Unraveling the molecular dynamics of Pseudomonas aeruginosa biofilms at the air-liquid interface.

Aim: The aim of this study was to probe the dynamics of Pseudomonas aeruginosa PA14 air-liquid interface (ALI) biofilms over time through global proteomic analysis. Materials & methods: P. aeruginosa PA14 ALI biofilm samples, collected over 48-144 h, underwent differential expression analysis to identify varying proteins at each time point. Results: A consistent set of 778 proteins was identified, with variable expression over time. Upregulated proteins were mainly linked to 'amino acid transport and metabolism'. Biofilm-related pathways, including cAMP/Vfr and QS, underwent significant changes. Flagella were more influential than pili, especially in early biofilm development. Proteins associated with virulence, transporters and iron showed differential expression throughout. Conclusion: The findings enhance our understanding of ALI biofilm development.

This study looks at how the bacteria Pseudomonas aeruginosa forms a community called a biofilm at the air­liquid interface (ALI), an important environment for bacterial growth. Biofilms at the ALI are resistant to external forces and contribute to antibiotic resistance. Over 48­144 h, we observed a noticeable increase in biofilm thickness. Our data suggested that the flagella, a sort of propeller of the bacterium, plays a crucial role, especially in the initial stages of ALI biofilm formation. Proteins associated with virulence, transporters and iron also showed their significance in ALI biofilms. These findings offer valuable insights into the protein changes and functions involved in P. aeruginosa ALI biofilms, improving our understanding of biofilm development.

Distinct proteomes and allergen profiles appear across the life cycle stages of Alternaria alternata.

BACKGROUND: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. OBJECTIVE: To determine differences between spore and hyphae proteomes and how allergens are distributed in A. alternata. METHODS: Data independent acquisition mass spectrometry to quantitatively compare the proteomes of asexual spores (non-germinating and germinating) with vegetative hyphae. RESULTS: We identified 4515 proteins in non-germinating spores, germinating spores and hyphae; most known allergens are more abundant in non-germinating spores. Comparing significant protein fold change differences between non-germinating spores and hyphae, 174 proteins were upregulated in non-germinating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. Comparing non-germinating and germinating spores, 25 proteins were upregulated in non-germinating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. CONCLUSION: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in non-germinating spores.

An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage, Quantitative Accuracy and Precision.

Tandem mass tags (TMT) are widely used in proteomics to simultaneously quantify multiple samples in a single experiment. The tags can be easily added to the primary amines of peptides/proteins through chemical reactions. In addition to amines, TMT reagents also partially react with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the analytical sensitivity and precision. Under alkaline conditions, reducing the TMT molar excess can partially mitigate overlabeling of histidine-free peptides, but has a limited effect on peptides containing histidine and hydroxyl groups. Here, we present a method under acidic conditions to suppress overlabeling while efficiently labeling amines, using only one-fifth of the TMT amount recommended by the manufacturer. In a deep-scale analysis of a yeast/human two-proteome sample, we systematically evaluated our method against the manufacturer's method and a previously reported TMT-reduced method. Our method reduced overlabeled peptides by 9-fold and 6-fold, respectively, resulting in the substantial enhancement in peptide/protein identification rates. More importantly, the quantitative accuracy and precision were improved as overlabeling was reduced, endowing our method with greater statistical power to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively. Mass spectrometric data have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD047052.

Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Metabolic and inflammatory perturbation of diabetes associated gut dysbiosis in people living with and without HIV infection.

BACKGROUND: Gut dysbiosis has been linked with both HIV infection and diabetes, but its interplay with metabolic and inflammatory responses in diabetes, particularly in the context of HIV infection, remains unclear. METHODS: We first conducted a cross-sectional association analysis to characterize the gut microbial, circulating metabolite, and immune/inflammatory protein features associated with diabetes in up to 493 women (~ 146 with prevalent diabetes with 69.9% HIV +) of the Women's Interagency HIV Study. Prospective analyses were then conducted to determine associations of identified metabolites with incident diabetes over 12 years of follow-up in 694 participants (391 women from WIHS and 303 men from the Multicenter AIDS Cohort Study; 166 incident cases were recorded) with and without HIV infection. Mediation analyses were conducted to explore whether gut bacteria-diabetes associations are explained by altered metabolites and proteins. RESULTS: Seven gut bacterial genera were identified to be associated with diabetes (FDR-q < 0.1), with positive associations for Shigella, Escherichia, Megasphaera, and Lactobacillus, and inverse associations for Adlercreutzia, Ruminococcus, and Intestinibacter. Importantly, the associations of most species, especially Adlercreutzia and Ruminococcus, were largely independent of antidiabetic medications use. Meanwhile, 18 proteins and 76 metabolites, including 3 microbially derived metabolites (trimethylamine N-oxide, phenylacetylglutamine (PAGln), imidazolepropionic acid (IMP)), 50 lipids (e.g., diradylglycerols (DGs) and triradylglycerols (TGs)) and 23 non-lipid metabolites, were associated with diabetes (FDR-q < 0.1), with the majority showing positive associations and more than half of them (59/76) associated with incident diabetes. In mediation analyses, several proteins, especially interleukin-18 receptor 1 and osteoprotegerin, IMP and PAGln partially mediate the observed bacterial genera-diabetes associations, particularly for those of Adlercreutzia and Escherichia. Many diabetes-associated metabolites and proteins were altered in HIV, but no effect modification on their associations with diabetes was observed by HIV. CONCLUSION: Among individuals with and without HIV, multiple gut bacterial genera, blood metabolites, and proinflammatory proteins were associated with diabetes. The observed mediated effects by metabolites and proteins in genera-diabetes associations highlighted the potential involvement of inflammatory and metabolic perturbations in the link between gut dysbiosis and diabetes in the context of HIV infection.

Integrated Transcriptome and Proteome Analysis Reveals That Cell Wall Activity Affects Phelipanche aegyptiaca Parasitism.

Phelipanche aegyptiaca can infect many crops, causing large agricultural production losses. It is important to study the parasitism mechanism of P. aegyptiaca to control its harm. In this experiment, the P. aegyptiaca HY13M and TE9M from Tacheng Prefecture and Hami City in Xinjiang, respectively, were used to analyze the parasitical mechanism of P. aegyptiaca by means of transcriptome and proteome analyses. The parasitic capacity of TE9M was significantly stronger than that of HY13M in Citrullus lanatus. The results showed that the DEGs and DEPs were prominently enriched in the cell wall metabolism pathways, including "cell wall organization or biogenesis", "cell wall organization", and "cell wall". Moreover, the functions of the pectinesterase enzyme gene (TR138070_c0_g), which is involved in the cell wall metabolism of P. aegyptiaca in its parasitism, were studied by means HIGS. The number and weight of P. aegyptiaca were significantly reduced when TR138070_c0_g1, which encodes a cell-wall-degrading protease, was silenced, indicating that it positively regulates P. aegyptiaca parasitism. Thus, these results suggest that the cell wall metabolism pathway is involved in P. aegyptiaca differentiation of the parasitic ability and that the TR138070_c0_g1 gene plays an important role in P. aegyptiaca's parasitism.

Exposure to 7,12-dimethylbenz[a]anthracene impacts ovarian DNA damage sensing and repair proteins differently in lean and obese female mice and weight loss may mitigate obesity-induced ovarian dysfunction.

Obesity impairs oocyte quality, fertility, pregnancy maintenance, and is associated with offspring birth defects. The model ovotoxicant, 7,12-dimethylbenz[a]anthracene (DMBA), causes ovarian DNA damage and follicle loss. Both DMBA-induced chemical biotransformation and the DNA damage response are partially attenuated in obese relative to lean female mice but whether weight loss could improve the DNA damage response to DMBA exposure has not been explored. Thus, at six weeks of age, C57BL/6 J female mice were divided in three groups: 1) Lean (L; n = 20) fed a chow diet for 12 weeks, 2) obese (O; n = 20) fed a high fat high sugar (HFHS) diet for 12 weeks and, 3) slim-down (S; n = 20). The S group was fed with HFHS diet for 7 weeks until attaining a higher body relative to L mice on week 7.5 and switched to a chow diet for 5 weeks to achieve weight loss. Mice then received either corn oil (CT) or DMBA (D; 1 mg/kg) for 7 d via intraperitoneal injection (n = 10/treatment). Obesity increased (P < 0.05) kidney and spleen weight, and DMBA decreased uterine weight (P < 0.05). Ovarian weight was reduced (P < 0.05) in S mice, but DMBA exposure increased ovary weight in the S mice. LC-MS/MS identified 18, 64, and 7 ovarian proteins as altered (P < 0.05) by DMBA in the L, S and O groups, respectively. In S and O mice, 24 and 8 proteins differed, respectively, from L mice. These findings support weight loss as a strategy to modulate the ovarian genotoxicant response.

Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

Frontiers in Plasma Proteome Profiling Platforms: Innovations and Applications.

Biomarkers play a crucial role in advancing precision medicine by enabling more targeted and individualized approaches to diagnosis and treatment. Various biofluids, including serum, plasma, cerebrospinal fluid (CSF), saliva, tears, pancreatic cyst fluids, and urine, have been identified as rich sources of potential for the early detection of disease biomarkers in conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders. The analysis of plasma and serum in proteomics research encounters challenges due to their high complexity and the wide dynamic range of protein abundance. These factors impede the sensitivity, coverage, and precision of protein detection when employing mass spectrometry, a widely utilized technology in discovery proteomics. Conventional approaches such as neat plasma workflow are inefficient in accurately quantifying low-abundant proteins, including those associated with tissue leakage, immune response molecules, interleukins, cytokines, and interferons. Moreover, the manual nature of the workflow poses a significant hurdle in conducting large cohort studies. In this study, our focus is on comparing workflows for plasma proteomic profiling to establish a methodology that is not only sensitive and reproducible but also applicable for large cohort studies in biomarker discovery. Our investigation revealed that the SeerProteographXT workflow outperforms other workflows in terms of plasma proteome depth, quantitative accuracy, and reproducibility while offering complete automation of sample preparation. Notably, SeerProteographXT demonstrates versatility by applying it to various types of biofluids. Additionally, the proteins quantified widely cover secretory proteins in peripheral blood, and the pathway analysis enriched with relevant components such as interleukins, tissue necrosis factors, chemokines, and B and T cell receptors provides valuable insights. These proteins, often challenging to quantify in complex biological samples, hold potential as early detection markers for various diseases, thereby contributing to the improvement of patient care quality.

Differences in the Synovial Fluid Proteome of Septic and Aseptic Implant Failure.

Implant loosening is a severe complication after total joint replacement. Here, differential diagnosis between septic and aseptic cases is crucial for further surgical treatment, but low-grade periprosthetic joint infections (PJIs) in particular remain a challenge. In this study, we analyzed the synovial fluid proteome of 21 patients undergoing revision surgery for septic (eight cases) or aseptic (thirteen cases) implant failure using LC-MS/MS to identify potential new biomarkers as future diagnostic tools. Staphylococci were found in four cases, Streptococci in two cases, Serratia marcescens and Cutibacterium acnes in one case. Proteomic analysis of the synovial fluid resulted in the identification of 515 different proteins based on at least two peptides. A statistical comparison revealed 37 differentially abundant proteins (p < 0.05), of which 17 proteins (46%) showed a higher abundance in the septic group. The proteins with the highest fold change included the known marker proteins c-reactive protein (7.57-fold) and the calprotectin components protein S100-A8 (4.41-fold) and protein S100-A9 (3.1-fold). However, the protein with the highest fold change was leucine-rich alpha-2-glycoprotein 1 (LRG1) (9.07-fold), a currently discussed new biomarker for inflammatory diseases. Elevated LRG1 levels could facilitate the diagnosis of PJI in the future, but their significance needs to be further investigated.

Scientometrics Evaluation of Published Scientific Papers on the Use of Proteomics Technologies in Mastitis Research in Ruminants.

The objective of this study was the presentation of quantitative characteristics regarding the scientific content and bibliometric details of the relevant publications. In total, 156 papers were considered. Most papers presented original studies (n = 135), and fewer were reviews (n = 21). Most original articles (n = 101) referred to work involving cattle. Most original articles described work related to the diagnosis (n = 72) or pathogenesis (n = 62) of mastitis. Most original articles included field work (n = 75), whilst fewer included experimental (n = 31) or laboratory (n = 30) work. The tissue assessed most frequently in the studies was milk (n = 59). Milk was assessed more frequently in studies on the diagnosis (61.1% of relevant studies) or pathogenesis (30.6%) of the infection, but mammary tissue was assessed more frequently in studies on the treatment (31.0%). In total, 47 pathogens were included in the studies described; most were Gram-positive bacteria (n = 34). The three bacteria most frequently included in the studies were Staphylococcus aureus (n = 55 articles), Escherichia coli (n = 31) and Streptococcus uberis (n = 19). The proteomics technology employed more often in the respective studies was liquid chromatography-tandem mass spectrometry (LC-MS/MS), either on its own (n = 56) or in combination with other technologies (n = 40). The median year of publication of articles involving bioinformatics or LC-MS/MS and bioinformatics was the most recent: 2022. The 156 papers were published in 78 different journals, most frequently in the Journal of Proteomics (n = 16 papers) and the Journal of Dairy Science (n = 12). The median number of cited references in the papers was 48. In the papers, there were 1143 co-authors (mean: 7.3 ± 0.3 co-authors per paper, median: 7, min.-max.: 1-19) and 742 individual authors. Among them, 15 authors had published at least seven papers (max.: 10). Further, there were 218 individual authors who were the first or last authors in the papers. Most papers were submitted for open access (n = 79). The median number of citations received by the 156 papers was 12 (min.-max.: 0-339), and the median yearly number of citations was 2.0 (min.-max.: 0.0-29.5). The h-index of the papers was 33, and the m-index was 2. The increased number of cited references in papers and international collaboration in the respective study were the variables associated with most citations to published papers. This is the first ever scientometrics evaluation of proteomics studies, the results of which highlighted the characteristics of published papers on mastitis and proteomics. The use of proteomics in mastitis research has focused on the elucidation of pathogenesis and diagnosis of the infection; LC-MS/MS has been established as the most frequently used proteomics technology, although the use of bioinformatics has also emerged recently as a useful tool.

Characterization of the Mitochondrial Proteome in the Ctenophore Mnemiopsis leidyi Using MitoPredictor.

Mitochondrial proteomes have been experimentally characterized for only a handful of animal species. However, the increasing availability of genomic and transcriptomic data allows one to infer mitochondrial proteins using computational tools. MitoPredictor is a novel random forest classifier, which utilizes orthology search, mitochondrial targeting signal (MTS) identification, and protein domain content to infer mitochondrial proteins in animals. MitoPredictor's output also includes an easy-to-use R Shiny applet for the visualization and analysis of the results. In this article, we provide a guide for predicting and analyzing the mitochondrial proteome of the ctenophore Mnemiopsis leidyi using MitoPredictor.

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept: a quantitative analysis based on 4D label-free technique.

AIM: To identify different metabolites, proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy (PDR) and resistance to anti-vascular endothelial growth factor (VEGF) drugs, and to provide biomarkers for the diagnosis and treatment of PDR. METHODS: Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analyses based on 4D label-free technology. Statistically differentially expressed proteins (DEPs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representation and protein interactions were analyzed. RESULTS: A total of 12 samples were analyzed. The proteomics results showed that a total of 58 proteins were identified as DEPs, of which 47 proteins were up-regulated and 11 proteins were down-regulated. We found that C1q and tumor necrosis factor related protein 5 (C1QTNF5), Clusterin (CLU), tissue inhibitor of metal protease 1 (TIMP1) and signal regulatory protein alpha (SIRPα) can all be specifically regulated after aflibercept treatment. GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR. In addition, protein-protein interaction (PPI) network evaluation revealed that TIMP1 plays a central role in neural regulation. In addition, CD47/SIRPα may become a key target to resolve anti-VEGF drug resistance in PDR. CONCLUSION: Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR. Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept, among which C1QTNF5, CLU, TIMP1 and SIRPα may become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad", defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington's and Alzheimer's disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, DNA and accessory structures of the sperm head and tail. Introduction of next generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Proteome, lysine acetylome and succinylome identify post-translational modification of STAT1 as a novel drug target in silicosis.

Inhalation of crystalline silica dust induces incurable lung damage, silicosis and pulmonary fibrosis. However, the mechanisms of the lung injury remain poorly understood, with limited therapeutic options aside from lung transplantation. Post-translational modifications can regulate the function of proteins and play an important role in studying disease mechanisms. To investigate changes in post-translational modifications of proteins in silicosis, combined quantitative proteome, acetylome, and succinylome analyses were performed with lung tissues from silica-injured and healthy mice using liquid chromatography-mass spectrometry. Combined analysis was applied to the three omics datasets to construct a protein landscape. The acetylation and succinylation of the key transcription factor STAT1 were found to play important roles in the silica-induced pathophysiological changes. Modulating the acetylation level of STAT1 with geranylgeranylacetone (GGA) effectively inhibited the progression of silicosis. This report revealed a comprehensive landscape of post-translational modifications in silica-injured mouse, presented a novel therapeutic strategy targeting the post-translational level for silica-induced lung diseases.

Bacterial proteome adaptation during fermentation in dairy environments.

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

Colonization of root endophytic fungus Serendipita indica improves drought tolerance of Pinus taeda seedlings by regulating metabolome and proteome.

Pinus taeda is an important forest tree species for plantations because of its rapid growth and high yield of oleoresins. Although P. taeda plantations distribute in warm and wet southern China, drought, sometime serious and long time, often occurs in the region. To explore drought tolerance of P. taeda and usage of beneficial microorganisms, P. taeda seedlings were planted in pots and were inoculated with root endophytic fungus Serendipita indica and finally were treated with drought stress for 53 d. Metabolome and proteome of their needles were analyzed. The results showed that S. indica inoculation of P. taeda seedlings under drought stress caused great changes in levels of some metabolites in their needles, especially some flavonoids and organic acids. Among them, the levels of eriocitrin, trans-aconitic acid, vitamin C, uric acid, alpha-ketoglutaric acid, vitamin A, stachydrine, coumalic acid, itaconic acid, calceolarioside B, 2-oxoglutaric acid, and citric acid were upregulated more than three times in inoculated seedlings under drought stress, compared to those of non-inoculated seedlings under drought stress. KEGG analysis showed that some pathways were enriched in inoculated seedlings under drought stress, such as flavonoid biosynthesis, ascorbate and aldarate metabolism, C5-branched dibasic acid metabolism. Proteome analysis revealed some specific differential proteins. Two proteins, namely, H9X056 and H9VDW5, only appeared in the needles of inoculated seedlings under drought stress. The protein H9VNE7 was upregulated more than 11.0 times as that of non-inoculated seedlings under drought stress. In addition, S. indica inoculation increased enrichment of water deficient-inducible proteins (such as LP3-1, LP3-2, LP3-3, and dehydrins) and those involved in ribosomal structures (such as A0A385JF23). Meanwhile, under drought stress, the inoculation caused great changes in biosynthesis and metabolism pathways, mainly including phenylpropanoid biosynthesis, cutin, suberine and wax biosynthesis, and 2-oxocarboxylic acid metabolism. In addition, there were positive relationships between accumulation of some metabolites and enrichment of proteins in P. taeda under drought stress. Altogether, our results showed great changes in metabolome and proteome in inoculated seedlings under drought stress and provided a guideline to further study functions of metabolites and proteins, especially those related to drought stress.

Comparative transcriptomics and proteomics analysis of the symbiotic germination of Paphiopedilum barbigerum with Epulorhiza sp. FQXY019.

Introduction: Paphiopedilum barbigerum is currently the rarest and most endangered species of orchids in China and has significant ornamental value. The mature seeds of P. barbigerum are difficult to germinate owing to the absence of an endosperm and are highly dependent on mycorrhizal fungi for germination and subsequent development. However, little is known about the regulation mechanisms of symbiosis and symbiotic germination of P. barbigerum seeds. Methods: Herein, transcriptomics and proteomics were used to explore the changes in the P. barbigerum seeds after inoculation with (FQXY019 treatment group) or without (control group) Epulorhiza sp. FQXY019 at 90 days after germination. Results: Transcriptome sequencing revealed that a total of 10,961 differentially expressed genes (DEGs; 2,599 upregulated and 8,402 downregulated) were identified in the control and FQXY019 treatment groups. These DEGs were mainly involved in carbohydrate, fatty acid, and amino acid metabolism. Furthermore, the expression levels of candidate DEGs related to nodulin, Ca2+ signaling, and plant lectins were significantly affected in P. barbigerum in the FQXY019 treatment groups. Subsequently, tandem mass tag-based quantitative proteomics was performed to recognize the differentially expressed proteins (DEPs), and a total of 537 DEPs (220 upregulated and 317 downregulated) were identified that were enriched in processes including photosynthesis, photosynthesis-antenna proteins, and fatty acid biosynthesis and metabolism. Discussion: This study provides novel insight on the mechanisms underlying the in vitro seed germination and protocorm development of P. barbigerum by using a compatible fungal symbiont and will benefit the reintroduction and mycorrhizal symbiotic germination of endangered orchids.

MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds. Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data. Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24 h post-transfection. Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.