Higher Abundance Of Proteins Research Articles

Characterization of Extracellular Vesicles from Streptococcus thermophilus 065 and Their Potential to Modulate the Immune Response.

Bacteria can release membrane-derived nanoparticles made of lipid bilayers, so-called extracellular vesicles (EVs), which can carry diverse cargo and are important for microbe-microbe and microbe-host interactions. Here, we studied the production of EVs by Streptococcus thermophilus 065, the protein composition of the EVs, and how the produced EVs impact the immune response in vitro. Cultures of S. thermophilus grown for 6h at 40°C in M17 broth with 2% lactose reached high biomass yields and a high level of EVs quantified by lipophilic fluorescent dye staining. Proteome analysis of the isolated EVs revealed a high abundance of membrane-associated binding proteins of ABC transporters, ribosomal proteins, and glycolytic enzymes. In addition, phage proteins were found to be present in the EVs, which suggests a low-level expression of prophage genes during growth most likely supporting the release of EVs without causing cell lysis. The role of prophage activation was confirmed in an experiment with the addition of mitomycin C resulting in the expression of phage proteins including holin and endolysin causing a drop in culture OD and concomitant EV release. Subsequent in vitro immune assays using non-activated and activated human peripheral blood mononuclear cells (PBMCs) showed immune regulation in both cases upon exposure to S. thermophilus EVs and producer cells. This study shows the capacity of S. thermophilus EVs to act as immune modulators and opens the possibility for their use as postbiotics.

Single cell proteomics by mass spectrometry reveals deep epigenetic insight into the actions of an orphan histone deacetylase inhibitor.

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than previously described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434.

Abstract 141: Lesion Site-specific Mass Spectrometry Analysis Of Human Carotid Plaques Reveals Strongest Association Of The Necrotic Core And Fibrous Cap Proteome With Plaque Stability

The spatial impact of the plaque proteome on atherosclerotic plaque stability remains largely unknown. In this study, we performed mass spectrometry-based proteomics of various carotid plaque subregions (media, necrotic core, fibrous cap), using guided laser capture micro-dissections (LCM) of FFPE tissue in a matched cohort of 112 patients undergoing carotid endarterectomy (CEA) to study the impact of the plaque proteome, not only on lesion stability, but also on subregional changes of expression. Further, we performed a histological analysis according to the AHA classification and combined the data with clinical information on risk factors, comorbidities, and patient symptoms. Our LCM mass spectrometry analysis yielded over 3000 proteins for each plaque subregion. Gene set enrichment analysis revealed a high abundance of extracellular proteins and vice versa for cellular proteins. Subregion analysis identified the necrotic core and the fibrous cap to display the most significant changes in proteins associated with the stability status, whereas the media proteome remained largely unaffected. Primary and secondary enrichment analysis revealed synthetic vascular smooth muscle cells as the major source of these proteins. In addition, we analyzed the serum proteome of more than 500 patients (including non-athero control patients) with overlap from the tissue cohort. We combined these with histological evaluation and a complete set of clinical data. We used these two datasets for developing tissue and serological markers associated with plaque stability. In summary, our approach utilized LCM mass spectrometry, histological evaluation, and clinical data to analyze the association of different lesion subregions on plaque stability. We are able to demonstrate a high through-put method to identify potential protein markers relevant to atherosclerotic plaque stability.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host’s immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-10, and TGF-β1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development.Key points• Preparation and characterization of P. multocida OMVs.• P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system.• P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Neoadjuvant chemo- or chemo-radiation-therapy of pancreatic ductal adenocarcinoma differentially shift ECM composition, complement activation, energy metabolism and ribosomal proteins of the residual tumor mass.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, often diagnosed at stages that dis-qualify for surgical resection. Neoadjuvant therapies offer potential tumor regression and improved resectability. Although features of the tumor biology (e.g., molecular markers) may guide adjuvant therapy, biological alterations after neoadjuvant therapy remain largely unexplored. We performed mass spectrometry to characterize the proteomes of 67 PDAC resection specimens of patients who received either neoadjuvant chemo (NCT) or chemo-radiation (NCRT) therapy. We employed data-independent acquisition (DIA), yielding a proteome coverage in excess of 3500 proteins. Moreover, we successfully integrated two publicly available proteome datasets of treatment-naïve PDAC to unravel proteome alterations in response to neoadjuvant therapy, highlighting the feasibility of this approach. We found highly distinguishable proteome profiles. Treatment-naïve PDAC was characterized by enrichment of immunoglobulins, complement and extracellular matrix (ECM) proteins. Post-NCT and post-NCRT PDAC presented high abundance of ribosomal and metabolic proteins as compared to treatment-naïve PDAC. Further analyses on patient survival and protein expression identified treatment-specific prognostic candidates. We present the first proteomic characterization of the residual PDAC mass after NCT and NCRT, and potential protein candidate markers associated with overall survival. We conclude that residual PDAC exhibits fundamentally different proteome profiles as compared to treatment-naïve PDAC, influenced by the type of neoadjuvant treatment. These findings may impact adjuvant or targeted therapy options.

High Mitochondrial Protein Expression as a Potential Predictor of Relapse Risk in Acute Myeloid Leukemia Patients with the Monocytic FAB Subtypes M4 and M5.

AML is a highly aggressive and heterogeneous form of hematological cancer. Proteomics-based stratification of patients into more refined subgroups may contribute to a more precise characterization of the patient-derived AML cells. Here, we reanalyzed liquid chromatography-tandem mass spectrometry (LC-MS/MS) generated proteomic and phosphoproteomic data from 26 FAB-M4/M5 patients. The patients achieved complete hematological remission after induction therapy. Twelve of them later developed chemoresistant relapse (RELAPSE), and 14 patients were relapse-free (REL_FREE) long-term survivors. We considered not only the RELAPSE and REL_FREE characteristics but also integrated the French-American-British (FAB) classification, along with considering the presence of nucleophosmin 1 (NPM1) mutation and cytogenetically normal AML. We found a significant number of differentially enriched proteins (911) and phosphoproteins (257) between the various FAB subtypes in RELAPSE patients. Patients with the myeloblastic M1/M2 subtype showed higher levels of RNA processing-related routes and lower levels of signaling related to terms like translation and degranulation when compared with the M4/M5 subtype. Moreover, we found that a high abundance of proteins associated with mitochondrial translation and oxidative phosphorylation, particularly observed in the RELAPSE M4/M5 NPM1 mutated subgroup, distinguishes relapsing from non-relapsing AML patient cells with the FAB subtype M4/M5. Thus, the discovery of subtype-specific biomarkers through proteomic profiling may complement the existing classification system for AML and potentially aid in selecting personalized treatment strategies for individual patients.

Comprehensive Matrisome Profiling of Human Adipose Tissue for Soft Tissue Reconstruction.

For effective translation of research from tissue engineering and regenerative medicine domains, the cell-instructive extracellular matrix (ECM) of specific tissues must be accurately realized. As adipose tissue is gaining traction as a biomaterial for soft tissue reconstruction, with highly variable clinical outcomes obtained, a quantitative investigation of the adipose tissue matrisome is overdue. In this study, the human adipose tissue matrisome is profiled using quantitative sequential windowed acquisition of all theoretical fragment ion spectra - mass spectrometry (SWATH-MS)proteomics across a cohort of 13 fat-grafting patients, to provide characterization of ECM proteins within the tissue, and to understand human population variation. There are considerable differences in the expression of matrisome proteins across the patient cohort, with age and lipoaspirate collection technique contributing to the greatest variation across the core matrisome. A high abundance of basement membrane proteins (collagen IV and heparan sulfate proteoglycan) is detected, as well as fibrillar collagens I and II, reflecting the hierarchical structure of the tissue. This study provides a comprehensive proteomic evaluation of the adipose tissue matrisome and contributes to an enhanced understanding of the influence of the matrisome in adipose-related pathologies by providing a healthy reference cohort and details an experimental pipeline that can be further exploited for future biomaterial development.

Venom comparisons of endemic and micro-endemic speckled rattlesnakes Crotalus mitchellii, C. polisi and C. thalassoporus from Baja California Peninsula

A high diversity of rattlesnake species can be found in the Baja California peninsula and the island of the Gulf of California, nevertheless, their venom has been poorly evaluated. The aim of this work was to present the first characterization of endemic Crotalus mitchellii, micro endemic C. polisi and C. thalassoporus venoms. All samples provoke human plasma coagulation showing doses in the rank of 2.3-41.0μg and also produce rapid hydrolysis of the alpha chain of bovine fibrinogen while the beta chain is attacked at larger incubation periods by C. polisi and especially by C. thalassoporus. Phospholipase activity ranging from 23.2 to 173.8 U/mg. The venoms of C. thalassoporus and C. polisi show very high hemorrhagic activity (from 0.03 to 0.31μg). A total of 130 toxin-related proteins were identified and classified into ten families. Crotalus mitchellii venom was characterized by high abundance of crotoxin-like and other phospholipase proteins (34.5%) and serine proteinases (29.8%). Crotalus polisi showed a similar proportion of metalloproteinases (34%) and serine proteinases (22.8%) components with important contribution of C-type lectins (14.3%) and CRiSP (14.0%) proteins. Venom of C. thalassoporus is dominated by metalloproteases that amount to more than 66% of total toxin proteins. These results provide a foundation for comprehending the biological, ecological and evolutionary significance of venom composition of speckled rattlesnake from the Baja California peninsula.

A proteomic approach to identify biomarkers of foal meat quality: A focus on tenderness, color and intramuscular fat traits

Foal meat is considered a healthy alternative to other meat sources and more environmentally sustainable. However, its quality is highly variable and there is lack of knowledge about the molecular mechanisms underlying its determination. Genotype and diet play a relevant role as the main factors that can allow a control of the final quality and the use of high-throughput analytical methods such as proteomics is a way to achieve this lofty goal. This research aimed to study-two breeds (Burguete and Jaca Navarra) supplemented with two different finishing diets: conventional concentrate and straw (C) vs silage and organic feed (S). The proteomic approach built a library of 294 proteins that were subjected to several statistical and bioinformatic analyses. Burguete breed finished with concentrate produced higher meat quality in terms of tenderness, intramuscular fat and color lightness mainly due to the high abundance of energy metabolic proteins. Tenderness was correlated to myofibrillar proteins (ACTA1, MYBPH, MYL1 and TNNC1) and energy metabolic proteins (ALDOA, CKM, TPI1 and PGMA2). Regarding color, the main pathways were energy metabolism, involving several glycolytic enzymes (ALDOA, PKM, PFKM and CKM). Oxidative stress and response to stress proteins (HSPA1A, SOD2 and PRDX2) were further involved in color variation. Moreover, we revealed that several proteins were related to the intramuscular fat accordingly to the breed. This study proposed several candidate protein biomarkers for foal meat quality that are worthy to evaluate in the future.

IFIT3 mediated the type I interferon antiviral response by targeting Senecavirus A entry, assembly and release pathways

Senecavirus A (SVA) is a newly emerging etiological agent of vesicular disease associated with sow abortion and acute piglet death, causing devastating economic consequences to global pig industry. IFN-induced protein with tetratricopeptide repeats (IFIT) genes are versatile in combating a variety of viruses, but the detailed mechanisms-of-action against SVA is unexplored. Transcriptomic analysis and immunoblot revealed high abundance of IFIT transcripts and proteins following SVA infection, initially implying the correlation between IFITs and SVA. Type I IFNs restricted SVA replication accompanied with substantial elevation of IFIT expression, potentializing IFITs as anti-SVA effectors downstream of IFN signaling. Gain-of-function assay demonstrated that IFIT3 rather than IFIT1/2 potently inhibited SVA replication, which was consistently verified by SVA strain SVV CH-FJ-2017 by TCID50 titration and an eGFP-tagged recombinant SVA using fluorescent microscopy. Afterwards, IFIT3 disrupted SVA life cycle at the early stage of virus binding and internalization, and at the late stage of virus assembly and release, as quantified by copy number and transmission electron microscopy, respectively. Directly transfecting SVA infectious RNA in IFIT3-overexpressed cells bypassed its antiviral activity, further suggesting that IFIT3 targeted viral life cycle beyond RNA replication. Further investigations showed that IFIT3 overexpression was incapable of regulating host immune responses against pathogens. Those results identified IFIT3 as a potent inhibitor of SVA and implicated IFIT3 pathway in the regulation of virus entry/assembly. In short, IFIT3 exerted significant inhibitory effects on the replication and spread of SVA, and played different functions in the life cycle of SVA.

A proteomic approach for in-depth characterization and understanding the impact of immunocastration on dry-cured ham of male and female pigs

Dry-cured ham is a high-quality product elaborated through a long and complex process. To ensure the success of the process, it is necessary to select the most suitable pork leg and one of the major factors is pig castration. Due to animal welfare, pig castration is becoming a paramount issue in recent years. The proteomic differences of dry-cured ham from immunecastrated pigs against entire females, as well as, between immunecastrated pigs and surgically castrated males were analysed. The identification and quantification of proteins were carried out by sequential window acquisition of all theoretical mass spectra (SWATH-MS). A total of 249 proteins were identified across the samples of dry-cured ham, resulting in 17 and 37 differentially abundant proteins in the case of males and females, respectively. In the case of males, a high abundance of structural proteins in dry-cured ham from surgically castrated animals as well as a high abundance of trypsinogen and proteosome subunit C9-like protein with protease activity in samples from immunocastrated males suggests that immunocastration impact on myofibrils of dry-cured ham. Regarding females, the immunocastration provoked an increase of abundance in several structural proteins of the myosin heavy chain (MYH7, MYH7B and MYH4) and a decrease in others (ACTN2, TNNT3, MYL3 and TCAP) concerning entire. Overall, MYH4 and ACT were found to a greater degree in immunocastrated males and females indicating a potential for biomarkers.

RNA isolation from micro-quantity of articular cartilage for quantitative gene expression by microarray analysis.

Isolation of quality RNA from articular cartilage has been challenging due to low cellularity and the high abundance of extracellular matrix and proteoglycan proteins. Recently developed methods for isolation of high quality RNA from cartilage are more applicable to larger cartilage specimens typically weighing at least 25 mg. While these methods generate RNA suitable for analysis, they are less successful with smaller tissue inputs. For the study of small focal defect cartilage specimens an improved RNA extraction method is needed. Here we report a protocol for direct RNA isolation from less than 3 mg of wet weight rabbit articular cartilage for quantitative microarray gene profiling. This protocol is useful for identifying differentially expressed genes in chondrocytes following focal cartilage repair and can potentially be adopted for gene expression analysis of cartilage biopsy specimens from human joints.

Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20μL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74g/L for IgG1, 0.24-4.46g/L for IgG2, 0.038-0.752g/L for IgG3, 0.025-0.435g/L for IgG4, and 0.62-15.5g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43g/L for IgG1, 0.22-3.76g/L for IgG2, 0.0387-0.721g/L for Ig3, and 0.0279-1.46g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29g/L for IgG1, 0.22g/L for IgG2, 0.019g/L for IgG3, and 0.0067g/L for IgG4.

The Fungus Metarhizium sp. BCC 4849 Is an Effective and Safe Mycoinsecticide for the Management of Spider Mites and Other Insect Pests.

Simple SummaryThe spider mite is a destructive pest of various crops during warm and dry conditions in tropical countries, including Thailand. The pest is difficult to control despite synthetic acaricide use. In the field, insect populations gradually develop resistance to synthetic pesticides over long-term use. The use of entomopathogenic fungi is more human- and environmentally friendly. We searched and identified a potential fungal isolate from a culture collection, focusing on the genus Metarhizium. Metarhizium sp. BCC 4849 not only controls the spider mite but also plays a significant role as a natural regulator of other insect pests. Here, we investigated its infection process on the mite, optimized a conidial formulation for extended shelf life, and conducted toxicological assays in animals to assess its biosafety in humans. The fungal genome has been sequenced. The genomic data indicated that oxidoreduction proteins; zinc-, heme-, and iron-binding proteins; and transmembrane transporters are abundant in the genome.Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92–99%, on 3rd–6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85–100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48–72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96–120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays.

Increased cellular detoxification, cytoskeletal activities and protein transport explain physiological stress in a lagoon sponge.

Tropical lagoon-inhabiting organisms live in highly irradiated ecosystems and are particularly susceptible to thermal stress resulting from climate change. However, despite living close to their thermal maxima, stress response mechanisms found in these organisms are poorly understood. We used a novel physiological-proteomic approach for sponges to describe the stress response mechanisms of the lagoon-inhabiting sponge Amphimedon navalis, when exposed to elevated seawater temperatures of +2°C and +4°C relative to a 26°C ambient temperature for 4weeks. After 4weeks of thermal exposure, the buoyant weight of the sponge experienced a significant decline, while its pumping rates and oxygen consumption rates significantly increased. Proteome dynamics revealed 50 differentially abundant proteins in sponges exposed to elevated temperature, suggesting that shifts in the sponge proteome were potential drivers of physiological dysfunction. Thermal stress promoted an increase in detoxification proteins, such as catalase, suggesting that an excess of reactive oxygen species in sponge cells was responsible for the significant increase in oxygen consumption. Elevated temperature also disrupted cellular growth and cell proliferation, promoting the loss of sponge biomass, and the high abundance of multiple α-tubulin chain proteins also indicated an increase in cytoskeletal activities within sponge cells, which may have induced the increase in sponge pumping rate. Our results show that sustained thermal exposure in susceptible lagoonal sponges may induce significant disruption of cellular homeostasis, leading to physiological dysfunction, and that a combined physiological-proteomic approach may provide new insights into physiological functions and cellular processes occurring in sponges.

Exosomes in serum‑free cultures of THP‑1 macrophages infected with Mycobacterium tuberculosis.

It has been shown from the isolation and characterization of exosomes from cell culture media supplemented with fetal bovine serum that both their quality and purity are affected. The high abundance of serum proteins, including bovine cell derived exosomes, is also a potential source of contaminants, which may result in appreciable yields of impure exosomes, thereby leading to artifacts. Isolation and characterization of exosomes from cells maintained under serum-free conditions should therefore ensure the high quality necessary for medical applications. To meet this end, the present study aimed to characterize exosomes released from THP-1 macrophages cultured in serum-free, ultra-centrifuged medium upon infection with the human pathogen Mycobacterium tuberculosis (Mtb). Macrophages differentiated from the human cell line THP-1 were infected at a multiplicity of infection (MOI) of 5. Macrophages were cultivated in CellGenix® GMP DC serum-free ultra-centrifuged medium for 4, 24 and 48 h at 37°C in a humidified atmosphere with 5% CO2. Total exosome isolation reagent was used to extract the exosomes from the cell culture supernatants of naïve and Mtb-infected THP-1 macrophages. The size and purity of the exosomes isolated were subsequently assessed by various methods, including nanoparticle tracking analysis, flow cytometry, MACSPlex exosome analysis, and western blotting. The serum-free, ultra-centrifuged medium was found to support the proliferation of the THP-1 cells successfully. The nanoparticle tracking analysis data revealed that the majority of the isolated particles were within the size range of exosomes (i.e., 30–150 nM). The MACSPlex exosome analysis confirmed the expression of the exosomal markers, CD9, CD63 and CD81. Furthermore, western blot analysis of the isolated exosomes indicated the presence of CD9, CD63, CD81 and lysosomal associated membrane protein-1 (LAMP-1), and also confirmed the absence of Mtb proteins. Taken together, these data provide evidence that serum-free, ultra-centrifuged CellGenix® GMP DC medium is suitable for application in exosome research, and may significantly advance such studies. Therefore, the use of serum-free medium for exosome isolation purposes could offer considerable advantages, and constitute a significant improvement in the growing field of extracellular vesicle research. The use of more sensitive methods represents an advance that will enable researchers to rule out the presence of Mtb pathogenic proteins in exosomes isolated from infected serum-free cell cultures.

ITS2 Sequencing and Targeted Meta-Proteomics of Infant Gut Mycobiome Reveal the Functional Role of Rhodotorula sp. during Atopic Dermatitis Manifestation.

Association between the gut mycobiome and atopic dermatitis was investigated in 9–12-month-old infants using metagenomics. Two groups of atopic dermatitis infants were classified according to their symptom development as outgrown (recovered) and persisted (still undergoing). The evenness and diversity of the mycobiome in the persisted group were higher than in the healthy and outgrown groups. Dysbiosis of the microbiome in the persisted group was observed by a reduction in the Ascomycota/Basidiomycota ratio. Five fungi were selected as markers from each sample group. In the persisted group, Rhodotorula sp. abundance increased significantly, while Wickerhamomyces sp. and Kodamaea sp. abundance increased in the healthy group, and Acremonium sp. and Rhizopus sp. abundance increased considerably in the outgrown group. Metaproteomic analysis revealed that the persisted group had a high abundance of fungal proteins, particularly those from Rhodotorula sp. Unique proteins such as RAN-binding protein 1 and glycerol kinase from Rhodotorula sp. were hypothesized to be related to atopic dermatitis manifestation in infants.

Rice mutants with tolerance to multiple abiotic stresses show high constitutive abundance of stress-related transcripts and proteins

Mutation breeding has a long track record in the development of crop cultivars with improved tolerance to abiotic stresses such as heat, salinity and drought. Oryza sativa L. cv IR64 is a very popular high yielding rice, but susceptible to major abiotic stresses, such as low and high temperatures, salinity and drought. We subjected IR64 to gamma irradiation and generated a mutant population (M3) with ~2,000 families. These were screened at the seedling stage for tolerance to high-temperature stress using hydroponics and controlled-environment chambers, resulting in the identification of three mutant lines showing a robust seedling phenotype. Under heat stress, higher CO2 assimilation (10-30%), higher spikelet fertility (40-45%) and higher antioxidant activity (15-20% catalase activity) confirmed superiority of the selected mutant lines over wild type plants at seedling and flowering stages. Upon exposure to salinity and drought stress, the three selected lines also exhibited better tolerance than wild type in terms of higher CO2 assimilation, stomatal conductance, transpiration and chlorophyll fluorescence. Transcript and protein abundance analyses confirmed higher constitutive levels of heat shock proteins and antioxidant enzymes in the mutant lines relative to wild type. Tolerance to multiple abiotic stresses was reflected in higher (25-30%) grain yield than wild type. It is anticipated that the mutant lines identified will be useful for developing new improved cultivars for dry and saline areas and may be exploited to dissect the molecular basis of multiple stress tolerance in crop plants

Metabolic and molecular basis for the salt and alkali responses of Suaeda corniculata

• Alkali stress largely influenced plant growth as compared to salt stress. • Protein and metabolite profiles showed alkali and salt stress cause distinct molecular effect on S. corniculata . • AsA-GSH cycle and Prx/Trx pathway mainly contributed to ROS removal upon stress condition. • Stress induced proteins involved in fatty acid metabolism probably modulate alkali tolerance. High salinity and alkalinity, two of the most widespread abiotic stressors which largely impact plant growth and development. Most researches focus on how plant cope with neutral salt stress, the underlying mechanism of plant response to alkali stress has not been systematically studied, and little information is available as reference. The halophyte Suaeda corniculata is an outstanding pasture with strong tolerance to salt and alkali stress. Here, the combined analysis of 1 H NMR-based metabolomics, 2DE-based proteomics and physiological observation were performed, to demonstrate the comparison on metabolic and molecular network of S. corniculata responsive to salt and alkali stress. Our results showed that the alkali stress induced more severe effect on the plant growth than salt stress. The metabolomic and proteomic characteristics showed distinct regulation patterns in response to salt and alkali in S. corniculata . The evaluation of reactive oxygen species (ROS)-scavenging systems, as well as abundance patterns of salt and alkali-responsive proteins or metabolites indicated that the hyper activation of ROS scavenging system, including APX dependent AsA-GSH cycle and Prx/Trx pathway, played a major role in maintaining redox homeostasis to alleviate oxidative damage. In addition, the accumulation of glycine betaine mainly contributes to the re-establishment of osmotic homeostasis. Importantly, the high abundance of germin-like proteins and enzymes involved in fatty acid metabolism might positively contribute to the alkali tolerance of S. corniculata . Together, this study provides novel insights into the physiological and metabolic features in halophyte S. corniculata to salt and alkali stress, and this deepens our understanding of how halophytes reprogram the molecular activities to cope with salinity and alkalinity in soil.