Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori).

Mercury (Hg) contamination poses a global threat to the environment, given its elevated ecotoxicity. Herein, we employed the lepidopteran model insect, silkworm (Bombyx mori), to systematically investigate the toxic effects of Hg-stress across its growth and development, histomorphology, antioxidant enzyme activities, and transcriptome responses. High doses of Hg exposure induced evident poisoning symptoms, markedly impeding the growth of silkworm larvae and escalating mortality in a dose-dependent manner. Under Hg exposure, the histomorphology of both the midgut and fat body exhibited impairments. Carboxylesterase (CarE) activity was increased in both midgut and fat body tissues responding to Hg treatment. Conversely, glutathione S-transferase (GST) levels increased in the fat body but decreased in the midgut. The transcriptomic analysis revealed that the response induced by Hg stress involved multiple metabolism processes. Significantly differently expressed genes (DEGs) exhibited strong associations with oxidative phosphorylation, nutrient metabolisms, insect hormone biosynthesis, lysosome, ribosome biogenesis in eukaryotes, and ribosome pathways in the midgut or the fat body. The findings implied that exposure to Hg might induce the oxidative stress response, attempting to compensate for impaired metabolism. Concurrently, disruptions in nutrient metabolism and insect hormone activity might hinder growth and development, leading to immune dysfunction in silkworms. These insights significantly advance our theoretical understanding of the potential mechanisms underlying Hg toxicity in invertebrate organisms.

The acetyltransferase BmCBP changes the acetylation modification of BmSP3 and affects its protein expression in silkworm, Bombyx mori.

BACKGROUND: Protein acetylation is an important post-translational modification (PTM) that widely exists in organisms. As a reversible PTM, acetylation modification can regulate the function of proteins with high efficiency. In the previous study, the acetylation sites of silkworm proteins were identified on a large scale by nano-HPLC/MS/MS (nanoscale high performance liquid chromatography-tandem secondary mass spectrometry), and a total of 11 acetylation sites were discovered on Bombyx mori nutrient-storage protein SP3 (BmSP3). The purpose of this study was to investigate the effect of acetylation level on BmSP3. METHODS AND RESULTS: In this study, the acetylation of BmSP3 was further verified by immunoprecipitation (IP) and Western blotting. Then, it was confirmed that acetylation could up-regulate the expression of BmSP3 by improving its protein stability in BmN cells. Co-IP and RNAi experiments showed acetyltransferase BmCBP could bind to BmSP3 and catalyze its acetylation modification, then regulate the expression of BmSP3. Furthermore, the knock-down of BmCBP could improve the ubiquitination level of BmSP3. Both acetylation and ubiquitination occur on the side chain of lysine residues, therefore, we speculated that the acetylation of BmSP3 catalyzed by BmCBP could competitively inhibit its ubiquitination modification and improve its protein stability by inhibiting ubiquitin-mediated proteasome degradation pathway, and thereby increase the expression and intracellular accumulation. CONCLUSIONS: BmCBP catalyzes the acetylation of BmSP3 and may improve the stability of BmSP3 by competitive ubiquitination. This conclusion provides a new functional basis for the extensive involvement of acetylation in the regulation of nutrient storage and utilization in silkworm, Bombyx mori.

Study on depolymerization kinetics of formic acid dimers in binary mixture.

In this study, polarization Raman spectra were collected for binary mixtures of formic acid/methanol and formic acid/acetonitrile with different volume fractions. The broad band of formic acid in the CO vibration region was divided into four vibration peaks, corresponding to CO symmetric and anti-symmetric stretching vibration from cyclic dimer, CO stretching from open dimer, and CO stretching from the free monomer. The experiments showed that as the volume fraction of formic acid in the binary mixture decreased, the cyclic dimer gradually converted to the open dimer, and at a volume fraction of 0.1, fully depolymerized into monomer form (free monomer, solvated monomer, and hydrogen bonding monomer clusters with solvent). The contribution percentage of the total CO stretching intensity of each structure at different concentrations was quantitatively calculated using high resolution infrared spectroscopy, and the results were consistent with the conclusions predicted by polarization Raman spectroscopy. Concentration-triggered 2D-COS synchronous and asynchronous spectra also confirmed the kinetics of formic acid diluted in acetonitrile. This work provides a spectroscopic method for studying the structure of organic compounds in solution and concentration-triggering kinetics in mixtures.

The intermolecular polar bond interaction and coupling induced spectral splitting phenomenon for a binary mixture.

To explain the polarization Raman noncoincidence effect of specific polar bonds and the noncoincidence phenomenon between FT-Raman and FT-IR spectra, aggregation-induced spectral splitting theory was proposed. In this paper, the vibration splitting theory was demonstrated using two strategies: improving the spectral resolution with cryogenic matrix isolation techniques and identifying cases where the coupling splitting is large enough to be distinguishable. The monomer and dimer splitting bands of acetone were detected when cryogenically isolated by the Ar matrix. Additionally, the polarization Raman and two-dimensional infrared spectra of a ß-propiolactone (PIL)/CCl4 binary mixture were collected at room temperature, and the spectral splitting phenomenon was clearly observed. The dynamic transformation between the monomer and dimer could be achieved and detected by adjusting the PIL concentration. The observed splitting phenomenon was further confirmed by theoretical DFT calculations based on the monomer and dimer of PIL, as well as the FT-IR and FT-Raman spectra of PIL. Concentration-triggered 2D-COS synchronous and asynchronous spectra also confirmed the splitting phenomenon and the dilution kinetics of PIL/CCl4.

BmCBP Catalyzes the Acetylation of BmApoLp-II Protein and Regulates Its Stability in Silkworm, Bombyx mori.

Acetylation is an important and reversible post-translational modification (PTM) of protein, which is involved in many cellular physiological processes. In previous studies, lots of nutrient storage proteins were found to be highly acetylated in silkworms, and acetylation can improve the stability of these proteins. However, the related acetyltransferase was not involved. In the present work, a Bombyx mori nutrient storage protein, apolipophorin II (BmApoLp-II), was further confirmed to be acetylated, and the acetylation could improve its protein expression. Furthermore, RNAi and Co-IP showed that the acetyltransferase BmCBP was found to catalyze the acetylation modification of BmApoLp-II, and thus affect its protein expression. Meanwhile, it was proved that acetylation could improve the stability of the BmApoLp-II protein by completing its ubiquitination. These results lay a foundation for further study on the mechanism of regulating nutrition storage and hydrolysis utilization of storage proteins by BmCBP and the acetylation in the silkworm Bombyx mori.

Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

The polarized Raman spectra of N-methylpyrrolidone-an effective method for determination of intermolecular interaction and H-bond formation.

The isotropic and anisotropic component Raman spectra and H NMR of N-methylpyrrolidone (NMP)/carbon tetrachloride and NMP/methanol binary mixture at different volume fractions have been collected. The polarization Raman frequencies and frequency differences of CO stretching vibration for NMP/methanol mixture show unique concentration-dependence and abrupt jump feature. It is found that the H-bond between solute and solvent does not destroy the noncoincidence (NCE) phenomenon, but has a significant synergistic effect on the NCE. Two distinctive clusters constrained by H-bond and intermolecular interactions were easily determined by means of linear extension method from abrupt jump curve. The experimental phenomena can be well explained by aggregation-induced splitting theory with the proposed dimer structure and H-bond cluster model. Applying the same methodology the conformation of NMP in water has been determined successfully. The establishment of this method will play an important role in the determination of biomolecule aggregation behavior and supramolecular self-assembly structure.

Aggregation induced spectral splitting and Fermi resonance of Ethylene Carbonate in binary mixture.

The vibration band of the ring stretching (ν14), the fundamental ring breathing (ν17) and the Fermi resonance band of carbonyl stretching mixing with the overtone of the ring breathing (ν5 + 2ν17) have been investigated in solid ethylene carbonate (EC) and EC/CH3CN and EC/CHCl3 binary mixture. Dimer structure with aggregation-induced spectral splitting model (AIS) was applied to calculate the vibration spectra using the B3LYP-D3/6-311+G (d,p) procedure. The noncoincidence effect (NCE) and concentration induced frequency shifts of the ν14 and ν5 could be well explained by AIS model based on the dimer structure. Four bands were observed with two in the isotropic and two in the anisotropic Raman spectra and their NCE value decreased with the decrease of EC volume fraction in the binary mixture, and finally disappeared. NCE value and the Fermi resonance constants of EC at different concentrations were calculated from the experimental data.

The Acetylation Modification of SP1 Regulates the Protein Stability in Silkworm.

Acetylation is a highly conservative and reversible post-translational modification. Acetylation modification can regulate gene expression by altering protein function and is widely identified in an increasing number of species. Previously, the acetylated proteome of silkworm was identified by combining acetylated polypeptide enrichment with nano-HPLC/MS/MS; the identification revealed that the SP proteins (SPs) were high acetylated. In this study, the acetylation of SP1, one of the SPs, was further confirmed using immunoprecipitation (IP) and Western blotting. Then, we found the acetylation could upregulate SP1 protein expression by enhancing the protein stability. Further research found that the acetylation of SP1 protein can competitively inhibit its ubiquitination and thus improve the stability and cell accumulation of SP1 protein by inhibiting the ubiquitin-mediated proteasome degradation pathway. This result provides a basis for acetylation to regulate the nutrient storage and utilization of silkworm.

The acetylation modification regulates the stability of Bm30K-15 protein and its mechanism in silkworm, Bombyx mori.

The 30 K proteins are the major silkworm hemolymph proteins and are involved in a variety of physiological processes, such as nutrient and energy storage, embryogenesis, immune response, and inhibition of apoptosis. The Bm30K-15 protein is one of the 30 K proteins and is abundant in the hemolymph of fifth instar silkworm larva. We previously found that the Bm30K-15 protein can be acetylated. In the present study, we found that acetylation can improve the protein stability of Bm30K-15. Further exploration confirmed that the increase in protein stability by acetylation was caused by competition between acetylation and ubiquitination. In summary, these findings aim to provide insight into the effect of acetylation modification on the protein level and stability of the Bm30K-15 and the possible molecular mechanism of its existence in silkworm, Bombyx mori.

The complete mitogenome sequence of the hawk moth, Theretra latreillii subsp. lucasii (Lepidoptera: Sphingidae) from Zhejiang Province, China.

The sphingid, Theretra latreillii subsp. lucasii is a common hawk moth distributed in southeast Asia and Australian regions. Although barcode analyses have been published, its complete mitogenome sequence has not been deciphered. In this study, the complete mitogenome of T. latreillii lucasii (GeneBank accession no. MW539688) was sequenced using Illumina HiSeq X Ten system for mitogenome-based phylogenetic analysis. The mitogenome was 15,354 bp in length and comprises 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 transfer RNAs (tRNAs) with the typical gene order and orientation of Sphingidae mitogenomes. The nucleotide composition of majority strand is 41.2% for A, 7.4% for G, 12.0% for C, and 39.4% for T, with an A + T content of 80.6%. Phylogenetic analysis using the 13 PCGs fully resolved T. latreillii lucasii in a clade with T. japonica, Macroglossum stellatarum, and Ampelophaga rubiginosa, with high nodal support both by Bayesian inference and maximum-likelihood methods, forming the Macroglossini monophyletic group.

Combination therapy with B7H3-redirected bispecific antibody and Sorafenib elicits enhanced synergistic antitumor efficacy.

Rationale: Current traditional treatment options are frequently ineffective to fight against ovarian cancer due to late diagnosis and high recurrence. Therefore, there is a vital need for the development of novel therapeutic agents. B7H3, an immune checkpoint protein, is highly expressed in various cancers, representing it a promising target for cancer immunotherapy. Although targeting B7H3 by bispecific T cell-engaging antibodies (BiTE) has achieved successes in hematological malignancies during recent years, attempts to use them for the treatment of solid cancers are less favorable, in part due to the heterogeneity of tumors. Sorafenib is an unselective inhibitor of multiple kinases currently being tested in clinical trials for several tumors, including ovarian cancer which showed limited activity and inevitable side effect for ovarian cancer treatment. However, it is able to enhance antitumor immune response, which indicates sorafenib may improve the efficiency of immunotherapy. Methods: We evaluated the expression of B7H3 in ovarian cancer using online database and validated its expression of tumor tissues by immunohistochemistry staining. Then, B7H3 expression and the effects of sorafenib on ovarian cancer cell lines were determined by flow cytometry. In addition, 2D and 3D ovarian cancer models were established to test the combined therapeutic effect in vitro. Finally, the efficiency of B7H3×CD3 BiTE alone and its combination with sorafenib were evaluated both in vitro and in vivo. Results: Our data showed that B7H3 was highly expressed in ovarian cancer compared with normal samples. Treatment with sorafenib inhibited ovarian cancer cell proliferation and induced a noticeable upregulation of B7H3 expression level. Further study suggested that B7H3×CD3 BiTE was effective in mediating T cell killing to cancer cells. Combined treatment of sorafenib and B7H3×CD3 BiTE had synergistic anti-tumor effects in ovarian cancer models. Conclusions: Overall, our study indicates that combination therapy with sorafenib and B7H3×CD3 BiTE may be a new therapeutic option for the further study of preclinical treatment of OC.

Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors.

Purpose: Given that heterogeneous expression and variants of antigens on solid tumors are responsible for relapse after chimeric antigen receptor (CAR)-T cell therapy, we hypothesized that combinatorial targeting two tumor-associated antigens would lessen this problem and enhance the antitumor activity of T cells. Methods: The co-expression level of CD70 and B7-H3 was analyzed in multiple tumor tissue samples. Further, two putative antigens were identified in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis database. Two CD70 targeted CARs with different antigen binding domain, truncated CD27 and CD70 specific single-chain antibody fragment (scFv), were designed to screen a more suitable target-antigen binding moiety. Accordingly, we designed a bivalent tandem CAR (TanCAR) and further assessed the anti-tumor efficacy of TanCAR-T cells in vitro and in vivo. Results: Our results indicated that co-expression of CD70 and B7-H3 was observed on multiple tumor types including kidney, breast, esophageal, liver, colon cancer, glioma as well as melanoma. The CD70 targeted CAR-T cells with binding moiety of CD70 specific scFv exhibit a higher affinity and antitumor effect against CD70+ tumor cells. TanCAR-T cells induced enhanced ability of cytolysis and cytokine release over unispecific CAR-T cells when encountering tumor cells expressing two target-antigens. Further, low doses of TanCAR-T cells could also effectively control the lung cancer and melanoma xenografts and improved overall survival of the treated animals. Conclusion: TanCAR-T cells targeting CD70 and B7-H3 exhibit enhanced antitumor functionality and improve the problem of antigenic heterogeneity and variant in the treatment against solid tumor and melanoma.

Bioactivity and safety of B7-H3-targeted chimeric antigen receptor T cells against anaplastic meningioma.

OBJECTIVE: We conducted a first-in-human study to evaluate the bioactivity and safety of B7-H3-targeted chimeric antigen receptor (CAR) autologous T cells for treating recurrent anaplastic meningioma. METHODS: Tumor tissues from a patient with recurrent anaplastic meningioma were evaluated for B7-H3 expression. B7-H3-targeted CAR-T cells were delivered into the intracranial tumor resection cavity using an Ommaya device at a maximum dose of 1.5 × 107 cells. Magnetic resonance imaging (MRI) screening and multiple serum indexes were regularly monitored. The patient received surgical intervention after three-cycle infusions, allowing analysis for CAR-T-cell infiltration and target antigen expression in post-CAR-T therapy tumor tissues. RESULTS: Immunochemical analysis demonstrated high and homogeneous B7-H3 expression in tumor samples. MRI results indicated that the tumor near the delivery device was relatively stable compared with the rapid progression of tumors distant from the device. We found CAR-T-cell trafficking to regions of B7-H3+ tumor tissues near the device, but not to tumor tissues distant from the device. Decreased B7-H3 expression was observed near the region of CAR-T-cell infiltration after therapy. The intracavitary delivery of B7-H3-targeted CAR-T cells was well-tolerated and not associated with any toxic effects of grade 3 or higher. CONCLUSION: Our results suggested that although intracavitary administration of B7-H3-targeted CAR-T cells was safe and resulted in local bioactivity, addressing antigen loss and CAR-T-cell trafficking may further enhance the applications of B7-H3-targeted CAR-T-cell therapy.

Rab9 defense against white spot syndrome virus by participation in autophagy in Marsupenaeus japonicas.

White spot syndrome virus (WSSV) is the main pathogen of shrimp and has led to considerable economic losses to the shrimp industry around the world. However, so far there are still no effective strategies to address this problem. In this paper, the tissue distribution of Rab9 as well as its defense mechanism against WSSV in Japanese shrimp (Marsupenaeus japonicas) was investigated. The results revealed that Rab9 had a higher expression in hemocyte and gill while expression was lower in heart, muscle, intestine, liver, indicating Rab9 was involved in the innate immune process. The results showed that the Rab9 expression increased when shrimp was challenged with WSSV compared with that of control, while the silence of Rab9 led to the increase of WSSV copies. In order to explore the antiviral mechanism of Rab9, it was demonstrated that the expression level of Rab9 changed during autophagy process, which indicated that Rab9 is participated in the autophagy procedure of shrimp. The fact that autophagy decreased after Rab9 silenced, may also suggest that Rab9 protein could affect autophagy. In short, the results showed Rab9 played a key role in antivirus through regulating autophagy. The results not only enlarge the limited views about molecular mechanism of Rab in invertebrate, but also help to enrich the immunological content in marine invertebrate.

B7-H3-Targeted CAR-T Cells Exhibit Potent Antitumor Effects on Hematologic and Solid Tumors.

Recently, B7-H3 was frequently reported to be overexpressed in various cancer types and has been suggested to be a promising target for cancer immunotherapy. In the present study, we analyzed the mRNA expression of B7-H3 in The Cancer Genome Atlas (TCGA) database and validated its expression across multiple cancer types. We then generated a novel B7-H3-targeted chimeric antigen receptor (CAR) and tested its antitumor activity both in vitro and in vivo. The B7-H3 expression heterogeneity and variation were frequent. Moderate or even high expression levels of B7-H3 were also observed in some tumor-adjacent tissues, but the staining intensity was weaker than that in tumor tissues. B7-H3 expression was absent or very low in normal tissues and organs. Flow cytometry indicated that the mean expression level of B7-H3 in eight bone marrow specimens from patients with acute myeloid leukemia (AML) was 57.2% (range 38.8-80.4). Furthermore, we showed that the B7-H3-targeted CAR-T cells exhibited significant antitumor activity against AML and melanoma in vitro and in xenograft mouse models. In conclusion, although B7-H3 represents a promising pan-cancer target, and B7-H3-redirected CAR-T cells can effectively control tumor growth, the expression heterogeneity and variation have to be carefully considered in translating B7-H3-targeted CAR-T cell therapy into clinical practice.

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm, Bombyx mori.

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H2 O2 . In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H2 O2 -induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.

Overexpression of B7-H3 as an opportunity for targeted therapy in head and neck cancers.

Head and neck cancers (HNCs) are the sixth most common type of cancer in the world. Despite the development of refined surgical techniques and precise targeted radiation, patients with HNCs have a dismal prognosis. Here, we examine the expression profile of B7-H3 in HNCs and verify whether B7-H3 can serve as a novel therapeutic target for HNCs via anti-B7-H3×CD3 bispecific antibodies (biAbs). We analyzed the expression level of B7-H3 in 274 HNC samples and evaluated the association between B7-H3 expression and clinicopathological parameters. Anti-B7-H3×CD3 biAbs were constructed, and the efficacy of these biAbs in targeting HNCs was assessed in vitro and in vivo. As a result, high expression of B7-H3 was detected in 66.1% of clinical HNC samples and was correlated with poor survival. Specific antitumor effects of anti-B7-H3×CD3 biAbs were confirmed in vitro using HNC cell lines. In xenograft HNC mouse model, anti-B7-H3×CD3 biAbs delayed tumor growth and prolonged survival. In conclusion, B7-H3 is frequently overexpressed in HNCs and could be a promising therapeutic target for biAb therapy.

Frequent B7-H3 overexpression in craniopharyngioma.

Craniopharyngiomas (CPs) are uncommon intracranial benign neoplasms that located in sellar/parasellar region with clinically challenging. B7-H3 is an immune checkpoint molecule highly expressed in many malignant tumors. In this study, we analyzed whether B7-H3 is expressed in 44 CPs samples (adamantinomatous CPs: n = 30 and papillary CPs: n = 14), and whether it could serve as an immunotherapy target in CPs. Immunohistochemical analysis showed that B7-H3 was highly expressed in adamantinomatous CPs (184.3 ±â€¯13.58) and papillary CPs (223.2 ±â€¯11.89), while almost undetectable in normal brain tissue (24 ±â€¯4.9). Besides, B7-H3 expression level was correlated with poor prognosis of patients with CPs. Immunofluorescence and Western blot analysis further suggested that ß-catenin co-localized with B7-H3 and could promote its expression in adaCPs. B7-H3 expression level was positively correlated with staining intensity of IBA1+ cells, but negatively with T cell infiltration in CPs, suggesting that B7-H3 might play a role in the regulation of tumor microenvironment in CPs. Moreover, B7-H3/CD3 bi-specific T cell engager (BiTE) efficiently inhibited the growth of human primary craniopharyngioma cells in a time- and dose-dependent manner. Our results revealed B7-H3 was highly expressed in CPs and targeting B7-H3 might therefore be an effective therapeutic strategy against craniopharyngioma.

Molecular Network-Based Drug Prediction in Thyroid Cancer.

As a common malignant tumor disease, thyroid cancer lacks effective preventive and therapeutic drugs. Thus, it is crucial to provide an effective drug selection method for thyroid cancer patients. The connectivity map (CMAP) project provides an experimental validated strategy to repurpose and optimize cancer drugs, the rationale behind which is to select drugs to reverse the gene expression variations induced by cancer. However, it has a few limitations. Firstly, CMAP was performed on cell lines, which are usually different from human tissues. Secondly, only gene expression information was considered, while the information about gene regulations and modules/pathways was more or less ignored. In this study, we first measured comprehensively the perturbations of thyroid cancer on a patient including variations at gene expression level, gene co-expression level and gene module level. After that, we provided a drug selection pipeline to reverse the perturbations based on drug signatures derived from tissue studies. We applied the analyses pipeline to the cancer genome atlas (TCGA) thyroid cancer data consisting of 56 normal and 500 cancer samples. As a result, we obtained 812 up-regulated and 213 down-regulated genes, whose functions are significantly enriched in extracellular matrix and receptor localization to synapses. In addition, a total of 33,778 significant differentiated co-expressed gene pairs were found, which form a larger module associated with impaired immune function and low immunity. Finally, we predicted drugs and gene perturbations that could reverse the gene expression and co-expression changes incurred by the development of thyroid cancer through the Fisher's exact test. Top predicted drugs included validated drugs like baclofen, nevirapine, glucocorticoid, formaldehyde and so on. Combining our analyses with literature mining, we inferred that the regulation of thyroid hormone secretion might be closely related to the inhibition of the proliferation of thyroid cancer cells.