Oviducts are the "traffic hubs" of the female reproductive system, serving as the crucial conduits for egg transportation. By performing LC-MS/MS proteomic detection together with transcriptomic analysis, 80 lateral oviduct-secreted proteins were identified, and 5 genes (NlOdsp, NlOdsp1, NlOdsp2, NlOdsp3 and NlOdsp4) specifically expressed in the oviducts of the brown planthopper Nilaparvata lugens, the most destructive rice pest, were authenticated. qRT-PCR analysis revealed that these genes and proteins were mainly/specifically expressed in the female reproductive system in adulthood. RNA interference (RNAi) against the 5 NlOdsp genes significantly affected the survival rates (3.4% - 68.7% of the control) and fecundities of female adults (3.9% - 57.6% of the control) at 8 d post injection (p.i.). In addition, the lack of NlOdsp1 caused decreases in the gel-like brown secretions inside the lateral oviducts, while increased secretions were found in the dsNlOdsp2-treated groups. In addition, NlOdsp3 is a pleiotropic gene involved in both oocyte development and egg movement through the lateral oviducts, similar to the role of NlOdsp in egg transportation. The results deepen our understanding of oviduct-secreted proteins in female insects and provide novel target genes for RNAi-based insect pest control. SIGNIFICANCE: Oviduct plays a vital role in animal reproductive processes and it serves as the crucial conduit for egg transportation. Though oviduct secretes have been well documented in high animals, the proteomic information of insect oviduct secretes remains poorly understood. The present study revealed 80 oviduct secreted proteins, including 19 unknown proteins, from the rice planthopper, the most destructive rice pest which lay eggs in plant tissues. Five of the 19 proteins were further functionally characterized. The results not only deepen our understanding of the oviduct secreted proteins in insect reproductive biology, but also provide basis for interaction between insects and host plants, and provide novel target genes for RNAi-based insect pest control.

Oxaliplatin (OXA)-induced peripheral neurotoxicity (OIPN) is a high-incidence and dose-dependent adverse reaction during OXA treatment. Its underlying mechanisms remain unclear, and no effective treatment or prevention therapies are currently available. Here, we employed a data independent acquisition (DIA)-based quantitative proteomic strategy to investigate the global proteome alterations in the dorsal root ganglion (DRG) tissues from mice injected with OXA for different periods. We identified 1128 differentially regulated proteins that were divided into six subclusters according to their alteration trends. Interestingly, these proteins were involved in cellular processes such as cell cycle, ribosomal stress, metabolism, and ion transport. In addition, OXA administration induced abundance changes of ion channels and proteins associated with mitochondrial function and reactive oxygen species production. Furthermore, we investigated the effects of diroximel fumarate (DRF), an FDA-approved oral fumarate drug for the treatment of relapsing forms of multiple sclerosis. Our findings showed that DRF could effectively ameliorate symptoms of OIPN and reduce the level of oxidative stress in mice. Taken together, our study systematically mapped the proteome alteration associated with the neural toxicity of OXA, and the findings could be leveraged to better understand the mechanisms of OIPN and to develop more effect treatment therapies. SIGNIFICANCE: Oxaliplatin (OXA)-induced peripheral neurotoxicity (OIPN) is a high-incidence and dose-dependent adverse reaction with unclear mechanism. Here we employed a data independent acquisition (DIA)-based quantitative proteomic strategy to explore the proteome changes in dorsal root ganglion (DRG) tissues from mice treated by OXA. The findings provided novel insights regarding the mechanisms of OIPN. For example, our data showed that OXA induced a broad disturbance in metabolism, particularly in glycolysis and amino acid metabolism. Additionally, we observed abundance changes of many ion channels and proteins associated with mitochondrial function and reactive oxygen species production. Furthermore, this study provided the first evidence for the possibility of repositioning diroximel fumarate (DRF) for treating OIPN.

Lysine malonylation, a novel identified protein posttranslational modification (PTM), is conservative and present in both eukaryotic and prokaryotic cells. Previous studies have reported that malonylation plays an important role in inflammation, angiogenesis, and diabetes. However, its potential role in cardiac remodeling remains unknown. Here, we observed a reduced lysine malonylation in hypertrophic mice hearts created by transverse aortic constriction (TAC) for 8 weeks. We also detected a decreased lysine malonylation in hypertrophic H9C2 cardiomyocytes induced by angiotensin II for 48 h. Using a proteomic method based on affinity purification and LC-MS/MS, we identified total 679 malonylated sites in 330 proteins in the hearts of sham mice and TAC mice. Bioinformatic analysis of the proteomic data revealed enrichment of malonylated proteins involved in cardiac structure and contraction, cGMP-PKG pathway, and metabolism. Specifically, we detected a decreased lysine malonylation in myocardial isocitrate dehydrogenase 2 (IDH2) by immunoprecipitation coupled with Western blotting both in vivo and in vitro. Together, our work suggests an important role and implication of protein lysine malonylation in cardiac hypertrophy, especially the IDH2. SIGNIFICANCE: Heart failure is the terminal stage of cardiac hypertrophy, which imposes an enormous clinical and economic burden worldwide. Despite our knowledge on the pathophysiology of the disease, current therapeutic approaches are still largely limited. Cardiac hypertrophy can be regulated at post-translational modifications (PTMs), and several PTMs have been reported in cardiac hypertrrophy and heart failure. In our study, we first reported a novel PTMs, lysine malonylation, in cardiac hypertophy. we found a reduced lysine malonylation in hypertrophic mice hearts in vivo and H9C2 cardiomyocytes after stimulating with angiotensinII for 48 h in vitro. Using affinity purification and LC-MS/MS, we identified 679 malonylated sites in 330 proteins in the hearts of sham and TAC mice. Compared to the sham group, 5 sites in 2 proteins were quantified as downregulated targets using a 2-fold threshold (downregulation <0.5-fold, P < 0.05). Functional analysis showed a significant enrichment in cardiac structure and contraction, cGMP-PKG pathway and metabolism. Notably, we identified a decreased Kmal level in isocitrate dehydrogenase 2 (IDH2), but the protein level of IDH2 has no changed in cardiac hypertrophy, These results highlight that lysine malonylation is associated with cardiac hypertrophy, and may be a new therapeutic target of the disease.

Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.

Sulfolobus islandicus is thermophilic archaea that live in an extreme environment of 75 °C-80 °C and pH 2-3. Currently, the molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. This study utilizes proteomics to analyze the differential expression of S. islandicus proteins at different temperatures. We found that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are all affected by temperature. Methylation modification of some proteins changed with temperature. Thermal proteome profiling (TPP) was used to analyze the thermal stability of proteins under 65 °C-85 °C growth conditions. It is suggested that the Tm values of proteins are mainly distributed around the optimum growth temperature (OGT). The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. The protein thermal stability of S. islandicus cultured under 65 °C and 85 °C was higher than that of 75 °C. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time. SIGNIFICANCE: The molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. Our proteomics study identified 477 differentially expressed proteins of S. islandicus at different temperatures, suggesting that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are affected by temperature. Meanwhile, we found that methylation modification of some proteins changed with temperature. To evaluate the thermal stability of the proteome, we performed thermal proteome profiling to analyze the Tm of proteins under 65 °C-85 °C growth conditions. Tm values of proteins are mainly distributed around the optimum growth temperature. The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time.

The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.

Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SIGNIFICANCE: Oesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of 'treatment response prediction assays' for stratification of OAC patients prior to chemotherapy.

Senescence is the inevitable biological processes and is also considered as the biggest risk factor for the development of age - related diseases (ARDs) and geriatric syndrome (GS). Senescence is also known as inflammaging because it is characterized by persistent, long-term, low-grade inflammation named senescence-associated secretory phenotype (SASP). However, the mechanism for the persistence of inflammaging remains largely unclear. To explore the role of extracellular vesicles (EVs) in senescence/inflammaging, we established the cellular senescence model and performed TMT-based comparative quantitative proteomics and parallel reaction monitoring (PRM) to reveal the changes of EVs between young cells and senescent cells. A total of 3966 proteins were quantifiable, of which 132 were up-regulated, 144 were down-regulated, compared with the young cells. Subsequently, we chose 19 proteins involved in inflammation or proliferation to carry out PRM validation analysis. The result indicated that proteins promoting NF-κB signal pathway were up-regulated, and proteins promoting cell proliferation were down-regulated. The study provided a comprehensive altered proteomics profiles of EVs from senescent cells, and the result showed that EVs could serve as information carrier for further research on the pathogenesis and progression of senescence/inflammaging. SIGNIFICANCE: The mechanism of inflammaging occurrence and development has yet been clear. Therefore, this study attempts to provide an improved understanding of inflammaging from the perspective of EVs. The proteomics analysis revealed that the most changed proteins were connected to inflammation signaling pathways, cell growth and cell death, and PRM analysis results showed that proteins involved in NF-κB signal pathway and cell proliferation were more changed. The research systematically analyzed the profiles of proteins in senescence cell model, and the result indicated that further research should focus on the relationship between EVs and senescence/inflammaging.

OBJECTIVE: To identify gingival recession-related biomarkers in orthodontic patients, we compared the proteome of gingival crevicular fluids (GCF) from healthy gingiva without orthodontic treatment (GH), healthy gingiva undergoing orthodontic treatment (OGH), and recessed gingiva undergoing orthodontic treatment (OGR). METHODS: GCF samples were obtained from the anterior teeth of 15 volunteers (n = 5/group). Quantitative proteomic analysis was performed using DIA-based liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate differentially expressed proteins (DEPs). Receiver-operating characteristic (ROC) analysis was performed to detect and filter biomarker candidates, while Protein-Protein Interaction (PPI) Networks were utilized to determine the interactions between these DEPs. RESULTS: A total of 253, 238, and 101 DEPs were found in OGR vs. OGH, OGR vs. GH, and OGH vs. GH groups, respectively. Based on the Venn diagram of three groups, 128 DEPs in OGR vs. OGH group were identified as specific proteins associated with progressive gingival recession (GR) during orthodontic treatment. Molecular function analysis showed that 128 DEPs were enriched in "molecular binding", including antigen binding, RNA binding, double-stranded RNA binding, cadherin binding involved in cell-cell adhesion, vinculin binding, S100 protein binding, and Ral GTPase binding. The majority of these DEPs were also involved in cytoskeletal regulation. In addition, biological process analysis showed an enrichment in translation, while cellular component analysis indicated that 128 DEPs were related to extracellular exosome. Furthermore, Ribosome and Phagosome were the top two terms in KEGG analysis. The results of ROC analysis demonstrated that 26 proteins could be potential biomarker candidates for GR. PPI networks analysis predicted that IQGAP1, ACTN1, TLN1, VASP, FN1, FERMT3, MYO1C, RALA, RPL35, SEC61G, KPNB1, and NPM1 could be involved in the development of GR via cytoskeletal regulation. CONCLUSIONS: In summary, we identified several GCF proteins associated with GR after orthodontic treatment. These findings could contribute to the prevention of GR in susceptible patients before the initiation of orthodontic treatment. SIGNIFICANCE: Orthodontic patients with GR often report esthetic defects or root hypersensitivity during orthodontic treatment, especially at the anterior teeth site. GCF, rich in protein, is an easily accessible source of potential biomarkers for the diagnosis of periodontal diseases; however, little is known about the changes in GCF proteome associated with GR in orthodontic patients. In this study we firstly used DIA-based LC-MS/MS to evaluate the proteome and to identify the biomarker candidates for GR in orthodontic patients. These findings will improve our understanding of GR during orthodontic treatment, and could contribute to an earlier diagnosis, or even prevention, of GR in susceptible populations before orthodontic treatment.

Current understanding of how odors impact intra-sex social behavior is based on those that increase intermale aggression. Yet, odors are often promoted to reduce fighting among male laboratory mice. It has been shown that a cage of male mice contains many proteins used for identification purposes. However, it is unknown if these proteins relate to social behavior or if they are uniformly produced across strains. This study aimed to compare proteomes from used nesting material and three sources (sweat, saliva, and urine) from three strains and compare levels of known protein odors with rates of social behavior. Used nesting material samples from each cage were analyzed using LC-MS/MS. Sweat, saliva, and urine samples from each cage's dominant and subordinate mouse were also analyzed. Proteomes were assessed using principal component analyses and compared to behavior by calculating correlation coefficients between PC scores and behavior proportions. Twenty-one proteins from nesting material either correlated with affiliative behavior or negatively correlated with aggression. Notably, proteins from the major urinary protein family, odorant binding protein family, and secretoglobin family displayed at least one of these patterns, making them candidates for future work. These findings provide preliminary information about how proteins can influence male mouse behavior. SIGNIFICANCE: Research on how olfactory signals influence same sex social behavior is primarily limited to those that promote intermale aggression. However, exploring how olfaction modulates a more diverse behavioral repertoire will improve our foundational understanding of this sensory modality. In this proteome analysis we identified a short list of protein signals that correspond to lower rates of aggression and higher rates of socio-positive behavior. While this study is only correlational, it sets a foundation for future work that can identify protein signals that directly influence social behavior and potentially identify new murine pheromones.

To gain a comprehensive and unbiased molecular understanding of the different skin colors of P. leopardus, we used Illumina HiSeq 2500 and TMT (Tandem Mass Tag) to compare transcription and protein levels between red and black skin of P. leopardus. We identified 797 upregulated and 314 downregulated genes (differentially expressed genes; DEGs) in red (RG) compared with black (BG) skin of P. leopardus. We also identified 377 differentially abundant proteins (DAPs), including 314 upregulated and 63 downregulated proteins. These DEGs and DAPs were significantly enriched in melanin synthesis (e.g., pyrimidine metabolism, Phenylalanine, tyrosine, and tryptophan biosynthesis, melanogenesis, phenylalanine metabolism, and tyrosine metabolism), oxidative phosphorylation (e.g., phosphonate and phosphinate metabolism, and oxidative phosphorylation), energy metabolism (e.g., HIF-1, glycolysis/gluconeogenesis, fatty acid biosynthesis, and fatty acid degradation), and signal transduction (e.g., Wnt, calcium, MAPK, and cGMP-PKG signaling pathways), etc. Further analysis of MAPKs showed that the activation levels of its main members JNK1 and ERK1/2 differed significantly between red and black skin colors. After RNAi was used to interfere with ERK1/2, it was found that the local skin of the tail of P. leopardus would turn black. Combined transcriptome and proteome analysis showed that most DEGs-DAPs in red skin were higher than in black skin (58 were upregulated, 1 was downregulated, and 4 were opposite). These DEGs-DAPs showed that the differences between red and black skin tissues of P. leopardus were related primarily to energy metabolism, signal transduction and cytoskeleton. These findings are not only conducive to understand the skin color regulation mechanism of P. leopardus and other coral reef fish, but also provide an important descriptive to the breeding of color strains. SIGNIFICANCE OF THE STUDY: The skin color of P. leopardus gradually darkens or blackens due to environmental factors such as changes in light intensity and human activities, and this directly affects its ornamental and economic value. In this study, RNAseq and TMT were used to conduct comparative quantitative transcriptomics and proteomics and analyze differences between red and black P. leopardus skin. The results showed that energy metabolism, signal transduction and cytoskeleton were the main metabolic pathways causing their skin color differences. These findings contribute to existing data describing fish skin color, and provide information about protein levels, which are of great significance to a deeper understanding of the skin color regulation mechanism in P. leopardus and other coral reef fishes.

BACKGROUND: Endometriosis (EM) leads to a decline in fertility, which is characterized by a decrease in the number and quality of follicles, and thus has a negative impact on in vitro fertilization (IVF) outcomes. However, the mechanism of how EM affects oocytes and leads to infertility remains unclear. As a potentially available sample directly related to oocyte growth, follicular fluid (FF) has important research value. Evaluating the association of FF content and EM-associated infertility through proteomics may helpful to explore the possible pathogenesis of EM-associated infertility. METHODS: In the present experimental study, from August 2019 to June 2020, FF samples were obtained as control group (CON-G; n = 10) from women with no one female factor of infertility and were undergoing IVF due to other reasons, 20 women with EM-associated infertility undergoing IVF with no other female factors were distributed into the EM group according to the time for IVF: (i) EM-group 1 (EM-G1, Stage I to Stage III, n = 10); (ii) EM-group 2 (EM-G2, Stage I to Stage III, n = 10). label-free quantitative proteomics (LFQP) technology and parallel reaction monitoring (PRM) approach were combined to aid in identifying and validating FF protein biomarkers for EM-associated infertility. In PRM analysis, another 20 subjects were enrolled as EM-associated infertility group (EM,Stage I to Stage III, n = 10) and controls (CON, n = 10) within the same time and inclusion criteria are the same as previously described. Finally, a potential protein biomarker panel of FF differential expressed proteins to EM-associated infertility was also evaluated by t-test and receiver operating characteristic (ROC) curve and binary Logistic regression models. RESULTS: 7 significant differential expressed proteins which closely related to EM-associated infertility were found by LFQP technology, among which immunoglobulin lambda variable 7-46 (IGLV7-46), Immunoglobulin heavy constant gamma 2 (IGHG2), glia-derived nexin (GDN) and Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) were significantly up-regulated (p < 0.05), while corticosteroid-binding globulin (CBG), angiotensinogen (AGT) and Fetuin-B (FETUB) were significantly down regulated (p < 0.05). Additionally, GDN and AGT was identified as a potential protein biomarker by further PRM analysis for EM-associated infertility according to ROC curve analysis and t-test (p < 0.05), the area under the curve (AUC) for GDN and AGT was 0.78 and 0.69 with optimum sensitivity of 50%, 70% and specificity of 100%, 90%, respectively. According to binary logistic regression and evaluated ROC analysis, the AUC for the combination of GDN and AGT was 0.80. CONCLUSIONS: To the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the FF of patients with EM-associated infertility. Combining LFQP technology and PRM method we found the abnormal of GDN and AGT in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility. Further experimental studies are required to confirm their mechanism in EM-associated infertility. The results of this study are also consistent with the previous conclusion that EM is a chronic inflammatory disease. SIGNIFICANCE: To the best of our knowledge, this is the first time that elevated GDN protein levels have been found in the follicular fluid of patients with EM-associated infertility. Combining LFQP technology and PRM methods we found the abnormal of GDN and AGT protein in FF may be the potential cause of EM-associated infertility which may help to better understand the physiological and pathological mechanism of EM-associated infertility. Clinically, it has been recognized that EM is related to infertility, but the mechanism remains unclear. Our study combines label-free quantitative proteomics technology and parallel reaction monitoring methods to identify and verify the FF protein biomarkers of EM-associated infertility, which provides a good research method for follow-up research.

Candida albicans biofilms are characterized by structural and cellular heterogeneity that confers antifungal resistance and immune evasion. Despite this, biofilm formation remains poorly understood. In this study, we used proteomic analysis to understand biofilm formation in C. albicans related to morphophysiological and architectural features. LC-MS/MS analysis revealed that 64 proteins were significantly modulated, of which 31 were upregulated and 33 were downregulated. The results indicate that metabolism (25 proteins), gene expression (13 proteins), stress response (7 proteins), and cell wall (5 proteins) composition are modulated. The rate of oxidative phosphorylation (OxPhos) and biosynthesis of UDP-N-acetylglucosamine, vitamin B6, and thiamine increased, while the rate of methionine biosynthesis decreased. There was a significant modification of the cell wall architecture due to higher levels of Sun41, Pir1 and Csh1 and increased glycosylation of proteins. It was observed that C. albicans induces hyphal growth by upregulating the expression of genes involved in cAMP-PKA and MAPK pathways. This study is significant in that it suggests an increase in OxPhos and alteration of cell wall architecture that could be contributing to the recalcitrance of C. albicans cells growing in biofilms. Nevertheless, a deeper investigation is needed to explore it further. SIGNIFICANCE: Candida sps is included in the list of pathogens with potential drug resistance threat due to the increased frequency especially colonization of medical devices, and tissues among the patients, in recent years. Significance of our study is that we are reporting traits like modulation in cell wall composition, amino acid and vitamin biosynthesis and importantly energy generation (OxPhos) etc. These traits could be conferring antifungal resistance, host immune evasion etc. and thus survival, in addition to facilitating biofilm formation. These findings are expected to prime the further studies on devising potent strategy against biofilm growth among the patients.

The freshwater pearl is one kind of valuable organic jewelry and traditional Chinese medicine (TCM). However, the molecular basis of matrix protein in pearl biomineralization and biomedical applications are largely unknown to date. In this study, the matrix proteins of water-soluble matrix, acid-soluble matrix and acid-insoluble matrix from the freshwater seedless pearl powder were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) respectively, and identified against the transcriptomic database of the pearl sac. The results showed that a total of 190 proteins were identified in pearl proteomics, which was divided into eight categories by their potential biomineralization functions. The composition of pearl matrix proteins and the high frequency conserved domains like carbonic anhydrase, von Willebrand factor type A, tyrosinase and chitin binding 2 in protein sequences, implying that the "chitin-silk fibroin gel proteins-acidic macromolecules" model was suitable for description the pearl biomineralization process. Meanwhile, ninety-one of pearl matrix proteins could be classified into seven categories by their potential medical functions including wound healing, osteogenic property, antioxidant activity, neuro-regulation effects, skin lightening effect, anti-inflammatory and anti-apoptotic effects and other immunomodulatory property. In general, these results provided valuable new insights into not only the diversity of pearl matrix protein for mollusc biomineralization, but the molecular basis of pearl matrix proteins responsible for their diverse biological properties in TCM application. SIGNIFICANCE: The significance of this study included the following points.

The 2020 global cancer registry has ranked breast cancer (BCa) as the most commonly diagnosed type of cancer and the most common cause of cancer-related deaths in women worldwide. Increasing resistance and significant side effects continue to limit the efficacy of anti-BCa drugs, hence the need to identify new drug targets and to develop novel compounds to overcome these limitations. Nature-inspired anti-cancer compounds are becoming increasingly popular since they often provide a relatively safe and effective alternative. In this study, we employed multi-omics techniques to gain insights into the relevant mechanism of action of two recently identified new nature-inspired anti-cancer compounds (SIMR3066 and SIMR3058). Discovery proteomics analysis combined with LC-MS/MS-based untargeted metabolomics analysis was performed on compound-treated vs DMSO-treated (control) MCF-7 cells. Downstream protein functional enrichment analysis showed that most of the responsive proteins were functionally associated with antigen processing and neutrophil degranulation, RNA catabolism and protein folding as well as cytoplasmic vesicle lumen and mitochondrial matrix formation. Consistent with the proteomics findings, metabolomic pathway analysis suggested that the differentially abundant compounds indicated altered metabolic pathways such as glycolysis, the Krebs cycle and oxidative phosphorylation. Furthermore, metabolomics-based enriched-for-action pathway analysis showed that the two compounds associate with mercaptopurine, thioguanine and azathioprine related pathways. Lastly, integrated proteomics and metabolomics analysis revealed that treatment of BCa with SIMR3066 disrupts several signaling pathways including p53-mediated apoptosis and the circadian entertainment pathway. Overall, the multi-omics approach we used in this study indicated that it is a powerful tool in probing the mechanism of action of lead drug candidates. SIGNIFICANCE: In this study we adopted a multi-omics (proteomics and metabolomics) strategy to learn more about the molecular mechanisms of action of nature-inspired potential anticancer drugs. Following treatment with SIMR3066 or SIMR3058, the integration of these multi-omics data sets revealed which biological pathways are altered in BCa cells. This study demonstrates that combining proteomics with metabolomics is a powerful method to investigate the mechanism of action of potential anticancer lead drug candidates.

Mitochondria remain active in postmortem muscles and can influence meat color via oxygen consumption. Previous studies have shown that dark-cutting compared with normal-pH beef has greater mitochondrial protein and DNA content per gram of muscle tissue. However, the mechanism regulating mitochondrial content in dark-cutting vs. normal-pH beef is still unknown. Therefore, the objective was to compare mitochondrial proteomes of dark-cutting vs. normal-pH beef using LC-MS/MS-based proteomics and mitochondrial respiratory capacity using a Clark oxygen electrode. Dark-cutting compared with normal-pH beef has up-regulation of proteins involved in mitochondrial biogenesis, oxidative phosphorylation, intracellular protein transport, and cellular calcium ion homeostasis. Mitochondria isolated from dark-cutting phenotypes showed greater mitochondrial complex II respiration and uncoupled oxidative phosphorylation. However, mitochondrial membrane integrity and respiration at complexes I and IV were not different between normal-pH and dark-cutting beef. These results indicate that dark-cutting beef has greater mitochondrial biogenesis proteins than normal-pH beef, increasing mitochondrial content and contributing to dark-cutting beef. SIGNIFICANCE: Defective glycogen metabolism resulting from chronic stress before slaughter coupled with the greater mitochondrial protein and DNA content per gram of muscle tissue promotes muscle darkening in dark-cutting phenotypes in beef. However, the mechanistic basis for this occurrence in dark-cutting phenotypes is still unknown. In this work, we show that dark-cutting beef phenotype is caused, in part, as a consequence of over-proliferation of mitochondria. This is supported by the up-regulation of proteins involved in mitochondrial biogenesis, mitochondrial electron transport, calcium homeostasis, and fatty acid metabolism. Hence, the study of mitochondrial proteome changes provides a set of mitochondrial biogenesis proteins that could be used as potential candidate markers for detecting changes in pre-slaughter developmental events contributing to dark-cutting phenotypes in beef.

Adipose tissue not only affects meat quality and animal productivity, but also participates in inflammation and immunity. Ningxiang pig is famous for their excellent meat quality, disease resistance and tolerance of roughage. It is not yet well known how proteins in adipose tissue is dynamically regulated during the growth of Ningxiang pig. This report studies the proteomic changes in subcutaneous adipose tissue in Ningxiang pigs to gain a better understanding of the molecular mechanism of fat development during the growth period. By TMT-labeled quantitative proteomic analysis of subcutaneous adipose tissue of 9 purebred Ningxiang pigs of different ages, we identified 2533 unique proteins and 716 differentially abundant proteins (DAPs), of which more than half of the DAPs were concentrated in the 90d-210d period. Retrograde endocannabinoid signaling was only significantly enriched in DAPs of N90d vs N30d, Alcoholism and Graft-versus-host disease were only significantly enriched in DAPs of N210d vs N90d. Proteins related to dilated cardiomyopathy was found to be an important pathway in fat development and lipid metabolism. A variety of novel DAPs involved in maintaining mitochondrial function and cell viability, such as NDUFS6, SDHB, COX5A, ATP5D and TNNT1, which play a role in controlling the prediction networks, may indirectly regulate the development and functional maintenance of adipocytes. SIGNIFICANCE: These age-dependent DAPs discovered in this study may help expand the understanding of the molecular mechanisms of the development, function maintenance and transformation of adipose tissue in Ningxiang pig for developing new strategies for improving meat quality and pig breeding in the future.

Lactobacillus rhamnosus can metabolize selenite into organic selenium and Se0. In this paper, label-free quantitative proteomics was applied to explore the mechanism of salt stress promoting selenium enrichment of L.rhamnosus. 397 proteins were up-regulated and 147 proteins were down-regulated of selenium-enriched L.rhamnosus under salt stress. The differentially expressed proteins (DEPs) were mainly involved in metabolism, membrane transport and genetic information processing. The results of quantitative real-time PCR showed that gene opuA, metN, trxR and ldh of Se-enriched L.rhamnosus with salt stress were significantly up-regulated. However, the expression levels of gene luxS, groEL, dnaK and pgk were down-regulated. It was indicated that L.rhamnosus promoted part of amino acids combining with selenium into selenoamino acids with salt stress. Secondly, sodium chloride stimulated the expression of key enzymes involved in metabolism to provide energy for the process of Se-enrichment. In addition, NaCl induced the expression of enzymes and genes involved in the synthesis of selenoproteins. SIGNIFICANCE: It was indicated that L.rhamnosus promoted part of amino acids combining with selenium into selenoamino acids with salt stress. Secondly, sodium chloride stimulated the expression of key enzymes involved in metabolism to provide energy for the process of Se-enrichment. In addition, NaCl induced the expression of enzymes and genes involved in the synthesis of selenoproteins.

The Xinjiang Uygur autonomous region has a high incidence of esophageal cancer. For the early diagnosis of patients with esophageal squamous cell carcinoma (ESCC), exosomes were isolated and quantified by liquid chromatography tandem mass spectrometry ((LC-MS/MS) with data independent acquisition (DIA) from the peripheral blood of patients with benign esophageal disease (BED), esophageal intraepithelial neoplasia (EIN) and ESCC. A total of 1117 proteins were identified in the above 9 samples. The proteomic results showed that the quantity of CD82 in exosomes of EIN was significantly higher than that in patients with BED and ESCC. Meanwhile, our ELISA test verified our proteomic results. In addition, the immunohistochemical results showed high CD82 expression in adjacent normal tissues and low expression in ESCC tissues. CD82 expression in ESCC tissues was negatively correlated with tumor stage and the expression of PKM2, and the high expression of CD82 combined with low expression of PKM2 in ESCC tissues suggested a good prognosis. To further clarify the tumor suppressive mechanism of CD82, the TIMER and TISDB databases were analyzed, and CD82 expression in tumor tissues was found to be related to the infiltration of immune cells. CD82 in exosomes is involved in the development of ESCC. SIGNIFICANCE: Xinjiang is a high incidence area of ESCC. When diagnosed in the middle and late stages of the disease, the prognosis of patients is poor. Exosomes provide the possibility of relatively noninvasive and early detection of esophageal carcinogenesis. To the best of our knowledge, this was the first study using the DIA technique to analyze the exosomal proteins of patients with different stages of ESCC. The proteins identified in the exosomes in these three groups could provide insights for understanding how exosomes promote the occurrence of ESCC, the antitumour mechanism of humans and the early diagnosis of ESCC.

Assessment of pain responses and inflammation during animal surgery is difficult because traditional methods, such as visual analogue scores, are not applicable while under anaesthesia. Acute phase proteins (APPs), such as C-reactive protein and haptoglobin, that are typically monitored in veterinary research, do not show a significant change until at least 2 h post-surgery and therefore, immediate pathophysiological changes are uncertain. The current study used sequential window acquisition of all theoretical mass spectra (SWATH-MS) to investigate plasma proteome changes that occur immediately following surgery in dogs and also to assess the efficacy of a novel transdermal ketoprofen (TK) formulation. Castration was chosen as surgical model in this study. The procedure was performed on twelve dogs (n = 6 in two groups) and blood samples were collected at 0 h, 1 and 2 h after surgery for proteomic analysis. Following surgery, there was a general downregulation of proteins, including complement C- 3, complement factor B, complement factor D, transthyretin, and proteins associated with lipid, cholesterol, and glucose metabolisms, reflecting the systemic response to surgical trauma. Many of these changes were diminished in the transdermal group (TD) since ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), inhibits prostanoids and the associated chemotactic neutrophil migration to site of tissue injury. SIGNIFICANCE: SWATH-MS Proteomic analysis revealed significant changes in plasma proteins, predominantly involved in early acute phase and inflammatory response at 1 & 2 h after surgery in castrated dogs. Pre-operative application of transdermal ketoprofen formulation had reduced the systemic immune response, which was confirmed by negligible alteration of proteins in transdermal treated group. A key outcome of this experiment was studying the efficacy of a novel transdermal NSAID formulation in dogs.

The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing and early detection, prevention as well as surveillance of the disease. We investigated whether targeted parallel reaction monitoring (PRM) quantification using high resolution Orbitrap instruments can provide the sensitivity and speed required for a high-throughput method that could be used for clinical diagnosis. We developed a high-throughput and sensitive PRM-MS assay that enables absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times by using isotopically labelled synthetic SARS-CoV-2 concatenated peptides. We established a fast and high-throughput S-trap-based sample preparation method and utilized it for testing 25 positive and 25 negative heat-inactivated clinical nasopharyngeal swab samples for SARS-CoV-2 detection. The method was able to differentiate between negative and some of the positive patients with high viral load. Moreover, based on the absolute quantification calculations, our data show that patients with Ct values as low as 17.8 correspond to NCAP protein amounts of around 7.5 pmol in swab samples. The present high-throughput method could potentially be utilized in specialized clinics as an alternative tool for detection of SARS-CoV-2 but will require enrichment of viral proteins in order to compete with RT-qPCR.

Incident light is a central modulator of plant growth and development. However, there are still open questions surrounding wavelength-specific plant proteomic responses. Here we applied tandem mass tag based quantitative proteomics technology to acquire an in-depth view of proteome changes in Arabidopsis thaliana response to narrow wavelength blue (B; 450 nm), amber (A; 595 nm), and red (R; 650 nm) light treatments. A total of 16,707 proteins were identified with 9120 proteins quantified across all three light treatments in three biological replicates. This enabled examination of changes in the abundance for proteins with low abundance and important regulatory roles including transcription factors and hormone signaling. Importantly, 18% (1631 proteins) of the A. thaliana proteome is differentially abundant in response to narrow wavelength lights, and changes in proteome correlate well with different morphologies exhibited by plants. To showcase the usefulness of this resource, data were placed in the context of more than thirty published datasets, providing orthogonal validation and further insights into light-specific biological pathways, including Systemic Acquired Resistance and Shade Avoidance Syndrome. This high-resolution resource for A. thaliana provides baseline data and a tool for defining molecular mechanisms that control fundamental aspects of plant response to changing light conditions, with implications in plant development and adaptation. SIGNIFICANCE: Understanding of molecular mechanisms involved in wavelength-specific response of plant is question of widespread interest both to basic researchers and to those interested in applying such knowledge to the engineering of novel proteins, as well as targeted lighting systems. Here we sought to generate a high-resolution proteomic profile of plant leaves, based on exposure to specific narrow-wavelength lights. Although changes in plant physiology in response to light spectral composition is well documented, there is limited knowledge on the roles of specific light wavelengths and their impact. Most previous studies have utilized relatively broad wavebands in their experiments. Such multi-wavelengths lights trigger diverse and complex signaling networks that pose major challenges in inference of wavelength-specific molecular processes that underly the plant response. Moreover, most studies have compared the effect of blue and red wavelengths comparing with FL, as control. As FL light consists the mixed spectra composition of both red and blue as well as numerous other wavelengths, comparing undeniably results in inconsistent and overlapping responses that will hamper effects to elucidate the plant response to specific wavelengths [1, 2]. Monitoring plant proteome response to specific wavelengths and further contrasting the changes with one another, rather than comparing plants proteome to FL, is thus necessary to gain detailed insights on underlying biological pathways and their consequences in plant physiology. Here, we employed narrow wavelength LED lights in our design to eliminate a potential overlap in molecular responses by ensuring non-overlapping wavelengths in the light treatments. We further applied TMT-labeling technology to gain a high-resolution view on the proteome changes. Our proteomics data provides an in-depth coverage suitable for system-wide analyses, providing deep insights on plant molecular response particularly because of the tremendous increase in the coverage of identified proteins which outreach the other biological data.

Silkworm is an economically important insect due to its efficient production of silk proteins. Silk itself and the silk trade have enriched human civilization through art and culture and contributed to early globalization in the Silk Road era for nearly two thousand years. Although a large number of studies on silk have been carried out, the mechanism of silk secretion in silkworms has not been thoroughly studied thus far. As the main component of fibroin, fibroin light chain (Fib-L) plays a key role in the secretion of silk. In this study, we constructed a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. Strikingly, fibrohexamerin (P25) was secreted almost normally in the homozygous mutant. Further analysis of cocoon proteins revealed that the mutant responded to greater environmental stress caused by a dramatic decrease in fibroin by significantly increasing the secretion of protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm. SIGNIFICANCE: This study generated a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. The analysis of the abundance of silk proteins in the cocoons revealed that P25 could be secreted almost normally. The analysis of the abundance of cocoon proteins other than silk proteins showed that the homozygous mutants responded to greater environmental stress by increasing the secretion of defense-related proteins, such as protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm.