the interaction between mechanical stress and chondrogenesis, more experimental evidences are required to verify these findings. Further experimental studies are planned confirm these analysis results, which will be featured in the near future.https://www.ncbi.nlm.nih.gov/geo) [The gene expression profiles of GSE18879 were downloaded from a public functional genomics data repository GEO database (gov/geo)  with thegov/geo) . This daThe CEL format files of raw data were converted into probe expression matrix, then underwent background adjustment, quantile normalisation, and ssummarisation using the Robust Multichip Average (RMA) in the RMAExpress software (version 1.2.0) . Then, ahttps://www.networkanalyst.ca), which is a visual analytics platform for comprehensive gene expression profiling and meta-analysis [p-value was corrected using the Benjamini-Hochberg test. Finally, the cut-off criterion of DEGs was set at the log2 fold change |log2FC| > 1.5 and adjusted as P < 0.05.The up-regulated and down-regulated DEGs between TGF-β3-induced MSCs specimens and TGF-β3/dynamic-compression-induced MSCs specimens were identified through the Limma package on the NetworkAnalyst 3.0 web tool  is an online functional enrichment analysis web tool that provides systematic annotation information for the biological function of large-scale gene list [The Database for Annotation, Visualisation, and Integrated Discovery  database [In order to understand the molecule mechanism and to study the interactions between dynamic compression and chondrogenesis, and between proteins encoded by DEGs and different proteins, the STRING (database  was utilP < 0.05). Similarly, irrelevant disease clusters in the KEGG pathway enrichment analysis were screened and removed before analysis and discussion.Another built-in APP Molecular Complex Detection (MCODE) was utilised to detect the dense functionally connected sub-clusters within the large PPI network. The parameters of network scoring and cluster finding were set as follows: degree cutoff = 2, node score cutoff = 0.2, k-core = 2, and max depth = 100. The top three sub-clusters identified by modularity analysis were then selected for GO and pathway enrichment analysis via DAVID (gene count > 2 and