Analysis of serum peptidome profiles of non-metastatic and metastatic feline mammary carcinoma using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Feline mammary carcinoma (FMC) is a common aggressive and highly metastatic cancer affecting female cats. Early detection is essential for preventing local and distant metastasis, thereby improving overall survival rates. While acquiring molecular data before surgery offers significant potential benefits, the current protein biomarkers for monitoring disease progression in non-metastatic FMC (NmFMC) and metastatic FMC (mFMC) are limited. The objective of this study was to investigate the serum peptidome profiles of NmFMC and mFMC using liquid chromatography-tandem mass spectrometry. A cross-sectional study was conducted to compare serum peptidome profiles in 13 NmFMC, 23 mFMC and 18 healthy cats. The liquid chromatography-tandem mass spectrometry analysis was performed on non-trypsinized samples. RESULTS: Out of a total of 8284 expressed proteins observed, several proteins were found to be associated with human breast cancer. In NmFMC, distinctive protein expressions encompassed double-stranded RNA-binding protein Staufen homolog 2 (STAU2), associated with cell proliferation, along with bromodomain adjacent to zinc finger domain 2A (BAZ2A) and gamma-aminobutyric acid type A receptor subunit epsilon (GABRE), identified as potential treatment targets. Paradoxically, positive prognostic markers emerged, such as complement C1q like 3 (C1QL3) and erythrocyte membrane protein band 4.1 (EPB41 or 4.1R). Within the mFMC group, overexpressed proteins associated with poor prognosis were exhibited, including B-cell lymphoma 6 transcription repressor (BCL6), thioredoxin reductase 3 (TXNRD3) and ceruloplasmin (CP). Meanwhile, the presence of POU class 5 homeobox (POU5F1 or OCT4) and laminin subunit alpha 1 (LAMA1), reported as metastatic biomarkers, was noted. CONCLUSION: The presence of both pro- and anti-proliferative proteins was observed, potentially indicating a distinctive characteristic of NmFMC. Conversely, proteins associated with poor prognosis and metastasis were noted in the mFMC group.

Comparison of proteomic profiles and biological properties for corneal wound healing of canine amniotic membrane and its extracts.

OBJECTIVE: The application of canine amniotic membrane (cAM) for corneal reconstruction is widely used in the veterinary field. However, the information on biological properties and alternative forms of cAM for corneal wound healing is limited. This study aimed to investigate the proteomic profiles and corneal wound healing properties of cAM, cAM extract (cAME), and lyophilized cAM extract (cAMX). ANIMAL STUDIED: A total number of 14 cAMs were sterilely harvested from healthy full-term puppies and randomly divided into three different forms: cAM (n = 14), cAME (n = 14), and cAMX (n = 14). PROCEDURES: Each form of cAMs was subjected to proteomic analysis using label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), followed by bioinformatic analysis. The proteins were classified into properties by comparing them with the literature search on human amniotic membrane (hAM) properties and the effect on corneal wound healing when given topically. RESULTS: The analyses identified 8136 proteins in cAM, 8211 proteins in cAME, and 7093 proteins in cAMX. A total number of 100 proteins were matched with proteins in hAM properties and were classified into anti-inflammatory, anti-fibrotic, anti-microbial, anti-angiogenic, promotion of epithelialization, analgesic, and support cell adhesion and growth properties. Furthermore, proteins with corneal wound healing effects were identified in cAME and cAMX. CONCLUSIONS: cAM and its extracts contain numerous proteins, including proteins related to corneal wound healing properties. Additionally, cAME and cAMX showed proteins involved in corneal wound healing and their potential benefits for topical use in ophthalmology.

Palmelloid Formation and Cell Aggregation Are Essential Mechanisms for High Light Tolerance in a Natural Strain of Chlamydomonas reinhardtii.

Photosynthetic organisms, such as higher plants and algae, require light to survive. However, an excessive amount of light can be harmful due to the production of reactive oxygen species (ROS), which cause cell damage and, if it is not effectively regulated, cell death. The study of plants' responses to light can aid in the development of methods to improve plants' growth and productivity. Due to the multicellular nature of plants, there may be variations in the results based on plant age and tissue type. Chlamydomonas reinhardtii, a unicellular green alga, has also been used as a model organism to study photosynthesis and photoprotection. Nonetheless, the majority of the research has been conducted with strains that have been consistently utilized in laboratories and originated from the same source. Despite the availability of many field isolates of this species, very few studies have compared the light responses of field isolates. This study examined the responses of two field isolates of Chlamydomonas to high light stress. The light-tolerant strain, CC-4414, managed reactive oxygen species (ROS) slightly better than the sensitive strain, CC-2344, did. The proteomic data of cells subjected to high light revealed cellular modifications of the light-tolerant strain toward membrane proteins. The morphology of cells under light stress revealed that this strain utilized the formation of palmelloid structures and cell aggregation to shield cells from excessive light. As indicated by proteome data, morphological modifications occur simultaneously with the increase in protein degradation and autophagy. By protecting cells from stress, cells are able to continue to upregulate ROS management mechanisms and prevent cell death. This is the first report of palmelloid formation in Chlamydomonas under high light stress.

FBPAII and rpoBC, the Two Novel Secreted Proteins Identified by the Proteomic Approach from a Comparative Study between Antibiotic-Sensitive and Antibiotic-Resistant Helicobacter pylori-Associated Gastritis Strains.

Helicobacter pylori infection is the leading cause of chronic gastritis, which can develop into gastric cancer. Eliminating H. pylori infection with antibiotics achieves the prevention of gastric cancer. Currently, the prevalence of H. pylori resistance to clarithromycin and metronidazole, and the dual resistance to metronidazole and clarithromycin (C_R, M_R, and C/M_R, respectively), remains at a high level worldwide. As a means of exploring new candidate proteins for the management of H. pylori infection, secreted proteins from antibiotic-susceptible and antibiotic-resistant H. pylori-associated gastritis strains were obtained by in-solution tryptic digestion coupled with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). A total of 583, 582, 590, and 578 differential expressed proteins were identified from C_R, M_R, C/M_R, and antibiotic-sensitive strain (S_S) samples, respectively. Of these, 23 overlapping proteins were found by Venn diagram analysis. Based on heat map analyses, the most and least differing protein expressions were observed from C/M_R strains and S_S strains, respectively. Of the proteins secreted by the S_S strain, only nine were found. After predicting the protein interaction with metronidazole and clarithromycin via the STITCH database, the two most interesting proteins were found to be rpoBC and FBPAII. After quantitative real-time reverse transcription PCR (qRT-PCR) analysis, a downregulation of rpoB from M_R strains was observed, suggesting a relationship of rpoB to metronidazole sensitivity. Inversely, an upregulation of fba from C_R, M_R, and C/M_R strains was noticed, suggesting the paradoxical expression of FBPAII and the fba gene. This report is the first to demonstrate the association of these two novel secreted proteins, namely, rpoBC and FBPAII, with antibiotic-sensitive H. pylori-associated gastritis strains.

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines.

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

Fungicidal Activity of Recombinant Javanicin against Cryptococcus neoformans Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes.

The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.

Protein secretion in wild-type and Othac1 mutant strains of thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC-MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors.

BACKGROUND: Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis. RESULTS: For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5-drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group. CONCLUSION: PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Insights into stress responses in mandarins triggered by Bacillus subtilis cyclic lipopeptides and exogenous plant hormones upon Penicillium digitatum infection.

KEY MESSAGE: Bacillus subtilis CLP extract activates defense gene expression and increases the unique protein production involving in pathways of ISR, SAR, ubiquitin-proteasome system, and glycolysis for stress responses in flavedo tissues. Cyclic lipopeptides (CLPs) of Bacillus subtilis ABS-S14 had ability to activate plant defensive pathways, increase resistance and control green mold rot caused by Penicillium digitatum in mandarin fruit. The current study investigated transcriptional and proteomic data to highlight the unique induction effect of CLPs produced by B. subtilis ABS-S14 on the defense mechanism of mandarins in response to P. digitatum attack, and their differences from those following the exogenous plant hormone application. The proteomic patterns of the flavedo tissues as affected by Bacillus CLP extract, salicylic acid (SA), methyl jasmonate (MeJA), and ethephon (Et) were explored. qPCR analysis revealed the great effects of CLP extract in enhancing the transcription of PAL, ACS1, GLU, POD, and PR1. Tryptic peptides by LC-MS analysis between treatments with and without fungal infection were compared. B. subtilis CLP extract empowered the plant's immune response to wound stress by the significant production of calmodulin-binding receptor-like cytoplasmic kinase 2, molybdenum cofactor sulfurase, and NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase. Ubiquitin carrier protein abundance was developed only in the treated flavedo with CLP extract coupled with P. digitatum infection. The gene expression and overall proteome findings involving pathways of ubiquitin proteasome system, ISR, SAR, and energy production provide a new insight into the molecular mechanisms of the antagonist B. subtilis ABS-S14 inducing resistance against green mold in mandarins.

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.

Hac1 function revealed by the protein expression profile of a OtHAC1 mutant of thermotolerant methylotrophic yeast Ogataea thermomethanolica.

In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.

Proteolytic effects of gingipains on trefoil factor family peptides.

OBJECTIVES: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis. MATERIALS AND METHODS: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides. RESULTS: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin. CONCLUSIONS: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides. CLINICAL RELEVANCE: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.

Comparative proteomic analysis of Chlamydomonas reinhardtii control and a salinity-tolerant strain revealed a differential protein expression pattern.

MAIN CONCLUSION: Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as proteolytic enzymes, were remarkably up-regulated in the salinity-tolerant strain of Chlamydomonas reinhardtii. Excessive concentration of NaCl in the environment can cause adverse effects on plants and microalgae. Successful adaptation of plants to long-term salinity stress requires complex cellular adjustments at different levels from molecular, biochemical and physiological processes. In this study, we developed a salinity-tolerant strain (ST) of the model unicellular green alga, Chlamydomonas reinhardtii, capable of growing in medium containing 300 mM NaCl. Comparative proteomic analyses were performed to assess differential protein expression pattern between the ST and the control progenitor cells. Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as protein degradation, were remarkably up-regulated in the ST cells, suggesting the importance of these processes in acclimation mechanisms to salinity stress. Moreover, 2-DE-based proteomic also revealed putative salinity-specific post-translational modifications (PTMs) on several important housekeeping proteins. Discussions were made regarding the roles of these differentially expressed proteins and the putative PTMs in cellular adaptation to long-term salinity stress.

Evidence of plasticity in the dengue virus: Host cell interaction.

Dengue virus (DENV) is the most important mosquito transmitted human viral pathogen. There are four different dengue viruses (DENV 1 to DENV 4) with multiple genotypes and strains. Whether there are significant differences in how these DENVs interact with and modulate the host cell proteome remains unclear. Using a panel of 12 DENVs representative of one isolate for each DENV from three different origins (lab adapted, low passage isolates from dengue fever patients, low passage isolates from dengue hemorrhagic fever patients) LLC-MK2 cells were equally infected and proteomic alterations compared by MALDI-TOF and principal component analysis and a sub-10 kDa peptidome analysis. There was no clear segregation of data with respect to either virus origin or serotype in either the MALDI-TOF or the peptidome analysis. The two isolates with the greatest variation from the other isolates in the MALDI-TOF analysis were a low passage DENV 3 dengue fever isolate and a low passage DENV 4 dengue hemorrhagic fever isolate. Analysis of the sub-10 kda protein fraction by LC-MS/MS identified 128 proteins of which only 28 (20%) were constantly expressed in all infections, while 80% showed variable expression, with no clear relationship with either serotype or virus origin. These results suggest that the interaction between DENV and the host cell is characterized by a degree of plasticity, whereby the end biological processes are not rigorously determined by specific proteome alterations, and that virus strain plays a role in determining the specific proteome changes.

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

KEY MESSAGE: Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

Comparative evaluation of 5-15-kDa salivary proteins from patients with different oral diseases by MALDI-TOF/TOF mass spectrometry.

OBJECTIVES: The present study aimed to determine the potential use of matrix-assisted laser desorption/ionization with time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for analyzing specific patterns of mass signals of low-molecular-weight proteins in saliva from patients with different oral diseases. MATERIALS AND METHODS: Unstimulated whole saliva samples were collected from healthy subjects (n = 30) and patients with oral diseases including oral cancer (n = 30), oral lichen planus (n = 30), and chronic periodontitis (n = 30). Proteomic profiles of 5,000-15,000-Da salivary proteins were evaluated by MALDI-TOF/TOF MS. Quantification of mass signals was performed by FlexAnalysis and ClinProTool software. RESULTS: In oral cancer, the percentages of mass signals at 5,592.26 and 8,301.46 Da were significantly increased as compared with other groups (p = 0.002 and p = 0.030, respectively). In oral lichen planus, the percentages of mass signals at 12,964.55 and 13,279.08 Da were significantly increased as compared with other groups (p < 0.001, and p < 0.001, respectively). In chronic periodontitis, the percentages of mass signals at 5,835.73 and 9,801.83 Da were significantly decreased as compared with other groups (p = 0.003 and p = 0.005, respectively). CONCLUSIONS: The present study demonstrated a potential use of MALDI-TOF/TOF as a rapid screening method to differentiate one oral disease from others by identifying specific patterns of mass signals in saliva from patients. However, MALDI-TOF/TOF has several limitations regarding the identification of the candidate mass signals. CLINICAL RELEVANCE: MALDI-TOF/TOF MS can be used as a rapid screening method to differentiate one oral disease from others with a caution concerning peptide identity.

Involvement of ATP synthase ß subunit in chikungunya virus entry into insect cells.

Chikungunya virus (CHIKV), the virus responsible for the disease chikungunya fever in humans, is transmitted by Aedes mosquitoes. While significant progress has been made in understanding the process by which CHIKV enters into mammalian cells, far less progress has been made in understanding the CHIKV entry process in insect cells. This study sought to identify mosquito-cell-expressed CHIKV-binding proteins through a combination of virus overlay protein binding assays (VOPBA) and mass spectroscopy. A 50-kDa CHIKV-binding protein was identified as the ATP synthase ß subunit (ATPSß). Co-immunoprecipitation studies confirmed the interaction, and colocalization analysis showed cell-surface and intracellular co-localization between CHIKV and ATPSß. Both antibody inhibition and siRNA-mediated downregulation experiments targeted to ATPSß showed a significant reduction in viral entry and virus production. These results suggest that ATPSß is a CHIKV-binding protein capable of mediating the entry of CHIKV into insect cells.