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Abstract Obesity is a health problem characterized by large expansion of adipose tissue. During this expansion, genotoxic stressors can be accumulated and negatively affect the mesenchymal stem cells (MSCs) of adipose tissue. Due to the oxidative stress generated by these genotoxic stressors, senescence phenotype might be observed in adipose tissue MSCs. Senescent MSCs lose their proliferations and differentiation properties and secrete senescence-associated molecules to their niche thus triggering senescence for the rest of the tissue. Accumulation of senescent cells in adipose tissue results in decreased tissue regeneration and functional impairment not only in the close vicinity but also in the other tissues. Here we hypothesized that declined tissue regeneration might be associated with loss of stemness markers in MSCs population. We analyzed the expression of several stemness-associated genes of in vitro cultured MSCs originated from adipose tissue of high-fat diet and normal diet mice models. Since the heterogenous MSCs population covers a small percentage of the pluripotent stem cells, which have roles in proliferation and tissue regeneration, we measured the percentage of these cells via TRA-1-60 pluripotent state antigen. Additionally, by conducting a shotgun proteomic approach using LC-MS/MS, whole cell proteome of the adipose tissue MSCs of high-fat diet and normal diet mice were analyzed and identified proteins were evaluated via gene ontology and PPI network analysis. MSCs of obese mice showed senescent phenotype and altered cell cycle distribution due to a hostile environment with oxidative stress in adipose tissue where they reside. Additionally, the number of pluripotent markers expressing cells declined in the MSC population of the high-fat diet mice. Gene expression analysis evidenced the loss of stemness with a decrease in the expression of stemness-associated genes. Of the proteomic comparison of the normal and the high-fat diet group, MSCs revealed that stemness-associated molecules were decreased while inflammation and senescence-associated phenotypes emerged in obese mice MSCs. Our results showed us that the MSCs of adipose tissue may lose their stemness properties due to obesity-associated stress conditions.

Summary: Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

BackgroundAcinetobacter baumannii is one of the most life-threatening multidrug-resistant pathogens worldwide. Currently, 50%–70% of clinical isolates of A. baumannii are extensively drug-resistant, and available antibiotic options against A. baumannii infections are limited. There is still a need to discover specific de facto bacterial antigenic proteins that could be effective vaccine candidates in human infection. With the growth of research in recent years, several candidate molecules have been identified for vaccine development. So far, no public health authorities have approved vaccines against A. baumannii.MethodsThis study aimed to identify immunodominant vaccine candidate proteins that can be immunoprecipitated specifically with patients’ IgGs, relying on the hypothesis that the infected person’s IgGs can capture immunodominant bacterial proteins. Herein, the outer-membrane and secreted proteins of sensitive and drug-resistant A. baumannii were captured using IgGs obtained from patient and healthy control sera and identified by Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS) analysis.ResultsUsing the subtractive proteomic approach, we determined 34 unique proteins captured only in drug-resistant A. baumannii strain via patient sera. After extensively evaluating the predicted epitope regions, solubility, transverse membrane characteristics, and structural properties, we selected several notable vaccine candidates.ConclusionWe identified vaccine candidate proteins that triggered a de facto response of the human immune system against the antibiotic-resistant A. baumannii. Precipitation of bacterial proteins via patient immunoglobulins was a novel approach to identifying the proteins that could trigger a response in the patient immune system.

Abstract Background The term mesenchymal stromal cells (MSCs) designates an assorted cell population comprised of stem cells, progenitor cells, fibroblasts, and stromal cells. MSCs contribute to the homeostatic maintenance of many organs through paracrine and long-distance signaling. Tissue environment, in both physiological and pathological conditions, may affect the intercellular communication of MSCs. Methods We performed a secretome analysis of MSCs isolated from subcutaneous adipose tissue (sWAT) and visceral adipose tissue (vWAT), and from bone marrow (BM), of normal and obese mice. Results The MSCs isolated from tissues of healthy mice share a common core of released factors: components of cytoskeletal and extracellular structures; regulators of basic cellular functions, such as protein synthesis and degradation; modulators of endoplasmic reticulum stress; and counteracting oxidative stress. It can be hypothesized that MSC secretome beneficially affects target cells by the horizontal transfer of many released factors. Each type of MSC may exert specific signaling functions, which could be determined by looking at the many factors that are exclusively released from every MSC type. The vWAT-MSCs release factors that play a role in detoxification activity in response to toxic substances and drugs. The sWAT-MSC secretome contains proteins involved in in chondrogenesis, osteogenesis, and angiogenesis. Analysis of BM-MSC secretome revealed that these cells exert a signaling function by remodeling extracellular matrix structures, such as those containing glycosaminoglycans. Obesity status profoundly modified the secretome content of MSCs, impairing the above-described activity and promoting the release of inflammatory factors. Conclusion We demonstrated that the content of MSC secretomes depends on tissue microenvironment and that pathological condition may profoundly alter its composition. Video abstract

Senotherapeutics are new drugs that can modulate senescence phenomena within tissues and reduce the onset of age-related pathologies. Senotherapeutics are divided into senolytics and senomorphics. The senolytics selectively kill senescent cells, while the senomorphics delay or block the onset of senescence. Metformin has been used to treat diabetes for several decades. Recently, it has been proposed that metformin may have anti-aging properties as it prevents DNA damage and inflammation. We evaluated the senomorphic effect of 6 weeks of therapeutic metformin treatment on the biology of human adipose mesenchymal stromal cells (MSCs). The study was combined with a proteome analysis of changes occurring in MSCs’ intracellular and secretome protein composition in order to identify molecular pathways associated with the observed biological phenomena. The metformin reduced the replicative senescence and cell death phenomena associated with prolonged in vitro cultivation. The continuous metformin supplementation delayed and/or reduced the impairment of MSC functions as evidenced by the presence of three specific pathways in metformin-treated samples: 1) the alpha-adrenergic signaling, which contributes to regulation of MSCs physiological secretory activity, 2) the signaling pathway associated with MSCs detoxification activity, and 3) the aspartate degradation pathway for optimal energy production. The senomorphic function of metformin seemed related to its reactive oxygen species (ROS) scavenging activity. In metformin-treated samples, the CEBPA, TP53 and USF1 transcription factors appeared to be involved in the regulation of several factors (SOD1, SOD2, CAT, GLRX, GSTP1) blocking ROS.

Objective: In the present study we aim to evaluate whether some of the proteins previously identified in the secretome of senescent bone marrow (BM)- and adipose (A)-mesenchymal stromal cells (MSCs) could be involved in regulation of senescence phenomena. Materials and Methods: Among the identified proteins, we selected Filamin B (FLNB) and Aminopeptidase N (CD13) that were exclusively found in the secretome of senescent cells. We silenced their mRNA expression in BM-MSCs by means of RNA interference technology and induced acute senescence by hydrogen peroxide treatment. Our goal was to evaluate if FLNB or CD13 silencing may affect onset of senescence. Results: Our preliminary data showed that CD13 protein could be a key factor involved in the regulation and determination of both primary and paracrine induced senescence in human BM-MSCs. On the other hand, Filamin B seems not to be involved in the determination of primary senescence whereas its presence could be part of the mechanism that regulates the senescence induction in human BM-MSCs by paracrine signaling. Conclusion: Our study provides a useful base for identifying the complex extracellular protein networks involved in the regulation of MSC cellular senescence.

There are only a few experimental studies which have investigated effects of glucose alone, and glucose in combination with insulin/insulin-like growth factors (IGF) on the growth of colon cancer. In the present study, we studied in vitro in human colorectal cancer cells originating from four Dukes’ stages of colorectal cancer the effects of glucose, insulin and IGFs on proliferation, migration, cell cycle progression and gene expression of the IGF system. Growth of colon cancer cells originating from a Dukes’ stage A was glucose-dependent, whereas growth of cancer cells from Dukes’ stage B, C and D was glucose-independent. Stimulatory effects of insulin and IGFs on cell growth were observed only in colon cancer cells originating from Dukes’ stage C and D. IGF-II stimulated migration in Dukes’ stage B cells only. The growth stimulatory effects in Dukes’ stage C and D colorectal cancer cells were accompanied by G2/M arrest and associated with an increased IGF-IR/IGF-II receptor ratio. In conclusion, our in vitro data suggest that the stimulating effects of glucose, IGFs and insulin on proliferation differ between colorectal cancer cells from early and late Dukes’ stages. Stimulatory effects of glucose on proliferation appear predominantly present in stage Dukes’ stage A colorectal cancer cells, while in contrast growth factor-mediated stimulation of cell proliferation is more pronounced in Dukes’ late stage (metastasized) colorectal cancer cells. Moreover, our study suggests that a stringent glucose control may be important to control tumor growth in early stages of colorectal cancer, while inhibition of the endocrine actions of the IGFs and insulin become more important in the late (metastasized) stages of colorectal cancer to restrain growth of colon cancer cells.

This study investigates in-vitro activities of phytochemicals in Callistemon citrinus against multi-resistant foodborne pathogens, alpha-glucosidase enzyme and MCF-7 cancer cell line. Assays were prepared with lyophilized extracts to determine antioxidant capacity, inhibition of α-glucosidase enzyme and growth of foodborne bacteria. Annexin-V detection kit was used for apoptosis detection and FT-IR spectroscopy to confirm structural and functional groups of phytochemicals. Cytotoxicity of the extracts against MCF-7 cells was monitored with xCELLigence Real-Time Cell Analyser. The result from FT-IR analysis gave a peak at 3295 cm−1 wavenumber, confirming the presence of O–H alcohol functional group. FT-IR analysis also showed the presence of different functional groups such as carboxylic acids, aromatics, alkanes, alcohols, aliphatic amines, alkenes and amine groups in the extracts. Callistemon exhibited strong antioxidant capacities with EC50 values of 0.474 ± 0.03 and 0.787 ± 0.15 mL sample/g of 2,2-diphenyl-1-picrylhydeazyl (DPPH) from leaf and flower extracts, respectively. Growth inhibition of most gram-positive foodborne bacteria by phytochemicals from flower extract appeared more promising as an alternative antimicrobial agent for food preservation. IC50 value of 3.69 ± 0.61 μg/mL obtained from leaf extract showed its inhibitory potential against α-glucosidase enzyme for managing diabetes type-2. A dose response obtained from real-time monitoring with xCELLigence system indicated higher cytotoxicity of the extracts against MCF-7 cell line at ≥200 μg/mL concentrations within 24 h of incubation. The versatility of phytochemicals in Callistemon observed in this study signifies its potential for enhancing feed or food functionality, moderating blood glucose level and inhibiting the growth of foodborne pathogens or invasive carcinoma in man.

The aim of this study was to investigate the antimicrobial and antioxidant activities of different extracts from Echinops emiliae Schwarz ex P. H. Davis. Antimicrobial activity was estimated against several common human pathogenic bacterial strains using the agar disc diffusion and minimal inhibitory concentration assays. Antioxidant activity was evaluated using the total flavanoid and total phenolic content methods. On the light of these experiments, E. emiliae would seem to be an important natural antioxidant. Antimicrobial and antioxidant of E. emiliae have not been reported up to now. The results of this study obviously reported that the antimicrobial and antifungal activity could be change with used extracts. Also, the micro dilution method was more sensitive than disk diffusion. This study is first report on the biological activity of E. emiliae as regarded endemic species from Turkey. The results show that E. emiliae could use in the treatment of some illness.