Hyperlipidemia, due to the practice of unhealthy lifestyles of modern people, has been a disturbance to a large portion of population worldwide. Recently, several scholars have turned their attention to Chinese medicine (CM) to seek out a lipid-lowering approach with high efficiency and low toxicity. This study aimed to explore the mechanism of Huatan Jiangzhuo decoction  in the treatment of hyperlipidemia and to determine the major regulation pathways and potential key targets involved in the treatment process.  Data on the compounds of HTJZD, compound-related targets (C-T), and known disease-related targets (D-T) were collected from databases. The intersection targets (I-T) between C-T and D-T were filtered again to acquire the selected targets (S-T) according to the specific index. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, as well as network construction, were applied to predict the putative mechanisms of HTJZD in treating hyperlipidemia. Thereafter, an animal experiment was conducted to validate the therapeutic effect of HTJZD. In addition, regulated differentially expressed genes (DEGs) were processed from the RNA sequencing analysis results. Common genes found between regulated DEGs and S-T were analyzed by KEGG pathway enrichment to select the key targets. Lastly, key targets were validated by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. P < 0.01).  A total of 210 S-T were filtered out for enrichment analysis and network construction. The enrichment results showed that HTJZD may exert an effect on hyperlipidemia through the regulation of lipid metabolism and insulin resistance. The networks predict that the therapeutic effect of HTJZD may be based on the composite pharmacological action of these active compounds. The animal experiment results verify that HTJZD can inhibit dyslipidemia in rats with hyperlipidemia, suppress lipid accumulation in the liver, and reverse the expression of 202 DEGs, which presented an opposite trend in the model and HTJZD groups. Six targets were selected from the common targets between 210 S-T and 202 regulated DEGs, and the qRT-PCR results showed that HTJZD could effectively reverse Srebp-1c, Cyp3a9, and Insr mRNA expression ( In brief, network pharmacology predicted that HTJZD exerts a therapeutic effect on hyperlipidemia. The animal experimental results confirmed that HTJZD suppressed the pathological process induced by hyperlipidemia by regulating the expression of targets involved in lipid metabolism and insulin resistance. Hyperlipidemia is a condition wherein lipid-glucose metabolic disruption occurs, and it is mainly characterized by the derangements of lipoproteins circulating in the blood, such as high levels of total cholesterol (TC), triglyceride (TG) and/or low-density lipoprotein cholesterol (LDL-c), or aberrant declined level of low-density lipoprotein cholesterol (HDL-c) , 2. GrowCurrently, statins, the first-line recommended therapy for lowering lipid, are the widely used drugs for hyperlipidemia . NeverthAlisma orientale could increase the HDL-c/LDL-c ratio and regulate abnormal cholesterol-related markers in high-fat diet (HFD)-fed rats [Poria cocos attenuates the perturbations of bile acid biosynthesis [Citrus reticulata extract could ameliorate HFD-induced obesity by initiating browning of beige adipocytes [Huatan Jiangzhuo decoction (HTJZD) was termed based on the therapeutic method of resolving phlegm and reducing turbidity of the CM theory. It includes the following nine herbs: Poria, Alismatis Rhizoma, Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, Pinelliae Rhizoma Praeparatum, Citri Reticulatae Pericarpium, Glycyrrhizae Radix et Rhizoma, Ginseng Radix et Rhizoma, and Citri Reticulatae Pericarpium Viride. It has been reported that hypolipidemic efficacy is found in a portion of ingredients in these herbs. For example, fed rats . Poria cynthesis . Moreoveipocytes . In addiipocytes , 20 haveThis study resorted to network pharmacology combined with RNA sequencing techniques to promote our understanding concerning the molecular mechanism of HTJZD in treating hyperlipidemia. Network pharmacology is a novel bioinformatic technology that is applied to elucidate the occurrence and development of diseases based on system biology and biological network equilibrium. It is also generally used to guide the discovery of new drugs and clarify the mechanism of CM . The RNAhttps://tcmspw.com/tcmsp.php). TCMSP is a composite website that contains information about active compounds, potential targets, related diseases, and pharmacodynamics data of CM. ADME parameters were used to evaluate the compounds, of which are oral bioavailability (OB)≥30% and drug likeness (DL) of ≥0.18 were the inclusion criteria used to select the active compounds of HTJZD in this study. Subsequently, the related targets of selected compounds were obtained from TCMSP, ETCM (http://www.tcmip.cn/ETCM/index.php/Home/Index/), and Swiss Target Prediction (http://www.swisstargetprediction.ch/). Candidate target genes from ETCM should meet the prediction confidence index of ≥0.80. Targets of compounds predicted by Swiss Target Prediction should attain the canonical SMILEs or InChI from PubChem (https://pubchem.ncbi.nlm.nih.gov/) first and then be pasted in the website to acquire ones with the probability of ≥0.The compounds of each herb in HTJZD were collected from TCMSP (https://www.disgenet.org/) and GeneCards (https://www.genecards.org/) with “hyperlipidemia” as the keyword. DisGeNET contains a large number of genes and variants related to human diseases and serves as a multifunctional platform to meet diverse research purposes. GeneCards is a composite database that provides known and predicted genes of Homo sapiens regarding multiple omics and functional information.Hyperlipidemia targets were collected from DisGeNET (https://www.uniprot.org/) based on H. sapiens genes to delete duplicate values. Then, the intersection targets (I-T) of C-T and D-T were searched and used to explore the correlation among I-T on STRING (https://www.string-db.org/). The minimum required interaction score was set as “highest confidence (0.900)” on STRING, and the simple tabular text output was downloaded from it. Lastly, the node  interaction meeting the criteria of “a combined score of ≥0.95” was screened out to construct the herb-compound-S-T network and S-T-pathway network in Cytoscape 3.7.2 for visualizing the results.Compound-related targets (C-T) and disease-related targets (D-T) were standardized in UniProtKB (https://biit.cs.ut.ee/gprofiler/gost) for KEGG signaling pathway enrichment analysis and GO functional annotation including biological process (BP), cellular component (CC), and molecular function (MF). The website, g:Profiler, updated in a timely manner, serves as a collection of functional enrichment analysis (g:Gost), gene ID conversion (g:Convert), and SNP id to gene name (g:SNPense). The GO results were depicted by GraphPad Prism 8, and the KEGG pathway results were painted by ggplot2 packages in R Studio (version 4.0.2).S-T were input to g:Profiler . HE staining and Oil Red O staining associated reagents were purchased from Sinopharm Chemical Reagent Co., Ltd  and Wuhan Servicebio Technology Co., Ltd , respectively. Bestar qPCR RT kit (batch number: 2220) and SYBR Green qPCR Master Mix kit (batch number: 2043) were produced by DBI Bioscience Co., Ltd. . Atorvastatin was diluted to 0.72 mg/mL in distilled water and preserved at 4°C.HTJZD is composed of Alismatis Rhizoma (10 g), Poria (15 g), Atractylodis Macrocephalae Rhizoma (15 g), Atractylodis Rhizoma (10 g), Pinelliae Rhizoma Praeparatum (10 g), Citri Reticulatae Pericarpium (10 g), Glycyrrhizae Radix et Rhizoma (10 g), Ginseng Radix et Rhizoma (10 g), and Citri Reticulatae Pericarpium Viride (10 g). All medicines were the herbal decoction pieces  purchaseForty male Sprague-Dawley (SD) rats  were purchased from Guangzhou Southern Medical University Laboratory Animal Science and Technology Development Co., Ltd., China (Certificate No. 44002100009585). All the animals were housed in the Laboratory Animal Center of Guangzhou University of Chinese Medicine  under the standard and controlled conditions, 21–25°C temperature, 50–70% humidity, and 12-h light-dark cycle. HFD was obtained from Guangdong Medical Laboratory Animal Center, China. HFD recipe consists of 52.2% maintenance feed, 0.4% premix feed, 20% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 10% casein, 0.6% calcium hydrophosphate, and 0.4% mountain flour.n= 10) and the HFD group (n = 30). The hyperlipidemia model establishment in way of feeding with HFD lasted for 30 days. Before collecting the blood samples, all the rats had to be fasted for 12 h. Serum lipids were measured using the kits to evaluate the establishment of hyperlipidemia model. In the intervention phase, the HFD group was continuously supplied with HFD but regrouped into three groups: model group  administered saline, atorvastatin group  administered 1.8 mg/kg per day of atorvastatin, and HTJZD group  administered 12.6 g/kg per day of HTJZD. Each rat was given an intragastric dose of 1 mL/100g.This research design is based on a previous exploratory experimental model , 24. AftAfter 4 weeks of intervention, all animals were sacrificed for blood sample and liver sample collection. The blood samples were centrifuged at 3,500 rpm for 10 minutes to obtain the isolated serum and stored at −20°C immediately for subsequent biochemical testing. The liver tissues were stored at −80°C. The protocol was approved by the Animal Experimental Ethics Committee of Guangzhou University of Chinese Medicine.After 30 days of feeding, blood samples were collected from the rats' posterior ocular venous plexus to judge the development of hyperlipidemia. Moreover, after 4 weeks of intervention, the animals were sacrificed for blood collection from abdominal aorta. Serum TC, TG, LDL-c, and HDL-c levels of each group were measured by detection kits (enzyme-labeling assay) according to the manufacture's operating instructions.The Evaluation Method of Auxiliary Hypolipidemic Function [P < 0.05) in the model group. As for the identification of treatment efficiency in the mixed hyperlipidemia model, conditions of the effective intervention wherein serum TC or LDL-c levels were significantly reduced (P < 0.05) were not accompanied by a significant increase in the HDL-c level or a decrease in the TG level in the intervention group when compared with the control group.According to Function  issued bμm slices for Oil Red O staining. All sections were observed by an upright optical microscope  and an image collection system  at 200× magnification.After dissection, liver specimens were fixed in 4% (w/v) paraformaldehyde for 24 h, followed by flushing with running water overnight. Moreover, tissues were dehydrated with graded ethanol and embedded in paraffin blocks. Thereafter, paraffin blocks were sectioned to 4 µm slices spread on the glass slides, which were then rehydrated and stained with HE. In addition, liver tissues were made into frozen samples. Fresh tissues were preserved in fixer for over 24 h, dehydrated with sucrose solution at 4°C, subsequently embedded on a molder with optimal cutting temperature freezing compound, and sectioned into 8 Total RNA was extracted from liver tissue samples of three groups  by using TRIzol reagent. Moreover, agar gel electrophoresis and NanoDrop were applied to test the purity and qualify the concentration of RNA. Agilent 2100 Bioanalyzer was facilitated to examine RNA quality. The transcriptomic library of cDNA was constructed and verified by Qubit 3.0 and Agilent 2100. After the library was purified, the sequencer HiSeq 2500  was used on all samples for paired-end sequencing. Clean reads were obtained according to the following filter criteria-eliminating low quality reads and reads that comprise adapters. Consequently, TopHat2 was applied in setting genome (Rnor_6.0) as sequencing alignment reference to clean reads used to attain the information from and beyond the referenced genome. Each gene expression was assessed by fragments per kilobase of transcript per million fragments mapped (FPKM). The computational method of FPKM is below:DESeq was chosen for analyzing differential expression of genes in three groups. Screening criteria concerning a false discovery rate (FDR) of <0.05 and fold change (FC) of >1.5 or <0.7 were used to identify significant DEGs. Venn diagram was painted by Draw.io, and the heatmap was processed by pheatmap package of R Studio (version 4.0.2).Rattus norvegicus genomes, S-T from H. Sapiens have to be mapped to the orthology of rat in the HUGO Gene Nomenclature Committee  in order to search out the common genes between regulated DEGs and S-T.Given that DEGs acquired from RNA-seq were based on μL reaction system containing cDNA, SYBR Green qPCR Master Mix, forward and reverse primers, and RNase-free ddH2O was placed into fluorescent qPCR analyzer Mx3000P , through a procedure of 40 cycles at 94°C for 2 min predegeneration, 94°C for 20 s degeneration, 58°C for 20 s renaturation, and 72°C for 20 s extension. Each sample was performed in triplicate with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. Gene primer sequences of Srebp-1c, Pcsk9, Insr, Gck, Cyp7a1, and Cyp3a9 used for reverse transcription polymerase chain reaction (PCR) are listed in Ct−ΔΔ method to qualify the relative expression levels of candidate genes.Total RNA was reversed to cDNA by Bestar qPCR RT kit . Subsequently, each cDNA sample was used to quantify the expression of respective gene by SYBR Green qPCR Master Mix kit . The 20 P-values of<0.05 were considered statistically significant. All the steps were performed by using SPSS .All the data had passed the normal distribution test. Moreover, one-way analysis of variance was used to test if data met variance homogeneity. If so, the LSD test was used for multiple comparisons; if not, Welch's analysis of variance combined with the Games–Howell test was applied. All results are expressed as mean ± SD. Based on the inclusive criteria of OB  ≥30% and DL ≥0.18, a total of 120 active compounds , the targets are mainly grouped into cellular response to steroid hormone stimulus, lipid oxidation, regulation of insulin secretion, regulation of fatty acid oxidation, and others. These targets were associated with apolipoprotein binding, bile acid binding, insulin receptor substrate binding, and others in MF annotation. In addition, it is predicted that HTJZD exerts a regulation effect on hyperlipidemia through the alteration of CC such as cellular organelle, organelle membrane, and lipoprotein particles. KEGG pathway enrichment analysis (P< 0.05) was performed as shown in P-value), respectively. A total of 210 S-T were mainly enriched in the insulin resistance pathway, nonalcoholic fatty liver disease pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, insulin signaling pathway, AMPK signaling pathway, etc.A total of 210 S-T, with strong connection among them, may play a key role for HTJZD in the treatment of hyperlipidemia. From BP annotations in the GO functional enrichment analysis  results We found that GO annotations and KEGG pathway enrichments showed a better view of the connections between targets and the underlying therapeutic mechanisms of HTJZD. From the enrichment results, lipid metabolism and insulin resistance processes presented a strong connection with the potential regulation effect of HTJZD in treating hyperlipidemia, which may shed light on the exploration of the efficacy of HTJZD.The interaction network of herb-compound-S-T  was compP-value) in The KEGG pathway enrichment results and interaction relation among 210 S-T from STRING provided original data to construct the S-T-pathway network . The netThe Evaluation Method of Auxiliary Hypolipidemic Function [P < 0.05). Compared with atorvastatin, HTJZD was more advantageous in lowering TG and increasing HDL-c levels  were screened out from M versus N, and 1,936  from M versus H. In addition, compared with the upregulation and downregulation parts, respectively, 202 (124 upregulated and 78 downregulated) were the regulated DEGs . FurtherR. norvegicus genomes in the HGNC. Furthermore, 10 common targets (P< 0.05), namely, steroid hormone biosynthesis pathway, insulin signaling pathway, cholesterol metabolism pathway, and linoleic acid metabolism pathway, involved in HTJZD in treating rats with hyperlipidemia. Moreover, six key targets, namely, Srebf1, Cyp7a1, Cyp3a9, Pcsk9, Insr, and Gck, were selected from the results for further validation.A total of 210 S-T were mapped to 301 targets  significantly in the M group compared with the N group, while the Cyp7a1, Cyp3a9, Insr, and Gck levels declined (P< 0.05), which indicated that HFD leads to the lipid–glucose metabolism disorders in SD rats. However, the mRNA expression in rats administered HTJZD showed a different picture. The expressions of Srebp-1c, Cyp3a9, and Insr mRNA were turning to a significant reversal (P< 0.01) in the H group when compared with the M group, which is consistent with the transcriptomic results, while Cyp7a1, Pcsk9, and Gck mRNA expression had no significant difference compared with rats with hyperlipidemia.Six genes of Srebp-1c, Cyp7a1, Cyp3a9, Pcsk9, Insr, and Gck, closely associated with lipid metabolism and insulin signaling pathway, were selected for qRT-PCR analysis . Srebp-1Hyperlipidemia is a chronic disorder generally caused by a long-term unbalanced diet, which is manifested pathologically with aberrant lipid accumulation and energy metabolism disruption –28. In a−/− mice [The results of active compounds collected from TCMSP indicated that HTJZD serves as a multicomponent prescription and especially contains the ingredients with lipid-lowering, glucose-reducing, and antioxidative effects. In the herb–compound–S-T network, wogonin, naringenin, beta-sitosterol, baicalein, and kaempferol are the potential compounds that may play a significant role in suppressing the progression of hyperlipidemia. Wogonin, screened out from the compounds of Atractylodis Rhizoma, has been reported to have an antioxidative effect and protect HFD-fed rats from developing nonalcoholic fatty liver disease . Naringe−/− mice . Kaempfe−/− mice , 35.P <  0.05) TC level  is significant transcription factor involved in regulating liposomal homeostasis.α-hydroxylase (Cyp7a1) were the key genes both enriched in the cholesterol metabolism pathways in hyperlipidemia rats treated with HTJZD. Evidence from genetic studies supports the role of Pcsk9, a serine protease, in the regulation of hepatic apolipoprotein B uptake and degradation of low-density lipoprotein receptor (LDLR) [It transcriptionally regulates the expression of its downstream lipogenic genes involving in the synthesis of fatty acid and triglyceride , 37. It r (LDLR) –43. Morer (LDLR) , 45. In r (LDLR) , 47. Howr (LDLR) , 49. We Insulin resistance, regarded as another representative pathological feature of metabolic disorders, aggravating on account of the inhibition of the insulin receptor substrates (Irs) pathway, could also be regulated by AMPK signaling pathway . AdditioTaken together, HTJZD acted as a hypolipidemic agent potentially via modulation of lipid metabolism and insulin resistance. An increasing number of studies report that the lipid metabolism-related signaling pathway and insulin signaling pathway could be regulated by natural compounds, which indicates that traditional herbal medicines are potential alternatives or supplements to treat metabolic diseases –55. In aNetwork pharmacology predicted that HTJZD may exert therapeutic effects on HLP. Moreover, the animal experiments verified that HTJZD effectively suppressed the pathological process of rat model fed with HFD. It is capable of regulating serum lipids of rats, reducing lipid deposition in rat's liver, and reversing the expression of genes which related to lipid metabolism and insulin resistence. Among six candidate targets , Srebp-1c and Insr may play central role in the treatment of hyperlipidemia.There are several limitations to the present study. Repeated experiments and prolonged intervention duration are needed in future studies. What has to be focused is whether the compensation mechanism, leading to the opposite expression of genes contrary to what was expected, exists in the liver sites with more severe lipid lesion. Furthermore, a blood glucose level test should be performed to further support the intervention effect on insulin resistance of HTJZD. We will pursue a more systematic comprehensive analysis of HTJZD intervening hyperlipidemia and relevant metabolic disorders in future study.


Astragalus and Angelica are often combined to treat male infertility, but the specific therapeutic mechanism is not clear. Therefore, this study applies a network pharmacology approach to investigate the possible mechanism of action of the drug pair Astragalus-Angelica (PAA) in the treatment of male infertility.  The traditional Chinese medicines  Relevant targets for PAA treatment of male infertility are obtained through databases. Protein-protein interactions (PPIs) are constructed through STRING database and screen core targets, and an enrichment analysis is conducted through the Metascape platform. Finally, molecular docking experiments were carried out to evaluate the affinity between the target protein and the ligand of PAA.  The active ingredients of 112 PAA, 980 corresponding targets, and 374 effective targets of PAA for the treatment of male infertility were obtained, which are related to PI3K-Akt signaling pathway, HIF-1 signaling pathway, AGE-RAGE signaling pathway, IL-17 signaling pathway, and thyroid hormone signaling pathway.  In this study, using a network pharmacology method, we preliminarily analyzed the effective components and action targets of the PAA. We also explored the possible mechanism of action of PAA in treating male infertility. They also lay a foundation for expanding the clinical application of PAA and provide new ideas and directions for further research on the mechanisms of action of the PAA and its components for male infertility treatment. Male sterility, a common disease in andrology, is defined as male factor infertility in which a couple have attempted to conceive for more than one year without success. Approximately 15% of couples do not achieve pregnancy within one year and seek medical treatment for infertility. One in eight couples encounters problems when attempting to conceive a first child, and one in six encounters problems when attempting to conceive a subsequent child. Three percent of women who are currently trying to conceive remain involuntarily childless, while 6% of parous women are not able to have as many children as they would wish . The etiAstragalus and Angelica are often used as a drug combination in the treatment of male infertility. Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge. or Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. [Angelica is the root of Angelica sinensis (Oliv.) [The treatment of male infertility with traditional Chinese medicine (TCM) has a long history and is now gaining popularity in western countries , 7. The ) Hsiao. . The eff (Oliv.) . The eff (Oliv.) , 11. HowNetwork pharmacology is a new discipline that has been used in recent years to study the pharmacological mechanisms of traditional Chinese medicines. Through integration of current traditional Chinese medicine pharmacology methods, high-throughput bioinformatics, and high-end data analysis software programs, comprehensive analyses of the mechanisms of action of traditional Chinese medicines can be carried out . It can Astragalus and Angelica were typed into the UniProt database for each target https://www.uniprot.org/ to obtain the standard number of targets, and the final results were counted into an Excel sheet.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)  is a relhttps://omim.org/) is a database of human genes and genetic phenotypes which focuses on molecular relationships between gene variation and dominant expression and contains information on more than 15,000 genes. GeneCards [https://www.genecards.org/) contains a wealth of biomedical data on genes and their products, including genomic, proteomic, and gene function-related information. DisGeNET [https://geneticassociationdb.nih.gov/), and DisGeNET (http://www.disgenet.org) to search for the targets related to male infertility and record them in an Excel sheet.Online Mendelian Inheritance in Man (OMIM) (DisGeNET  is a mulDisGeNET  is a stahttp://bioinformatics.psb.ugent.be/web-tools/Venn/). Finally, the targets at the intersection were considered the effective targets of PAA in the treatment of male infertility. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) can be used to judge the confidence of PPIs through different evidence scoring systems so that we will know which proteins play a major role in the treatment of diseases. The targets obtained after intersection analysis were input into the STRING to construct a PPI network, and the results were imported into Cytoscape software (version 3.7.2) [The effective components, targets, and disease targets of the PAA were deduplicated and imported into a Venn diagram program (n 3.7.2) . Accordin 3.7.2)  plug-in,The disease-drug-active ingredient-target network of PAA in the treatment of male infertility was visualized by using Cytoscape software. In the network, each node represents a data element, such as a disease, drug, active ingredient, or target. In the scale-free network, the index node degree reflects the participation of each node, and the size of the degree is directly proportional to the participation of the node. After importing the data, the Network Analyzer module was used to obtain the degree of each data element.http://metascape.org/) is a powerful tool for gene function annotation analysis that can help users apply the current popular bioinformatics analysis methods to batch gene and protein analysis in order to understand gene or protein functions. In this study, we used the Metascape database to perform gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. GO analysis was used to describe the functions of gene targets in terms of cellular component (CC), molecular function (MF), and biological process (BP). KEGG analysis was used to identify the signaling pathways associated with the gene targets, and the analysis results with statistical significance were selected (P < 0.05). A network of the interactions between the core targets and signaling pathway was drawn with Cytoscape software to obtain the comprehensive information for each node.Metascape . The proteins selected as .pdb format were converted into .pdbqt format via AutoDock (http://autodock.scripps.edu/). Docking was performed in AutoDock with kaempferol, stigmasterol, and quercetin.The information of six proteins  in the PPI network which were most closely related to male infertility was obtained through the RCSB PDB  and TNF-α  in sperm; alleviate sperm damage [A total of 112 effective components, 980 corresponding targets, and 4108 disease targets of the PAA pair were screened from the component database, and 374 potential targets for male infertility were obtained after mapping. This finding shows that PAA has multicomponent and multitarget characteristics in the treatment of male infertility. The drug pair-component-target network diagram shows that kaempferol, stigmasterol, beta sitosterol, quercetin, and canavanine are the top active ingredients. The association of these several components with male infertility is as follows. Kaempferol is a flavonoid that can significantly increase the levels of antioxidants such as SOD (superoxide dismutase), CAT , and GPX (glutathione peroxidase) in the sperm of diabetic rats; reduce the levels of inflammatory factors such as NF-m damage ; and sigm damage . Quercetm damage . Relevanm damage . Stigmasm damage . TherefoAngelica and Astragalus in the treatment of male infertility include TP53 (tumor protein P53), mapk1 (mitogen-activated protein kinase 1), IL-6 (interleukin-6 receptor), ANXA1 (annexin-A1), EGF , and EGFR . Here, the relationship between these six targets and male infertility is described. The TP53 gene, an important tumor suppressor gene, can regulate cell growth, differentiation, aging, and the immune response. Meiosis is crucial in spermatogenesis, and TP53 mRNA and protein are expressed in primary spermatocytes and play important roles in spermatogenic cell apoptosis, suggesting that TP53 participates in meiosis [The results showed that the potential targets of  meiosis , 31. A g meiosis . However meiosis . ANXA1,  meiosis . Studies meiosis , and the meiosis . IL-6 is meiosis . EGF is  meiosis . In addi meiosis . As showKEGG enrichment analysis showed that the target pathways of PAA in the treatment of male infertility included the PI3K-Akt, IL-17 (interleukin 17), cAMP, FOXO, MAPK, and other signaling pathways. A mechanism diagram is shown in In conclusion, this study uses a network pharmacology method to study the therapeutic effects of PAA on male infertility at multiple levels and finds that TP53, MAPK1, IL-6, ANXA1, EGF, EGFR, and other genes may be key targets. The PI3K-Akt, IL-17 cAMP, FOXO, and MAPK signaling pathways may be key pathways that mainly play anti-inflammatory and antioxidant roles and promote cell proliferation and cell ATP production to treat male infertility. A possible molecular mechanism by which this drug pair treats male infertility has been revealed. A limitation of this study is that the pharmacologically active components and targets discovered through network pharmacology are predictions; thus, the results should be verified experimentally. Subsequent research will verify the findings in animal experiments and clinical studies to improve the rationality and scientific basis of clinical PAA application.


It has shown that schizophrenia (SCZ) is associated with a higher chance of myocardial infarction (MI) and increased mortality. However, the underlying mechanism is largely unknown. Here, we first constructed a literature-based genetic pathway linking SCZ and MI, and then we tested the expression levels of the genes involved in the pathway by a meta-analysis using nine gene expression datasets of MI. In addition, a literature-based data mining process was conducted to explore the connection between SCZ at different levels: small molecules, complex molecules, and functional classes. The genetic pathway revealed nine genes connecting SCZ and MI. Specifically, SCZ activates two promoters of MI (IL6 and CRP) and deactivates seven inhibitors of MI , suggesting that no protective role of SCZ in MI was detected. Meta-analysis showed that one promoter of MI (CRP) presented no significant increase, and six out of seven genetic inhibitors of MI demonstrated minor to moderately increased expression. Therefore, the elevation of CRP and inhibition of the six inhibitors of MI by SCZ could be critical pathways to promote MI. Nine other regulators of MI were influenced by SCZ, including two gene families (inflammatory cytokine and IL1 family), five small molecules , and two complexes (CaM kinase 2 and IL23). Our results suggested that SCZ promotes the development and progression of MI at different levels, including genes, small molecules, complex molecules, and functional classes. Schizophrenia (SCZ) is one of the most chronically disabled mental illnesses . The earAn increasing amount of literature has discussed the strong correlations between mental disorders and increased MI mortality, especially in patients with SCZ . A studyDespite the clinical outcomes that support the relationship between MI and SCZ , the undThis study is organized as follows. First, we conducted a Natural Language Processing (NLP)-based literature data-mining  to const1version 12.3), we conducted an NLP-based large-scale literature data mining to identify common genes that were downstream targets of SCZ and up-regulators of MI. That is, each gene was identified as influenced by SCZ, and was also regulating MI, forming a SCZ→Gene→MI relationship. For each relationship identified, there were at least three independent supporting references, which were provided in the supplementary material SCZ_MI→Ref4GeneticPathway, including the title, DOI/PMID, and the sentences where the relationship was identified. The process was conducted by using MedScan . Results from using both the random-effects model and fixed-effect model were compared following the statistics estimation used by iT is the deviation of each study, iW is the inverse variance of each study, and k is the total number of studies.Where Where k is the total number of studies.df is the degree of freedom defined in Equation (2).Where Q is the total variance defined by Equation (1), and P-values for each of these factors.To estimate the possible influence of several factors  on the gene expression in MI patients, we conducted a multiple linear regression (MLR) analysis and reported the 3. The analysis was first performed to identify functional gene class, small molecule, complex molecules, and cells that induced MI’s pathological development, and then these types of entities regulated by SCZ were also identified. The overlapped entities were used to construct SCZ→MI network. To ensure high confidence in the identified relationship, we used the confidence level of three (identified entities were supported by at least three references) to filter the relationships. We presented the identified entities and the corresponding relationships in SCZ_MI→Ref4SMPathway and SCZ_MI→Ref4CellPathway.To explore SCZ-driven MI regulators at other levels, we conducted another NLP-based literature data mining assisted by the network building module of Pathway StudioAs shown in We conducted a meta-analysis using nine MI-expression datasets to test the expression variation of the genes involved in SCZ-driven genetic pathways for MI. We presented the major results of the meta-analysis and MLR analysis in p-value = 3e–16 and 6e–6, respectively), and study age seems to be an influential factor for IL6 alone . These results suggested the complexity of the disease of MI, which could be influenced by multiple factors.As shown in p-value = 0.021). The other MI promoter (CRP) presented no significant expression change . Therefore, the activation of CRP could be a required course where SCZ promotes MI’s pathological development.As shown in p-value = 0.10 and 0.24, respectively). Therefore, inhibiting these two genes’ activity could be another path that SCZ contributes to the promotion of MI. Most of the other MI inhibitors demonstrated minor elevated expression except CTNNB1 (LFC = −0.069). The increased expressions of all these inhibitors of MI were beneficial in the progression MI. Thus, by deactivating these MI inhibitors, SCZ could worsen the progression of MI.We identified two inhibitors of MI, which got moderate elevation in their expression levels, including ADIPOQ and SOD2 . Six small molecules satisfied our data mining criteria and formed the small molecule pathway, as shown in Besides small molecules, we also identified two gene families (inflammatory cytokine and IL1 family) and two complexes (CaM kinase 2 and IL23) that were promoters of MI and stimulated by SCZ. To note, the gene family and complex pathways shown in This study explored the SCZ influenced MI regulators at multiple levels: genetic, gene family, small molecule, and complex. Corresponding pathways were constructed with a meta-analysis to test the gene expression within the genetic pathway in MI patients. The pathway built suggested a negative role of SCZ in MI’s pathological development, which is consistent with previous studies . Howeverp-value = 0.021). Therefore, the SCZ-IL6-MI could be one of the pathological paths where SCZ promotes MI.Firstly, we identified the potential association between SCZ and MI at the genetic level through the connection with nine common genes . Most ofThe meta-analysis also showed that the expression of CRP, a promoter of MI, was not significantly elevated in the nine MI-datasets employed in the meta-analysis . HoweverHowever, we noted that the early stage of SCZ could play some protective role in MI progression through the up-regulation of TXN (an MI inhibitor) . The ove+2 release pathways through Calmodulin Kinase 2 (CamKII) and inositol triphosphate  is tied to cardiomyopathies due to structural changes in heart muscle . Beta-caWe also identified six SCZ-driven small molecules that influence MI’s advance, as shown in Moreover, our study also uncovered four MI promoters , includiThis study has several limitations that need to be addressed in future work. First, the identification of the SCZ driven MI-regulators was filtered to have support by at least three references. While this decreased the identified entities’ false-positive ratio, some vital information might be lost between SCZ and MI connection, which needs further consideration. Second, the pathways and relations were constructed based on previous studies that were conducted in different backgrounds. Biology experiments are needed to validate any of the relationships identified in this study. Third, the sample size of the MI datasets employed in this study presented significant variance, which influenced the meta-analysis results. Forth, the relationships identified in this study were mainly quantitative changes at the gene/protein expression level. Other types of relation  may add new insights into the understanding of the SCZ-MI relationship. Fifth, due to the limitation of the NLP technique employed in this study, we did not separate different study types  when building the pathway given in We identified 19 SCZ-driven MI-regulators at different biological levels, and SCZ exerts an overall negative influence on MI through the regulation of most of them (18 out of 19). Our results indicated the complexity of the connection between SCZ and MI and may add new insights into the understanding of the negative role that SCZ plays in the pathology of MI.https://www.ncbi.nlm.nih.gov/geo/.Publicly available datasets were analyzed in this study. This data can be found here: XY, YC, and YL developed the study design, analyzed the data, and wrote the original manuscript. All authors read and approved the final manuscript.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.


The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. Many aspects of the Leak Pathway, such as its molecular mechanism, its properties, and its regulation, remain controversial. In this review, we provide a historical background to the evolution of the Leak Pathway concept from the initial examinations of paracellular permeability. We then discuss current information about the properties of the Leak Pathway and present current theories for the Leak Pathway. Finally, we discuss some recent research suggesting a possible molecular basis for the Leak Pathway. La+3 ions are not transported into cells and can be precipitated into the electron dense lanthanum sulfate which can be detected by electron microscopy [+3 permeates the tight junctions of both intestinal and gallbladder epithelia, supporting the idea that the tight junction is the site of the epithelial tissue paracellular permeability pathway. It was previously shown that lanthanum ions did not permeate into the tight junctions of toad urinary bladder epithelia [The concept of a barrier, the “terminal bar”, that restricts the movement of solutes and water through the paracellular pathway between epithelial cells dates back at least to Bizzozero . Identifd Palade . Their pd Palade  demonstrd Palade  used lancroscopy . They depithelia  and frogpithelia . Based opithelia  and Frömpithelia  proposedA molecular basis for the paracellular barrier function was suggested by the finding that freeze fracture electron microscopy revealed arrays of anastomosing strands composed of small particles running through the tight junction region at the immediate subapical region of the plasma membrane of epithelial cells ,9. MultiThe tight junction is the structure which forms the epithelial paracellular permeability barrier and regulates the paracellular movement of solutes, including the paracellular movement of macromolecules. As a first step to understanding this process, many groups have begun to compile a list of proteins associated with the tight junction. The first protein shown to be associated with the tight junction structure was Zonula Occludens-1, ZO-1 . ZO-1 iscis and trans interactions . Marvel D3 also exhibits cis-heterophilic binding interactions with occludin and tricellulin. Some claudins exhibit cis-oligomerization with TAMPs. JAM-A exhibits homophilic cis-binding interactions [trans binding interactions [cis and trans binding events, both homophilic and heterophilic, support the hypothesis that the tight junction structure is a highly organized supramolecular complex of transmembrane and cytoplasmic proteins that is crosslinked to the actin cytoskeleton and, likely, to the microtubule network . ractions . Occludiractions  and homoractions , as wellractions . ZO protractions ,19,38. Zractions ,39), triractions , and JAMractions ,41). ZO ractions ), actin-ractions ) and corractions ), and otractions ,46). Thee, e.g., ,47,48). e, e.g., ,52,53,54In addition to proteins, a role for lipids in forming the paracellular permeability barrier has been suggested . cis interactions, form antiparallel double row strands within the plasma membrane . Theembranes ,61. Thesembranes , as evidembranes . In addid breaks . All of d breaks ,64,65,66trans claudin-claudin interactions increased paracellular permeability to all solutes. Expression of these mutant claudin-5 proteins did not alter strand morphology, indicating that barrier disruption can be achieved by more subtle alterations in claudin-claudin interactions. Overexpression of Protein Kinase D in airway epithelial cells decreased claudin-1 expression [As described above, evidence supports the hypothesis that the tight junction strands are a central structure for the paracellular permeability barrier. There are at least three competing theories about what comprises the observed strands . As stapression . This wapression . This wapression . The celFurther support for a central role for claudins is provided by a recent study which demonstrated that genetic knockout of five claudins expressed in MDCK II cells resulted in loss of the tight junction strands . The stuII phase [A second theory proposes that the strands are lipid structures organized in the cylindrical hexagonal HII phase ,81. ThesII phase . These pII phase . This thII phase , which wII phase . It is sII phase structures and protein components that mediate paracellular permeation across the lipid barrier. In support of this hypothesis, both lipids and tight junction proteins are co-localized to the tight junction structure . As dese, e.g., ,56,57,58e, e.g., ,88), maye, e.g., ).The possibility of multiple pathways mediating solute movement across epithelia was first suggested by Durbin et al. . They reA third pathway for particles to cross the epithelial barrier has been described, the Unrestricted Pathway . This pStudies from many groups have contributed to the consensus that the Pore Pathway permeability is mediated by a subset of claudins which bind to complementary claudins on the apposing membrane of adjacent epithelial cells to form size- and charge-selective pores through the tight junction. For example, MDCK C7 (Type I) renal epithelial cells do not express claudin-2 and exhibit a high TER. Expression of claudin-2, a cation pore-forming claudin, in these cells decreased TER (increased Pore Pathway permeability) without altering the permeability to mannitol or lactulose (larger solutes) . This de1 renal epithelial cell line. They found no consistent concordance in the effects of these compounds on the permeabilities of the Pore Pathway and the Leak Pathway. In one study, RhoA activation, a small G protein that regulates actin organization, both increased TER (decreased Pore Pathway permeability) and increased mannitol flux (increased Leak Pathway permeability) in MDCK II cells [1 cells with low concentrations of hydrogen peroxide increased Leak Pathway permeability without altering TER [Barring the action of substrate-selective transporters, any opening that allows the passage of macromolecules will also allow the passage of small ions and small solutes. From this, it must be concluded that the Leak Pathway should mediate the passage of both macromolecules and small ions and solutes. Consistent with this conclusion, many studies have reported parallel changes in Pore Pathway permeability and Leak Pathway permeability in response to different modulators in a variety of epithelia , the baCell Damage/Death Model: One hypothesis is that the Leak Pathway represents sites of epithelial cell damage/death where a cell detaches from the surrounding cells in the epithelial sheet. This would allow the permeation of both large and small solutes but, if the damage/death is rare within the cell layer, the permeation of small ions and solutes might be insufficient to alter the global flux rate for small ions and solutes while allowing some level of macromolecule permeation. The ability to regulate reversibly Leak Pathway permeability without evidence of cell damage/death, however, seems inconsistent with this hypothesis. For example, we were unable to demonstrate a change in cell damage/death via multiple pathways under conditions that produced an increase in Leak Pathway permeability, but not Pore Pathway permeability, of MDCK II cells . TreatmeTricellular Junction Model: Another hypothesis is that the Leak Pathway is localized to the tricellular junction, composed of tricellulin and angulin family proteins . ModeratB is the Boltzmann’s constant, T is the temperature, and η is the viscosity of the solution.Some results, however, are inconsistent with the hypothesis that the tricellular junction can completely account for Leak Pathway permeability. Multiple studies have reported that paracellular permeability does not drop off dramatically for solutes larger than 20–30 Å 2 cell surface area.3).From direct observation, the number of tricellular junctions per microscopic field of post-confluent MDCK II cells averaged 35 tricellular junctions/3600 μmmeasured = 3.7125 Å/s for 4 kDa fluorescein-dextran across MDCK II cell monolayers.P(r)app that was approximately 30% of the measured Papp. Thus, the tricellular pores cannot account for even a majority of the permeation of these solutes across MDCK II cell monolayers. They cannot constitute the entirety of the Leak Pathway, although they may contribute significantly to the permeability of some solutes under certain conditions.This calculation does not assume any hindrance of solute passage, some level of which would be expected for these solutes passing through pores with a radius of 50 Å, which would decrease the permeability from this maximal rate. This theoretical upper limit for the solute permeability yielded a PStrand Break Model: A third hypothesis is that Leak Pathway permeability is mediated by transient localized breaks in the tight junction barrier. Since this barrier appears to be formed by the anastomosing strand network, the macromolecules would traverse the tight junction by passing through sequential breaks in individual strands until the macromolecule emerges on the other side of the tight junction . AsA recent study, however, suggests an added complexity to the paracellular permeability to macromolecules, i.e., Leak Pathway permeability. As described above, Otani et al.  knocked Strand Break-Tension Model: As discussed below, multiple studies have indicated that altered tension on the tight junction can increase Leak Pathway permeability. This suggests an enhancement of the “strand break” model to incorporate altered tension on the tight junction as a mechanism through which strand breaks are induced. Altered tension on the claudin strands, either due to altered cytoskeletal force or to decreased structural stability, would lead to localized breaks in the strands with a resultant increase in Leak Pathway permeability. If the alterations are global in nature, this would result in detectable increases in the permeability to both small and large molecules. If the alterations are more localized, this could result in increases in the permeability to macromolecules but, possibly, without a measurable increase in small ion and solute permeability. The report that the ZO protein-actin binding is a relatively weak interaction  could prWithin the “strand break” model, one could imagine two extremes to how this might function. In one extreme, the paracellular passage of a macromolecule across the tight junction structure would be through its progressive movement through intra-tight junction aqueous compartments via successive strand breaks, as described above a. Each oWhile an understanding of exactly what is the Leak Pathway remains unclear, many studies have identified components that, when manipulated in some fashion, can modulate Leak Pathway permeability. An exhaustive discussion of this topic is not possible due to the substantial literature related to this topic. We will, instead, attempt to provide examples of the different implicated components and the various manipulations of them that modulate Leak Pathway permeability.Claudins: Claudins appear to be central to the formation of a paracellular permeability barrier and the small ion- and small solute-selective pathways across this barrier Post-translational modification of claudins that alter claudin content or claudin-protein interactions have also be reported to regulate paracellular permeability. Claudin phosphorylation has been demonstrated in multiple studies . In OVCTricellulin: As described above, studies have indicated that manipulation of tricellulin content can alter permeability to, at least some, large solutes in multiple epithelial cell types . ThOccludin: Occludin preferentially, but not exclusively, localizes to strand branch points which appear to be a major locus for strand breaks . OccludiThe presence or absence of occludin may not directly alter Leak Pathway permeability. It may, however, alter the stability of the claudin strands, making them more or less susceptible to disruption upon imposition of tension/stress on the macromolecular tight junction structure. Consistent with this proposal, the ability of hydrogen peroxide to increase Leak Pathway permeability in MDCK II cells was enhanced by occludin knockdown and diminished by occludin overexpression . HydrogeXenopus renal epithelial cells (A6) increased occludin turnover and, thereby, decreased occludin content in the tight junction [Studies examining the effect of expression of occludin mutants or protein fragments on paracellular permeability also provide support for a role in regulating Leak Pathway permeability. The occludin COOH-terminal cytoplasmic tail has been shown to mediate inter-protein interactions as well as being involved in targeting occludin to the tight junction and development and maintenance of barrier function Staphylococcus aureus infection of airway epithelial cells [Many studies have demonstrated the post-translational modification of occludin and its association with modulation of paracellular permeability, including Leak Pathway permeability al cells . Leak PaIn aggregate, these results support a role for occludin in regulating Leak Pathway permeability. Further investigation of these observations is required to clarify the basis for the apparent regulatory action. The preferential localization of occludin to strand branch points, which are also preferential sites for strand breaks, suggests one possible mechanism through which occludin could regulate Leak Pathway permeability.Junctional Adhesion Molecules: Knockdown of JAM-1 (JAM-A) in SK-CO15 colonic epithelial cells increased permeability via both the Pore Pathway (decreased TER) and the Leak Pathway (increased permeability to 4 kDa fluorescein-dextran) . ConsistZO Proteins: The ZO proteins crosslink the tight junction membrane proteins to create a macromolecular lattice and connect them to the underlying actin cytoskeleton ,161,162.Actin Cytoskeleton: Many studies support a role for the actin cytoskeleton in regulation of epithelial barrier function Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) activated RhoA, rac1, and cdc42 and increased paracellular permeability to both small ions and macromolecules [Xenopus laevis gastrula divided. This likely increased tension on the tight junction structure at these sites, which exhibited dilution of tight junction proteins, ZO-1 and occludin, at the site of membrane elongation. Repair was initiated by localized RhoA activation, which was followed by localized actin polymerization, membrane contraction, and recruitment of ZO-1, occludin, and claudin-6 proteins to the breach site. Recruitment of these proteins led to reinforcement of the tight junction structure and repair of the paracellular permeability barrier breach.Small G Proteins: Small GTPases, Rho, cdc42, and rac, regulate actin cytoskeletal dynamics to modulate multiple cellular processes olecules . Changesolecules . Stephenolecules  reportedReflecting on apical junctional integrity, it is interesting to note the marked parallels between regulation of tight junction and adherens junction integrity. These include junctional tensile stress  tension , exhibit tension . As descIt is important to note that changes in Leak Pathway permeability have been noted under conditions that both increase and decrease tight junction tensile stress. One possible and, at present, highly speculative explanation relates to the direction of the tensile stress being modulated. The cortical actin ring might tug on tight junction proteins laterally along the membrane, i.e., parallel to the plane of the cell membrane. In contrast, the branching actin network and actin stress fibers might tug on tight junction membrane proteins in the direction of the cell body, i.e., perpendicular to the plane of the cell membrane. It is currently unclear how changes in tension in these different directions might affect tight junction integrity and paracellular permeability. Differences may also relate to the level of imposed stress. Lower levels of tension might induce minor damage to the tight junction structure that triggers repair mechanisms that actually enhance the tight junction structure and, thereby, decrease paracellular permeability. Higher levels of tension might induce more substantial damage that cannot be repaired sufficiently leading to increased paracellular permeability. Alternatively, the tight junction might be under optimal tension under normal conditions. Either increasing or decreasing the level of tension might lead to disruption of tight junction barrier integrity. Clearly, much more work needs to be done to investigate how stress on the tight junction structure affects paracellular permeability, in particular the Leak Pathway.Since openings that permit the passage of large solutes must also allow the passage of small solutes, the results presented above raise the question of why increased Leak Pathway permeability is not always paralleled by increased Pore Pathway permeability. This apparent inconsistency can be reconciled by considering two different scenarios. In the first scenario, there is global disruption in the paracellular permeability barrier of most, if not all, cells within the cell population. This would result in a dramatic increase in the permeability of the epithelial barrier to both small ions and macromolecules. This scenario would be consistent with the effects of experimental conditions that are reported to increase permeability via both the Pore Pathway and the Leak Pathway. In this case, however, the increased permeability to small ions would not be primarily via the claudin pores (classic Pore Pathway) but, rather, would reflect the greater movement of small ions through the extensive and larger openings of the Leak Pathway. Consistent with this hypothesis, it was reported that treatment of HT-29/B6 cells with Tumor Necrosis Factor-α, which increases permeability to both small ions (Pore Pathway) and macromolecules (Leak Pathway), markedly increased the number of sites of macromolecule passage within the cell monolayer . In the second scenario, there are relatively rare, highly localized barrier disruptions between some cells within a cell population. This would lead to a detectable increase in the permeability to macromolecules (Leak Pathway) since permeability to macromolecules is very low under normal conditions. For epithelial cell populations exhibiting a relatively high level of Pore Pathway permeability under normal conditions, however, the relatively minor contribution of small ions and molecules moving through a limited number of highly localized sites of barrier disruption might not be evident on top of this high “background” Pore Pathway permeability. Epithelial cells exhibiting a low Pore Pathway permeability may be more likely to exhibit an increase in Pore Pathway permeability from these rare barrier disruptions, but this remains to be determined.Xenopus laevis gastrula were discrete, limited in number, and transient, remaining open for no more than 5 min. Richter et al. [BBE cells, and HT-29/B6 cells was localized to specific transient sites, being “open” for 30 min or less. At any time, small regions around only some cells within an epithelial cell population exhibited macromolecule permeation. The locations of these permeation sites changed over time.Two recent studies demonstrating a small number of discrete sites of macromolecule passage within an epithelial cell population support the hypothesis that these can be rare events ,199. SteIt is worth returning briefly to the TAMPs and their potential involvement in maintaining the tight junction structure and regulating Leak Pathway permeability. As described above, there are currently three TAMPs, occludin, tricellulin, and Marvel D3. Under normal conditions, both occludin and Marvel D3 are localized primarily to bicellular junctions, whereas, tricellulin is localized primarily to tricellular junctions. At present, there are no data suggesting that Marvel D3 affects Leak Pathway permeability, although this has not been exhaustively investigated. In contrast, and as described above, both occludin and tricellulin appear to have roles in regulating Leak Pathway permeability. In most studies, knockdown of occludin does not alter Leak Pathway permeability ,132,134.It is tempting to speculate that, while claudins form the physical barrier of the strand structure, occludin and tricellulin regulate the complexity of this structure through modulation of claudin strand branching/connections. As described by Tervonen et al. , less crThe epithelial paracellular permeability pathway for macromolecules, the Leak Pathway, has received increasing attention and experimental investigation in recent years. Initial studies suggested the Leak Pathway represented sites of unrestricted paracellular passage, possibly due to cell damage. While this cell damage pathway, the Unrestricted Pathway, is still recognized, studies from many laboratories around the world have demonstrated that the Leak Pathway is a distinct paracellular permeability pathway localized to tight junctions and exhibiting dynamic regulation. Studies have described the regulation of Leak Pathway permeability by a wide variety of conditions and compounds. Responses are, in some cases, cell type-specific, arguing for caution about generalizing responses across epithelia. Tension/Stress on the tight junction structure may provide a unifying mechanism underlying various regulatory events in different epithelia, but this requires further study. Multiple tight junction proteins and cellular proteins have been identified that can influence Leak Pathway permeability under various conditions and in different epithelial cell types.Although much information about the Leak Pathway has been reported, many fundamental questions remain. For example, it is still unclear whether the Leak Pathway opening is comprised of a specific macromolecular complex of tight junction and cellular proteins, similar to the claudin pores or the gap junction, or is simply a disrupted region of the tight junction structure. Perhaps, Leak Pathway openings represent weak points in the overall tight junction structure with a distinct composition/organization that make them more easily disrupted by imposed cellular or external tension. Other fundamental questions relate to the size of the Leak Pathway opening and its dynamic behavior. While some studies have reported a size limit for the Leak Pathway of ~60 Å, this does not explain the ability of larger macromolecules to cross the epithelium. It is likewise unclear whether the Leak Pathway represents a progressive movement of macromolecules through the tight junction structure or the movement of macromolecules through a disruption of the entire tight junction structure or both modes of permeation. While these questions have intrinsic value for acquiring a better understanding of the Leak Pathway, they are also highly relevant for understanding the role of the Leak Pathway in the normal physiology and pathophysiology of various epithelial tissues. In addition, developing strategies to deliver macromolecules across epithelial barriers has been a major interest of drug companies. That interest has only increased with development of macromolecule pharmaceuticals (biologics) that currently are delivered through infusion. A greater understanding of the Leak Pathway may enable the development of novel approaches for macromolecule drug delivery.To date, most studies of the Leak Pathway have examined the bulk transepithelial movement of macromolecules at the cell population level. While these approaches still have utility, the questions raised above likely will also require new analytical approaches that yield information at a more molecular level. Imaging methodologies, such as super-resolution microscopy and single molecule tracking, may be required to define the properties and dynamics of individual Leak Pathway openings. Identification of the Leak Pathway opening structure and its constituents may require novel approaches to visualize the sites of transepithelial macromolecule passage and to identify the individual components at these sites. The use of tight junction proteins modified to sense and report changes in tension, such as the ZO-1 tension sensor construct , may ena


Wastewater stabilization ponds are a natural form of wastewater treatment. Their low operation and maintenance costs have made them popular, especially in developing countries. In these systems, effluents are retained for long periods of time, allowing the microbial communities present in the ponds to degrade the organic matter present, using both aerobic and anaerobic processes. Even though these systems are widespread in low income countries, there are no studies about the microorganisms present in them and how they operate. In this study, we analised the microbial communities of two serial full-scale stabilization ponds systems using whole genome shotgun sequencing. First, a taxonomic profiling of the reads was performed, to estimate the microbial diversity. Then, the reads of each system were assembled and binned, allowing the reconstruction of 110 microbial genomes. A functional analysis of the genomes allowed us to find how the main metabolic pathways are carried out, and we propose several organisms that would be key to this kind of environment, since they play an important role in these metabolic pathways. This study represents the first genome-centred approach to understand the metabolic processes in facultative ponds. A better understanding of these microbial communities and how they stabilize the effluents of dairy industries is necessary to improve them and to minimize the environmental impact of dairy industries wastewater. The wastewater generated by the cheese industries is composed mainly by different dilutions of milk , and washing water containing alkaline and acidic chemicals after the cleaning of bottles, tanks and equipment  sequencing. We report the reconstruction of over 100 metagenome assembled genomes (MAGs) using a binning strategy that allowed us to perform a genome-centred analysis of genes and metabolic pathways. The analysis of those MAGs provides the opportunity to improve the knowledge about the metabolic processes carried out in the environment and to look for key organisms specific to these treatment systems. Knowledge about the community composition and functional potential is key to maintain and improve the efficiency and stability of the process and provides the base for future studies regarding the use of stabilization ponds for waste treatment and the environmental impact of dairy industries wastewater in low income countries.http://siga2.inta.gov.ar/) indicate that the average temperature in the region showed no significant differences when comparing those months.Samples were collected from six full-scale stabilization ponds belonging to two small dairy industries, named CYC and AUR, located in the center of Santa Fe province in Argentina. CYC has four serial treatment ponds of 35 × 50 m. The first two are 2.5 m in depth, and the next two are 1.5 m. AUR possesses two ponds, the first is 80 × 40 m and 1.5 m in depth and the second 90 × 40 m and 2.5 m in depth. For each pond, three samples were taken from different sampling points, to avoid spatial variation. Each time water was collected from the bottom of the pond to its surface, to sample the complete water column. Later, all three samples were combined into one composite sample per pond. CYC samples were taken in April 2013 and AUR samples in September 2013. Historic temperatures measured by the Sistema de Información y Gestión Agrometeorológica  in Tris 250 mM pH 8 was added and incubated until melting, and then 45 min on ice. A solution containing 0.5% SDS, 50 mM Tris pH 7.5, 0.4 M EDTA and 1 mg/ml proteinase K was added (120 ul) and incubated at 50 °C for 1 h, mixing by inversion every 10 min. The suspension was then transferred to tubes containing the bead mix from PowerWater® Isolation kit , and vortexed for 5 min. Debris was separated by centrifugation at 13,000 RPM for 1 min and supernatant was passed to a clean tube. Finally, a classic phenol-chloroform extraction and ethanol precipitation was followed . DNA quaThe six samples were sequenced using a Illumina HiSeq 1,500 platform (INDEAR), generating 2 × 150 bp paired-end reads. All reads were trimmed using Trimmomatic v0.36; , with deFirst, a taxonomic assignment of the reads was performed with KRAKEN2 v2.0.6;  against Once the final set was obtained, we compared the MAGs between system using the average nucleotide identity ANI, , calculai.e., RuBisCo genes for carbon fixation).Genes were predicted in all MAGs, and not binned contigs also, using PRODIGAL  and annoAfter quality trimming, around 1% of the WGS reads were dropped . The aveProteobacteria was the dominant phyla, averaging 70% of every sample. On the other hand, in AUR1 pond both Proteobacteria (38%) and Actinobacteria (32%) were the predominant lineages, while in AUR2, Actinobacteria was the most represented phylum (62%). The other well represented phyla in all samples were Firmicutes (between 2% and 12%) and Bacteroidetes (between 2% and 7%). Overall, there were no major differences between ponds from the same system.Using KRAKEN2, only an average of 22% of the reads per sample were classified, with CYC1 having the most classified reads (30%). A total of 58 different phyla were found, but 50 never represented more than 1% of any given sample . In CYC The assembly of the 60 Gbp of whole-genome sequences generated 238,725 contigs with length ≥1,000 bp . Also, tThe binning process generated 264 bins , since their small genome size often leads to marker losses , except for the aforementioned Actinobacteria and INTA.AUR.003, classified as member of the Verrucomicrobia phylum. This last phylum had five members in the AUR dataset, some with high coverage, but was almost absent in the CYC dataset .Clostridia class; a pair of Chromatiales, a pair of Bacilli and two Spirochaetes complete the MAGs that were shared between pond systems.We calculated the ANI between MAGs of each data set, to see if there were any species shared between both systems. Only six pairs had aligned fractions over 50% , with ANPatescibacteria phylum (formerly known as Candidate Phyla Radiation or CPR) using GTDBtk. In order to corroborate these results, we used ANVI’O, which classified as CPR all these MAGs, but 2 (INTA.AUR.015 and INTA.AUR.064). These two genomes showed high levels of completeness (85% and 84% respectively) but also high levels of redundancy, hence were classified as “Inconclusive” and required manual inspection and contamination removal. The taxonomic classification showed that the MAGs were distributed across four classes, being Saccharimonadia the most abundant (7 MAGs), followed by ABY1 (5 MAGs).We found a total of 16 MAGs that were classified as members of the recently described Gracilibacteria; however, this was the only MAG that showed enzymes to utilize ribose. Also, only INTA.CYCY.027 had complete modules related to the synthesis of amino acids: threonine (M00018), serine (M00020), and methionine (M00035).On average, 33% of the genes of each MAG were annotated using the KEGG database. All MAGs lacked a complete TCA cycle, but almost all of them showed evidence of other central carbohydrate metabolism modules, namely M00002 (the core glycolysis module) and M00003 . The only MAG that lacked these two modules was INTA.CYC.027, the only one classified as Over two million genes were predicted between both data sets , with CYIn MAGs, on average, 40% of the genes were annotated using one of these databases. On the other hand, only a small fraction (<15%) of genes outside MAGs were annotated, since most of these genes were partial (~80%), which makes it difficult to correctly assign a function to them.For the hydrolysis of complex molecules, like carbohydrates and proteins, CAZymes and proteases were searched. A total of 21,196 CAZymes were found, from which 38.3% were glycosyl hydrolases (GH), 12.7% carbohydrate esterases (CE), and 1.6% polysaccharide lyases (PL). Every MAG had an average of 28 GH, and 8 CE. On the other hand, a total of 2,787 proteases were found, although the classification of these enzymes is based on the catalytic residues, not substrates nor activity, and does not allow a direct functional analysis.Several MAGs (63/110) had one or more ways to obtain ammonia from amino acids. For the nitrification stage, the first step of the process (methane/ammonia monooxygenase) was not found in either data set. However, there was evidence of the following steps. On the other hand, a total of 14 MAGs presented the module for dissimilatory nitrate reduction (M00530), while 4 MAGs would be able to denitrify nitrate (M00529), and 4 MAGs have the complete fixation module (M00175).Most MAGs showed one or more modules related to the acidogenesis stage of the carbon cycle. The acetogenesis stage is divided in two stages in KEGG, the conversion of pyruvate into acetyl-CoA (M00307) and the conversion of acetyl-CoA into acetate that can be catalyzed by five different enzymes. The complete or partial pyruvate oxidation module was found in many MAGs (41), but only seven of them also presented any of the enzymes needed for the next step.Finally, none of the methanogenesis modules were found complete neither in the MAGs nor among unbinned contigs. However, parts of the pathway for the acetoclastic methanogenesis (M00357), and the hydrogenotrophic methanogenesis (M00567) were found in the unbinned contigs of the CYC data set, and some of the genes from methanol methanogenesis pathway (M00356) were found in INTA.CYC.1002, the only archaeal MAG reconstructed.2 fixation pathways. In 4 MAGs, the first half of the Calvin cycle (M00166) was found complete, including both RuBisCO subunits (K01601 and K01602). The second half (M00167) was not complete in any of these MAGs, but many of the genes of the module were present . Interestingly, these 4 MAGs were also the only ones to have a complete module for a photosynthetic system . Enzymes for the Arnon-Buchanan cycle (M00173) were found in 33 MAGs, but all of them lacked the ATP-citrate lyase (K15230 and K15230), which is the key gene for this pathway. Some genes for the Wood-Ljungdahl pathway (M00377) and the dicarboxylate-hydroxybutyrate cycle (M00374) were found, but there were many steps missing  to be considered present.Evidence was found for 4 COProteobacteria, except for INTA.AUR.004, classified as Bacteroidetes, which only presented the assimilatory reduction pathway.The three sulfur metabolism modules described in KEGG were found in the MAGs. The full dissimilatory sulfate reduction module (M00596) was present in five MAGs, and partially present in another one. As for the assimilatory sulfate reduction module (M00176), only one MAG had it complete, and other 4 MAGs presented some evidence of it. Finally, in five MAGs was detected the complete SOX complex, related to the oxidation of sulfur compounds, plus another MAG where it was found partially. All these MAGs belonged to Finally, genes related to the phosphorus metabolism were searched. We looked for the poly-beta-hydroxybutyrate (PHB) synthesys cassette , that isIn this study, we present the analysis of two facultative stabilization ponds systems. These systems receive the effluents from dairy industries, mainly milk derived by-products, and the microbial communities in these environments are tasked with the degradation of this organic waste, combining both aerobic and anaerobic processes. We based the analysis on a genome-centred approach, attempting to reconstruct the complete genome of these microbes and focusing on complete metabolic pathways, and not just single gene markers.Actinobacteria and they both showed a significantly higher abundance in the second pond. The same situation was observed in KRAKEN2 results. Since the presence of Actinobacteria in activated sludge was linked to operational problems  were both classified as problems , we can Saccharimonadia , a lineage that was almost absent according to KRAKEN2 . Since KRAKEN2 uses the information available in the NCBI genome database, groups like the recently described Patescibacteria phylum  was classified by GTDB-Tk as a phylum , which iIt is also worth noting that only a small fraction of reads was classified using KRAKEN (13.9–30.8%), therefore the results may be useful to have a raw estimation of variation among the samples of a given set, but strong conclusions should not be drawn from them. We consider that a binning strategy followed by a phylogenomic classification, such as GTDB-Tk, is a more powerful strategy to determine the microbial diversity of an environment .2 or methane, going through different steps: acidogenesis, acetogenesis, and methanogenesis; and carbon fixation, obtaining complex compounds from CO2.The functional gene annotation was based on KEGG modules, which are a collection of manually defined gene sets in a metabolic pathway . The twoBacteroidetes, described as Bacteroidota in the GTDB, is one of the most represented classes among CYC MAGs (12), second only to Clostridia (13). These organisms have been reported to be present in different reactors because of their ability to degrade organic compounds into monomers  in the whole set, suggesting that they may be better suited to incorporate carbon from the environment (present mainly in the form of lactose). Therefore, Bacteroidetes are probably key members of the communities in facultative ponds .Clostridia. We also found members of the Spirochaetes class to be present in both samples. These MAGs show the module for the pyruvate oxidation, which agrees with previous reports  presented one of the enzymes required to convert acetyl-CoA into acetate, but only seven had both, most of them classified as  reports  that sug2  have an ANI of 99.97%. Given their crucial contribution to the carbon fixation in these environments and their conservation in both pond systems, we consider that these microorganisms play a key role in the system.From the six major systems used by autotrophs to fix CO2 , genes r2 . On top Desulfovibrionales and Sulfurospirillaceae. We were able to reconstruct 3 MAGs of the former (2 in AUR and 1 in CYC), and 2 MAGs of the later (1 in each pond). Members of the Desulfovibrionales family have shown the potential for the dissimilatory reduction of both sulfur and ammonia, so they would be important for the nitrogen cycle and to control the levels of sulfate that may be present in the ponds. Meanwhile, the 2 Sulfurospirillaceae also have presented the module for the dissimilatory reduction of ammonia.The other important metabolic process carried out in this type of environment is the nitrogen cycle, which was not found complete in any of the pond systems. In both cases, the missing enzyme was the methane/ammonia monooxygenase , which catalyses the first step of nitrification. The ammonia-oxidizing microorganisms fall in a few taxonomic groups  and, eveClostridiales (8 in AUR dataset and 13 in CYC). Clostridia have been suggested to be able to fermentate amino acids , have a limited metabolic potential, having notably fewer genes related to the synthesis of lipids, and nucleic acids . FurtherThere are other taxa shared between both systems, but that are present in low coverage. We found in both datasets microorganisms that have been previously reported to be involved in the degradation of different organic compounds, like Verrucomicrobia , SynergiWe were able to carry out a genome-centred analysis of the communities in two wastewater stabilization ponds systems. This approach allowed us to reconstruct 110 MAGs, and study the genes and metabolic pathways present in them. This kind of analysis allows a deeper understanding of the metabolic processes in a given environment, and expands the scope that a single marker gene, either functional or taxonomical, can provide. We found seven key taxa present in both pond systems studied, that would play important roles in the degradation of organic compounds in facultative stabilization ponds. A better understanding of the microbial communities and their metabolic potential is necessary to identify more key actors, and could contribute to the improvement of this kind of systems, so common in developing countries, and minimize their environmental impact.10.7717/peerj.12772/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12772/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12772/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12772/supp-4Supplemental Information 4Genome statistics and taxonomical classification (GTDBtk) for each bin obtained. Coverage for each bin was calculated as the sum of the coverage of each contig in a bin, divided by the number of number of contigs in that bin. To normalize values, each coverage was divided by the number of reads in each dataset over one million.Click here for additional data file.10.7717/peerj.12772/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12772/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12772/supp-7Supplemental Information 7Click here for additional data file.


Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities.Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response.Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.The online version contains supplementary material available at 10.1186/s12864-021-08226-5. Vertebrates have extensive variation in regenerative capacity, and identifying conserved vertebrate regenerative mechanisms holds promise both for developing novel strategies for therapeutic regeneration of tissues following injury or disease and for understanding and treating cancer. Non-traditional model systems that possess extreme regenerative phenotypes are particularly powerful for identifying the molecular basis of such phenotypes and may provide valuable insight into regenerative capacities and pathways in vertebrates. Most known examples of vertebrate regeneration involve regrowth of appendages, including fin regeneration in fish –3, limb Python bivittatus), increases its small intestine wet mass two-fold, intestinal mucosa thickness three-fold, and intestinal microvillus length five-fold [Multiple heavily bodied lineages of snakes  that experience months-long fasts between meals have evolved the ability to downregulate and atrophy energetically costly digestive organs during fasting, only to subsequently regenerate organ tissue and function at unequaled rates and magnitudes after feeding. For example, within 24 h of consuming a meal, the Burmese python , that experiences post-feeding regenerative intestinal growth [To address these gaps in our knowledge and advance models of vertebrate regeneration, we integrate multiple “omics” datasets and physiological data from a heavy bodied, infrequently feeding snake, the boa constrictor (l growth . We sampp < 0.05) increase five- to seven-fold by 12hrpf , 1dpf, 3dpf, and 6dpf. Within 6 days following feeding, the small intestine wet mass increases by over 100%, mucosal width doubles, and enterocyte volume doubles by 6dpf  of transport, stress response, and metabolic functions. Signals at 3dpf and 6dpf highlight major changes to tissue form as cell junction organization, blood vessel development, and mitosis increase. These patterns correspond to changes measured in the intestine, where we find early peaks in uptake and metabolic activity while microvillus, cell, and tissue growth do not reach their peak levels until 3dpf and 6dpf.To further investigate the functional significance of differentially expressed genes, we conducted Upstream Regulatory Molecule (URM) and Canonical Pathway activity predictions using Ingenuity Pathway Analysis    for Cluster 1  and do not (water snakes) regenerate following feeding . We evalInferences of URM and canonical pathway activation highlight many signaling patterns that are shared among regenerating species with absent or opposing activation in the non-regenerating water snake. These results implicate a core set of pathways and molecules central to the onset (1dpf) of the regenerative response across species Figs. b-c. OnlyWe present new evidence for a coordinated ‘switch’ in signaling activity derived from the interactions of nutrient uptake, stress, and growth pathways that direct the extreme regenerative response in snake small intestine Fig. c. Our fiPrevious genomic analysis in Burmese pythons identified positive selection in a number of genes relevant to extreme regeneration , and traIn addition to the central roles of major stress response pathways, inflammation is another key stress response with downstream effects on tissues that can be detrimental if not mediated, and links between inflammatory and regenerative responses are known from other examples of vertebrate regeneration , 60. WouIn some cases, we identify activation or inhibition of pathways that seem to conflict with our expectations. For example, loss of PTEN signaling leads to hyperproliferation, and we measure decreased PTEN early in the regenerative response when we predict low cell division while also finding simultaneous signaling events produce decreased proliferation. These results suggest that several complex networks interact to modulate early regeneration without tumorigenesis or other problematic growth under high stress conditions. Additionally, heterogeneity in the intestinal epithelium might also manifest in different cell populations that vary in stress levels and responses at a given time, resulting in some cells that continue division while others are extremely stressed or dying.Our new understanding of the molecular interactions that orchestrate the post-feeding regeneration response in the snake intestine highlights an intriguing and largely novel model that entails interactions between nutrient uptake, stress response and apoptosis, and growth pathways that direct tissue regeneration in a vertebrate model. This highly integrated molecular signaling model provides new valuable perspectives on how these pathways may be synergistically capable of directing regenerative capacity in other vertebrates, including humans, that naturally have limited capacity for regeneration , yet mayn = 5 per group; total n = 25) to provide a time series of the regenerative response. Sample sizes are consistent with previous experiments of snake regenerative physiology [Animal care and experimentation were conducted under an approved IACUC protocol at the University of Alabama 14-06-0075) in AAALAC-accredited facilities. Boa constrictors were obtained captive-bred from Strictly Reptiles  and acclimated for at least 3 months in a standard rack housing system and 12:12 light cycle, with meals offered weekly. Before experimentation, they were fasted until in post-absorptive Phase III fasted states and then fed meals sized to approximately 25% of their body mass. Snakes were humanely euthanized by severing the spinal cord immediately behind the head at five time points: while fasted and then 12hrpf, 1dpf, 3dpf, and 6dpf . Transmission electron microscopy was used to study ultrastructural changes to the intestinal brush border membrane. We fixed pieces of intestinal mucosa in 2.5% gluteraldehyde, postfixed them in 1% osmium tetroxide, dehydrated them in graded series of ethanol, and embedded them in Spurr’s epoxy resin. We cut ultra-thin sections (~ 80 nm) which were placed on copper mesh grids and viewed using a Hitachi (H-7650) transmission electron microscope at 20,000X magnification . We imaged sections of microvillus membrane with the scope’s high-definition (Orca) camera using Advanced Microscopy Techniques Corp. image analysis software.We examined post-prandial changes in intestinal tissue thickness and enterocyte size and assessed ultrastructural changes to the intestinal brush border membrane using transmission electron microscopy following previous studies , 63. We We followed the everted-sleeve technique protocols from previous studies , 63 to mn = 4), 12hrpf (n = 5), 1dpf (n = 5), 3dpf (n = 3), and 6dpf (n = 3) timepoints  (Qiagen Inc.) to determine upstream regulatory molecule (URM) activation, canonical pathway expression, mechanistic networks, and predicted downstream effects on function.Raw de-multiplexed RNAseq reads were filtered and trimmed using Trimmomatic v 0.32 . We quannscripts  as a refnscripts  then impnscripts . Counts nscripts , and painscripts  for indep < 0.05. Because genes were annotated based on homology to human protein-coding genes, we used the entire set of human protein-coding genes as a background for enrichment analyses. To determine overlapping gene networks, we intersected expressed genes of all pathways at fasted vs. 12hrpf and filtered to only connections in which at least 50% of expressed genes in one of the pathways overlap. The network was manually annotated for growth and stress response pathway groupings. This was repeated for overlap of significant pathways for fasted vs. each other timepoint and filtered to 60% overlap.Normalized counts were used in TCseq v 1.8.0  to identn = 4), 12hrpf (n = 4), 1dpf (n = 4), and 3dpf (n = 3) snakes with the T-PER Tissue Protein Extraction Reagent , using the same samples as were used for RNA extraction  based on manufacturer instructions. Briefly, the column was activated using a washing and binding buffer. The digested peptides passed through an activated filter and again washed with binding buffer. After removing all excess buffer, enriched phosphopeptides were eluted with elution buffer (50 μL) twice. Enriched peptides were dried in a speed-vacuum and reconstituted with 0.1% formic acid in water for liquid chromatography-mass spectrometry (LC-MS/MS) analysis.To isolate phosphopeptides, extracted proteins in T-PER buffer were reduced in 50 mM dithiothreitol (DTT) at 56 °C for 30 min and then alkylated in 30 mM iodoacetamide at room temperature (~ 25 °C) for 30 min in dark condition. Samples were digested with proteolytic enzyme trypsin (enzyme-to-proteins ratio of 1:50 at 37 °C for 16 h). Digested peptides were desalted through C18 cartridges  and dried by Speed Vacuum . The digBoa constrictor genome [A Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer  coupled to an UltiMate 3000 UHPLC  was used to analyze digested peptides and phosphopeptides as previously described . We perfr genome , 79 withr genome . For phor genome  node is r genome .For protein quantification, counts from peptide spectrum matches (PSM) were normalized, log2-transformed, and hierarchically clustered by expression patterns through the time series in R. These clusters were analyzed for functional and pathway enrichment analyses in WebGestalt, using the entire protein-coding gene set as a background . Kinase Additional file 1: Supplementary figures S1-S17 detailing gene expression results.Additional file 2: Table S1. Differentially expressed genes between timepoints in boa constrictor small intestine after feeding .Additional file 3: Table S3. Target molecules of NFE2L2 and their directional changes in the RNAseq dataset from fasted vs. 12hrpf.Additional file 4: Table S2. Target molecules of XBP1 and their directional changes in the RNAseq dataset from fasted vs. 12hrpf.Additional file 5: Table S5. Number of reads per sample in multiple species analysis.Additional file 6: Table S4. Description of snakes sampled for analyses.