The Potential of Eight Plasma Proteins as Biomarkers in Redefining Leptospirosis Diagnosis.

Leptospirosis, a notifiable endemic disease in Malaysia, has higher mortality rates than regional dengue fever. Diverse clinical symptoms and limited diagnostic methods complicate leptospirosis diagnosis. The demand for accurate biomarker-based diagnostics is increasing. This study investigated the plasma proteome of leptospirosis patients with leptospiraemia and seroconversion compared with dengue patients and healthy subjects using isobaric tags for relative and absolute quantitation (iTRAQ)-mass spectrometry (MS). The iTRAQ analysis identified a total of 450 proteins, which were refined to a list of 290 proteins through a series of exclusion criteria. Differential expression in the plasma proteome of leptospirosis patients compared to the control groups identified 11 proteins, which are apolipoprotein A-II (APOA2), C-reactive protein (CRP), fermitin family homolog 3 (FERMT3), leucine-rich alpha-2-glycoprotein 1 (LRG1), lipopolysaccharide-binding protein (LBP), myosin-9 (MYH9), platelet basic protein (PPBP), platelet factor 4 (PF4), profilin-1 (PFN1), serum amyloid A-1 protein (SAA1), and thrombospondin-1 (THBS1). Following a study on a verification cohort, a panel of eight plasma protein biomarkers was identified for potential leptospirosis diagnosis: CRP, LRG1, LBP, MYH9, PPBP, PF4, SAA1, and THBS1. In conclusion, a panel of eight protein biomarkers offers a promising approach for leptospirosis diagnosis, addressing the limitations of the "one disease, one biomarker" concept.

Isoglutaminyl Cyclase Overexpression Enhances KYSE30 Cancer Cell Proliferation and Migration via the MAPK Signaling Pathway.

To understand how upregulated isoglutaminyl cyclase (isoQC) is involved in the initiation of diseases such as cancer, we developed a human KYSE30 carcinoma cell model in which isoQC was stably overexpressed. GO and KEGG analysis of the DEGs (228) and DEPs (254) respectively implicated isoQC on the proliferation invasion and metastasis of cells and suggested that isoQC might participate in the regulation of MAPK, RAS, circadian rhythm, and related pathways. At the functional level, isoQC-overexpressing KYSE30 cells showed enhanced proliferation, migration, and invasion capacity. Next, we decided to study the precise effect of isoQC overexpression on JNK, p-JNK, AKT, p-AKT, ERK, p-ERK, and PER2, as RNA levels of these proteins are significantly correlated with signal levels indicated in RNA-Seq analysis, and these candidates are the top correlated DEPs enriched in RT-qPCR analysis. We saw that only p-ERK expression was inhibited, while PER2 was increased. These phenotypes were inhibited upon exposure to PER2 inhibitor KL044, which allowed for the restoration of p-ERK levels. These data support upregulated isoQC being able to promote cancer cell proliferation and migration in vitro, likely by helping to regulate the MAPK and RAS signaling pathways, and the circadian protein PER2 might be a potential mediator.

Proteo-metabolomic Dissection of Extracellular Matrix Reveals Alterations in Cell Wall Integrity and Calcium Signaling Governs Wall-Associated Susceptibility during Stem Rot Disease in Jute.

The plant surveillance system confers specificity to disease and immune states by activating distinct molecular pathways linked to cellular functionality. The extracellular matrix (ECM), a preformed passive barrier, is dynamically remodeled at sites of interaction with pathogenic microbes. Stem rot, caused by Macrophomina phaseolina, adversely affects fiber production in jute. However, how wall related susceptibility affects the ECM proteome and metabolome remains undetermined in bast fiber crops. Here, stem rot responsive quantitative temporal ECM proteome and metabolome were developed in jute upon M. phaseolina infection. Morpho-histological examination revealed that leaf shredding was accompanied by reactive oxygen species production in patho-stressed jute. Electron microscopy showed disease progression and ECM architecture remodeling due to necrosis in the later phase of fungal attack. Using isobaric tags for relative and absolute quantitative proteomics and liquid chromatography-tandem mass spectrometry, we identified 415 disease-responsive proteins involved in wall integrity, acidification, proteostasis, hydration, and redox homeostasis. The disease-related correlation network identified functional hubs centered on α-galactosidase, pectinesterase, and thaumatin. Gas chromatography-mass spectrometry analysis pointed toward enrichment of disease-responsive metabolites associated with the glutathione pathway, TCA cycle, and cutin, suberin, and wax metabolism. Data demonstrated that wall-degrading enzymes, structural carbohydrates, and calcium signaling govern rot responsive wall-susceptibility. Proteomics data were deposited in Pride (PXD046937; PXD046939).

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Adaptive response of Pseudomonas aeruginosa under serial ciprofloxacin exposure.

Understanding the evolution of antibiotic resistance is important for combating drug-resistant bacteria. In this work, we investigated the adaptive response of Pseudomonas aeruginosa to ciprofloxacin. Ciprofloxacin-susceptible P. aeruginosa ATCC 9027, CIP-E1 (P. aeruginosa ATCC 9027 exposed to ciprofloxacin for 14 days) and CIP-E2 (CIP-E1 cultured in antibiotic-free broth for 10 days) were compared. Phenotypic responses including cell morphology, antibiotic susceptibility, and production of pyoverdine, pyocyanin and rhamnolipid were assessed. Proteomic responses were evaluated using comparative iTRAQ labelling LC-MS/MS to identify differentially expressed proteins (DEPs). Expression of associated genes coding for notable DEPs and their related regulatory genes were checked using quantitative reverse transcriptase PCR. CIP-E1 displayed a heterogeneous morphology, featuring both filamentous cells and cells with reduced length and width. By contrast, although filaments were not present, CIP-E2 still exhibited size reduction. Considering the MIC values, ciprofloxacin-exposed strains developed resistance to fluoroquinolone antibiotics but maintained susceptibility to other antibiotic classes, except for carbapenems. Pyoverdine and pyocyanin production showed insignificant decreases, whereas there was a significant decrease in rhamnolipid production. A total of 1039 proteins were identified, of which approximately 25 % were DEPs. In general, there were more downregulated proteins than upregulated proteins. Noted changes included decreased OprD and PilP, and increased MexEF-OprN, MvaT and Vfr, as well as proteins of ribosome machinery and metabolism clusters. Gene expression analysis confirmed the proteomic data and indicated the downregulation of rpoB and rpoS. In summary, the response to CIP involved approximately a quarter of the proteome, primarily associated with ribosome machinery and metabolic processes. Potential targets for bacterial interference encompassed outer membrane proteins and global regulators, such as MvaT.

The polyextremophile Natranaerobius thermophilus adopts a dual adaptive strategy to long-term salinity stress, simultaneously accumulating compatible solutes and K.

The bacterium Natranaerobius thermophilus is an extremely halophilic alkalithermophile that can thrive under conditions of high salinity (3.3-3.9 M Na+), alkaline pH (9.5), and elevated temperature (53°C). To understand the molecular mechanisms of salt adaptation in N. thermophilus, it is essential to investigate the protein, mRNA, and key metabolite levels on a molecular basis. Based on proteome profiling of N. thermophilus under 3.1, 3.7, and 4.3 M Na+ conditions compared to 2.5 M Na+ condition, we discovered that a hybrid strategy, combining the "compatible solute" and "salt-in" mechanisms, was utilized for osmotic adjustment dur ing the long-term salinity adaptation of N. thermophilus. The mRNA level of key proteins and the intracellular content of compatible solutes and K+ support this conclusion. Specifically, N. thermophilus employs the glycine betaine ABC transporters (Opu and ProU families), Na+/solute symporters (SSS family), and glutamate and proline synthesis pathways to adapt to high salinity. The intracellular content of compatible solutes, including glycine betaine, glutamate, and proline, increases with rising salinity levels in N. thermophilus. Additionally, the upregulation of Na+/ K+/ H+ transporters facilitates the maintenance of intracellular K+ concentration, ensuring cellular ion homeostasis under varying salinities. Furthermore, N. thermophilus exhibits cytoplasmic acidification in response to high Na+ concentrations. The median isoelectric points of the upregulated proteins decrease with increasing salinity. Amino acid metabolism, carbohydrate and energy metabolism, membrane transport, and bacterial chemotaxis activities contribute to the adaptability of N. thermophilus under high salt stress. This study provides new data that support further elucidating the complex adaptation mechanisms of N. thermophilus under multiple extremes.IMPORTANCEThis study represents the first report of simultaneous utilization of two salt adaptation mechanisms within the Clostridia class in response to long-term salinity stress.

iTRAQ-based proteomic study on monocyte cell model discovered an association of LAMP2 downregulation with HIV-1 latency.

BACKGROUND: Patients with immunodeficiency virus-1 (HIV-1) infection are challenging to be cured completely due to the existence of HIV-1 latency reservoirs. However, the knowledge of the mechanisms and biomarkers associated with HIV-1 latency is limited. Therefore, identifying proteins related to HIV-1 latency could provide new insights into the underlying mechanisms of HIV-1 latency, and ultimately contribute to the eradication of HIV reservoirs. METHODS: An Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-labeled subcellular proteomic study was performed on an HIV-1 latently infected cell model (U1, a HIV-1-integrated U937 cell line) and its control (U937). Differentially expressed proteins (DEPs) were analyzed using STRING-DB. Selected DEPs were further evaluated by western blotting and multiple reaction monitoring technology in both cell model and patient-derived cluster of differentiation 4 (CD4)+ T cells. Finally, we investigated the relationship between a specific DEP lysosome-associated membrane glycoprotein 2 (LAMP2) and HIV-1 reactivation by panobinostat or lysosome regulation by a lysosomotropic agent hydroxychloroquine in U1 and U937 cells. RESULTS: In total, 110 DEPs were identified in U1 cells comparing to U937 control cells. Bioinformatics analysis suggested associations of the altered proteins with the immune response and endosomal/lysosomal pathway. LAMP2, leukocyte surface antigen CD47, CD55, and ITGA6 were downregulated in HIV-1 latent cells. Downregulated LAMP2 was further confirmed in resting CD4+ T cells from patients with latent HIV-1 infection. Furthermore, both HIV-1 reactivation by panobinostat and stimulation with hydroxychloroquine upregulated LAMP2 expression. CONCLUSIONS: Our results indicated the involvement of the endosomal/lysosomal pathway in HIV-1 latency in macrophage cell model. The down-modulation of LAMP2 was associated with HIV latency, and the restoration of LAMP2 expression accompanied the transition of viral latency to active infection. This study provides new insights into the mechanism of HIV-1 latency and potential strategies for eradicating HIV-1 reservoirs by targeting LAMP2 expression.

Differentially Expressed Proteins in the Serum of Elderly Patients Who Experienced Perioperative Neurocognitive Disorders Following Transurethral Resection of the Prostate.

OBJECTIVE: Perioperative neurocognitive disorders (PND) are a group of prevalent neurological complications that often occur in elderly individuals following major or emergency surgical procedures. The etiologies are not fully understood. This study endeavored to investigate novel targets and prediction methods for the occurrence of PND. METHODS: A total of 229 elderly patients diagnosed with prostatic hyperplasia who underwent transurethral resection of the prostate (TURP) combined with spinal cord and epidural analgesia were included in this study. The patients were divided into two groups, the PND group and non-PND group, based on the Z-score method. According to the principle of maintaining consistency between preoperative and intraoperative conditions, three patients from each group were randomly chosen for serum sample collection. isobaric tags for relative and absolute quantification (iTRAQ) proteomics technology was employed to analyze and identify the proteins that exhibited differential expression in the serum samples from the two groups. Bioinformatics analysis was performed on the proteins that exhibited differential expression. RESULTS: Among the 1101 serum proteins analyzed in the PND and non-PND groups, eight differentially expressed proteins were identified in PND patients. Of these, six proteins showed up-regulation, while two proteins showed down-regulation. Further bioinformatics analysis of the proteins that exhibited differential expression revealed their predominant involvement in cellular biological processes, cellular component formation, as well as endocytosis and phagocytosis Additionally, these proteins were found to possess the RING domain of E3 ubiquitin ligase. CONCLUSION: The iTRAQ proteomics technique was employed to analyze the variation in protein expression in serum samples from patients with PND and those without PND. This study successfully identified eight proteins that exhibited differential expression levels between the two groups. Bioinformatics analysis indicates that proteins exhibiting differential expression are primarily implicated in the biological processes associated with microtubules. Investigating the microtubule formation process as it relates to neuroplasticity and synaptic formation may offer valuable insights for enhancing our comprehension and potential prevention of PND. CLINICAL TRIAL REGISTRATION: Registered (ChiCTR2000028836). Date (20190306).

Impact of miR-29c-3p in the Nucleus Accumbens on Methamphetamine-Induced Behavioral Sensitization and Neuroplasticity-Related Proteins.

Methamphetamine (METH) abuse inflicts both physical and psychological harm. While our previous research has established the regulatory role of miR-29c-3p in behavior sensitization, the underlying mechanisms and target genes remain incompletely understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique in conjunction with Ingenuity pathway analysis (IPA) to probe the putative molecular mechanisms of METH sensitization through miR-29c-3p inhibition. Through a microinjection of AAV-anti-miR-29c-3p into the nucleus accumbens (NAc) of mice, we observed the attenuation of METH-induced locomotor effects. Subsequent iTRAQ analysis identified 70 differentially expressed proteins (DEPs), with 22 up-regulated potential target proteins identified through miR-29c-3p target gene prediction and IPA analysis. Our focus extended to the number of neuronal branches, the excitatory synapse count, and locomotion-related pathways. Notably, GPR37, NPC1, and IREB2 emerged as potential target molecules for miR-29c-3p regulation, suggesting their involvement in the modulation of METH sensitization. Quantitative PCR confirmed the METH-induced aberrant expression of Gpr37, Npc1, and Ireb2 in the NAc of mice. Specifically, the over-expression of miR-29c-3p led to a significant reduction in the mRNA level of Gpr37, while the inhibition of miR-29c-3p resulted in a significant increase in the mRNA level of Gpr37, consistent with the regulatory principle of miRNAs modulating target gene expression. This suggests that miR-29c-3p potentially influences METH sensitization through its regulation of neuroplasticity. Our research indicates that miR-29c-3p plays a crucial role in regulating METH-induced sensitization, and it identified the potential molecular of miR-29c-3p in regulating METH-induced sensitization.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Exosomes Secreted by Wharton's Jelly-Derived Mesenchymal Stem Cells Promote the Ability of Cell Proliferation and Migration for Keratinocyte.

Mesenchymal stem cells (MSCs) isolated from Wharton's jelly (WJ-MSCs) and adipose tissue (AD-MSCs) are alternative sources for bone marrow-derived MSCs. Owing to their multiple functions in angiogenesis, immune modulation, proliferation, migration, and nerve regeneration, MSC-derived exosomes can be applied in "cell-free cell therapy". Here, we investigated the functional protein components between the exosomes from WJ-MSCs and AD-MSCs to explain their distinct functions. Proteins of WJ-MSC and AD-MSC exosomes were collected and compared based on iTRAQ gel-free proteomics data. Results: In total, 1695 proteins were detected in exosomes. Of these, 315 were more abundant (>1.25-fold) in AD-MSC exosomes and 362 kept higher levels in WJ-MSC exosomes, including fibrinogen proteins. Pathway enrichment analysis suggested that WJ-MSC exosomes had higher potential for wound healing than AD-MSC exosomes. Therefore, we treated keratinocyte cells with exosomes and the recombinant protein of fibrinogen beta chain (FGB). It turned out that WJ-MSC exosomes better promoted keratinocyte growth and migration than AD-MSC exosomes. In addition, FGB treatment had similar results to WJ-MSC exosomes. The fact that WJ-MSC exosomes promoted keratinocyte growth and migration better than AD-MSC exosomes can be explained by their higher FGB abundance. Exploring the various components of AD-MSC and WJ-MSC exosomes can aid in their different clinical applications.

Improvement of the viability of Tetragenococcus halophilus under acidic stress by forming the biofilm cell structure based on RNA-Seq and iTRAQ analyses.

BACKGROUND: Tetragenococcus halophilus is a halophilic lactic acid bacterium (LAB) isolated from soya sauce moromi. During the production of these fermented foods, acid stress is an inevitable environmental stress. In our previous study, T. halophilus could form biofilms and the cells in the biofilms exhibited higher cell viability under multiple environmental stresses, including acid stress. RESULTS: In this study, the effect of preformed T. halophilus biofilms on cell survival, cellular structure, intracellular environment, and the expression of genes and proteins under acid stress was investigated. The result showed that acid stress with pH 4.30 for 1.5 h reduced the live T. halophilus cell count and caused cellular structure damage. However, T. halophilus biofilm cells exhibited greater cell survival under acid stress than the planktonic cells, and biofilm formation reduced the damage of acid stress to the cell membrane and cell wall. The biofilm cells maintained a higher level of H+ -ATPase activity and intracellular ammonia concentration after acid stress. The RNA-Seq and iTRAQ technologies revealed that the genes and proteins associated with ATP production, the uptake of trehalose and N-acetylmuramic acid, the assembly of H+ -ATPase, amino acid biosynthesis and metabolism, ammonia production, fatty acid biosynthesis, CoA biosynthesis, thiamine production, and acetoin biosynthesis might be responsible for the stronger acid tolerance of T. halophilus biofilm cells together. CONCLUSION: These findings further explained the mechanisms that allowed LAB biofilm cells to resist environmental stress. © 2023 Society of Chemical Industry.

iTRAQ-based proteomic analysis reveals proteins associated with cold stress response in two different populations of Manila clam (Ruditapes philippinarum).

Water temperature is one of the key environmental factors for marine ectotherms and a change in temperature beyond and organism's capacity limits can cause a series of changes to physiological state and damage to the organism. Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. Ruditapes philippinarum is an ecologically and scientifically important marine bivalve species. To uncover the molecular mechanisms of acclimation of R. philippinarum to low-temperature stress, iTRAQ-based quantitative proteomics was conducted to compare the proteomes of the north and south populations of R. philippinarum under low-temperature stress. The results showed a total of 6355 and 6352 proteins were identified in two populations, respectively. Among these, 94 and 83 were differentially abundant proteins (DAPs), and most of DAPs were related to oxidation-process, protein binding, or an integral component of membrane. According to the results of KEGG pathway enrichment analysis, most of DAPs in both populations are involved in immune-related pathways, while other population-specific significant abundance proteins of south population and north population were enriched in biosynthesis of amino acids (Enolase, Glutamine synthetase) and unsaturated fatty acids pathways (3-ketoacyl-CoA thiolase, Stearoyl-CoA desaturase), respectively, indicating that two population of clams may have different cold-stress regulation mechanisms. Our study provides new insights into different cold stress tolerance mechanisms in northern and southern populations of R. philippinarum using iTRAQ-based proteomics. This work contributes to a better understanding of molecular basis on cold stress response and adaptations, which shed lights on evolutionary biology and general ecophysiology of R. philippinarum.

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

Vinculin Identified as a Potential Biomarker in Hand-Arm Vibration Syndrome Based on iTRAQ and LC-MS/MS-Based Proteomic Analysis.

Local vibration can induce vascular injuries, one example is the hand-arm vibration syndrome (HAVS) caused by hand-transmitted vibration (HTV). Little is known about the molecular mechanism of HAVS-induced vascular injuries. Herein, the iTRAQ (isobaric tags for relative and absolute quantitation) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was applied to conduct the quantitative proteomic analysis of plasma from specimens with HTV exposure or HAVS diagnosis. Overall, 726 proteins were identified in iTRAQ. 37 proteins upregulated and 43 downregulated in HAVS. Moreover, 37 upregulated and 40 downregulated when comparing severe HAVS and mild HAVS. Among them, Vinculin (VCL) was found to be downregulated in the whole process of HAVS. The concentration of vinculin was further verified by ELISA, and the results suggested that the proteomics data was reliable. Bioinformative analyses were used, and those proteins mainly engaged in specific biological processes like binding, focal adhesion, and integrins. The potential of vinculin application in HAVS diagnosis was validated by the receiver operating characteristic curve.

iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm.

BACKGROUND: Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. RESULTS: 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which expressed 1.72-fold higher in good freezability ejaculate (GFE) compared to poor freezability ejaculate (PFE) sperms, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1 mg/L PRDX6 was added compared with blank control. CONCLUSIONS: Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.

iTRAQ-Based Proteomics Analysis of Human Cytomegalovirus Latency and Reactivation in T98G Cells.

Human cytomegalovirus (HCMV) establishes a persistent/latent infection after primary infection, and the host factor(s) plays a key role in regulating HCMV infection status. The spread of reactivated HCMV via the hematogenous or neural route usually results in severe diseases in newborns and immunocompromised individuals. As the primary reservoirs in vivo, cells of myeloid lineage have been utilized extensively to study HCMV infection. However, the molecular mechanism of HCMV latency/reactivation in neural cells is still poorly understood. We previously showed that HCMV-infected T98G cells maintain a large number of viral genomes and support HCMV reactivation from latency upon cAMP/IBMX treatment. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics to characterize cellular protein changes during HCMV latency and reactivation in T98G cells. A total of 168 differentially expressed proteins (DEPs) were identified, including 89 proteins in latency and 85 proteins in reactivation. Bioinformatics analysis showed that a few biological pathways were associated with HCMV latency or reactivation. Moreover, we validated 16 DEPs by both mRNA and protein expression profiles and further evaluated the effects of ApoE and the phosphatidylinositol 3-kinase (PI3K) pathway on HCMV infection. ApoE knockdown reduced HCMV loads and virus release, whereas overexpressing ApoE hampered HCMV latent infection, indicating a role in HCMV latency establishment/maintenance. Blocking the PI3K pathway by LY294002, a PI3K inhibitor, induced HCMV reactivation from latency in T98G cells. Overall, this comparative proteomics analysis delineates the cellular protein changes during HCMV latency and reactivation and provides a road map to advance our understanding of the mechanism(s) in the context of neural cells. IMPORTANCE Human cytomegalovirus (HCMV) is a highly transmissible betaherpesvirus that has a prevalence of 60% to 90% worldwide. This opportunist pathogen poses a significant threat to newborns and immunosuppressed individuals. One major obstacle for developing effective therapeutics is a poor understanding of HCMV latency/reactivation mechanisms. This study presents, for the first time, a systemic analysis of host cell protein expression changes during HCMV latency establishment and reactivation processes in neural cells. We showed that ApoE was downregulated by HCMV to facilitate latent infection. Also, the proteomics analysis has associated a few PI3K pathway-related proteins with HCMV reactivation. Altogether, this study highlights multiple host proteins and signaling pathways that can be further investigated as potential druggable targets for HCMV-related diseases, especially brain disorders.

Comparative proteomic and physiological analyses reveal tribenuron-methyl phytotoxicity and nontarget-site resistance mechanisms in Brassica napus.

The application of herbicides is the most effective strategy for weed control and the development of herbicide-resistant crops will facilitate the weed management. The acetolactate synthase-inhibiting herbicide, tribenuron-methyl (TBM), is broadly used for weed control. However, its application in rapeseed field is restricted since rapeseed is sensitive to TBM. Herein, an integrated study of cytological, physiological and proteomic analysis of the TBM-resistant rapeseed mutant M342 and its wild-type (WT) plants was conducted. After TBM spraying, M342 showed improved tolerance to TBM, and proteins implicated in non-target-site resistance (NTSR) to herbicides had a significantly higher level in M342 as compared with the WT. Differentially accumulated proteins (DAPs) between these two genotypes were enriched in glutathione metabolism and oxidoreduction coenzyme metabolic process, which protected the mutant from oxidative stress triggered by TBM. Important DAPs related to stress or defence response were up-accumulated in M342 regardless of the TBM treatment, which might serve as the constitutive part of NTSR to TBM. These results provide new clues for further exploration of the NTSR mechanism in plants and establish a theoretical basis for the development of herbicide-resistant crops.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.