Supernatant Medium Research Articles

Plasmonic nanocrystals on polycarbonate substrates for direct and label-free biodetection of Interleukin-6 in bioengineered 3D skeletal muscles

Abstract The development of nanostructured plasmonic biosensors has been widely widespread in the last years, motivated by the potential benefits they can offer in integration, miniaturization, multiplexing opportunities, and enhanced performance label-free biodetection in a wide field of applications. Between them, engineering tissues represent a novel, challenging, and prolific application field for nanostructured plasmonic biosensors considering the previously described benefits and the low levels of secreted biomarkers (≈pM–nM) to detect. Here, we present an integrated plasmonic nanocrystals-based biosensor using high throughput nanostructured polycarbonate substrates. Metallic film thickness and incident angle of light for reflectance measurements were optimized to enhance the detection of antibody–antigen biorecognition events using numerical simulations. We achieved an enhancement in biodetection up to 3× as the incident angle of light decreases, which can be related to shorter evanescent decay lengths. We achieved a high reproducibility between channels with a coefficient of variation below 2% in bulk refractive index measurements, demonstrating a high potential for multiplexed sensing. Finally, biosensing potential was demonstrated by the direct and label-free detection of interleukin-6 biomarker in undiluted cell culture media supernatants from bioengineered 3D skeletal muscle tissues stimulated with different concentrations of endotoxins achieving a limit of detection (LOD) of ≈ 0.03 ng/mL (1.4 pM).

Characterization of active lead molecules from Lissocarinus orbicularis with potential antimicrobial resistance inhibition properties

BackgroundMarine organisms are the potential contributors of novel bioactive molecules. Nevertheless, their biodiversity and the versatility of bioactive metabolites have not been fully explored. Hence, the aim of the present study was to investigate the potentials of gut associated bacteria from a marine crab for the production of novel antibacterial compound. MethodsAerobic gut autochthonous bacteria isolated from marine crab (Lissocarinus orbicularis) collected from Pazhayar coastal area in Nagapattinam district of Tamil Nadu, India were screened for antibacterial activity. Optimization for bacterial growth and antimicrobial compound production, extraction, purification and characterization were studied. ResultsIn the present study, eight morphologically distinct colonies of L. orbicularis gut associated aerobic bacterial isolates (Iso1-Iso8) on Zobell marine agar plate were selected. Isolates were screened for antimicrobial activity against human bacterial pathogens such as Salmonella paratyphi, Vibrio cholera, Vibrio parahaemolyticus, Aeromonas hydrophila and Listeria monocytogenes. On the basis of screening results, isolate 5 (Iso5) was selected as the most potential strain and identified as Paenibacillus polymyxa using biochemical and 16S rRNA sequencing methods. The sequence data was submitted to NCBI (Gene bank Accession No: MK583465). Optimization of P. polymyxa for growth and antimicrobial compound production revealed incubation period (36 h), agitation (150 rpm), pH 8.0, 35 °C, 2.5% salinity, 2% glucose and 1% yeast extract as carbon and nitrogen sources respectively were the ideal conditions and mass culture was done with these parameters. Antimicrobial compound from the cell free supernatant of mass culture medium was extracted using ethanol. The lowest minimum inhibitory concentration (MIC) of 16 μg/ml was observed against of both V. parahaemolyticus and V. cholerae. GC–MS analysis of the active ethanol fraction showed the presence of different components such as dodecane (96.72%), Tridecane (1.69%), Undecane, 2,6-dimethyl- (1.69%), Tetradecane (1.12%) and Dodecane, 2,6,11-trimethyl- (1.12%). ConclusionThe present study showed that the gut associated autochthonous bacteria of marine crabs are one of the potential sources of antibacterial compound. However, further studies are needed for the identification of the antimicrobial compound.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin.

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Mesenchymal Stem Cells in the Treatment of COVID-19, a Promising Future.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all tissues; they have a potent self-renewal capacity and can differentiate into multiple cell types. They also affect the ambient tissue by the paracrine secretion of numerous factors in vivo, including the induction of other stem cells’ differentiation. In vitro, the culture media supernatant is named secretome and contains soluble molecules and extracellular vesicles that retain potent biological function in tissue regeneration. MSCs are considered safe for human treatment; their use does not involve ethical issues, as embryonic stem cells do not require genetic manipulation as induced pluripotent stem cells, and after intravenous injection, they are mainly found in the lugs. Therefore, these cells are currently being tested in various preclinical and clinical trials for several diseases, including COVID-19. Several affected COVID-19 patients develop induced acute respiratory distress syndrome (ARDS) associated with an uncontrolled inflammatory response. This condition causes extensive damage to the lungs and may leave serious post-COVID-19 sequelae. As the disease may cause systemic alterations, such as thromboembolism and compromised renal and cardiac function, the intravenous injection of MSCs may be a therapeutic alternative against multiple pathological manifestations. In this work, we reviewed the literature about MSCs biology, focusing on their function in pulmonary regeneration and their use in COVID-19 treatment.

In vitro biosynthesis of organic selenium by Lactobacillus casei from inorganic selenium forms

The bioconversion of selenium in the form of sodium selenite (Na2 SeO3 ) (SeIV) or sodium selenate (Na2 SeO4 ) (SeVI) to organic form by Lactobacillus casei (L. casei) was investigated in vitro. MRS media was supplemented with 1, 2, 5, 10 or 20 ppm of Se, inoculated with 2% starter culture with about 105 cfu/ ml of L. casei and then incubated up to 24 hrs at 37o C. Increasing of selenite “Se(IV)” concentrations in the media markedly reduced the bacterial growth compared to selenate “Se(VI)” indicating cytotoxic effect of selenite. The media supplemented with 5 ppm or more of Se(IV) became reddish after 24 hr of incubation as a result of the formation of 100-200 nanometer particles of selenium (SeNPs). Se speciation of the cultured media supernatants and its corresponding cell fractions was carried out by HPLC-ICP-MS technique. The bioconversion rate of Se to organic form by L. casei was extremely higher in Se(IV) than Se(VI) in both fractions, however the media supernatant contained the highest content. Increasing the media Se content resulted in gradual increase of organic Se concentration in both cells and supernatant fractions. The medium supplemented with 1 ppm Se(IV) was completely depleted from the inorganic Se as it completely converted to organic form. Although the cell fractions from all Se(VI) supplemented media contained only organic Se, the media supernatant contained significant residual amount of the inorganic form. Our results demonstrate the ability of L. casei to convert Se(IV) or Se(VI) up to 20 ppm to organic form(s) either in the cultured media or inside the bacterial cells. However, Se(IV) but not Se(VI), at a limit concentration of 1 ppm, was completely converted and accumulated in an organic form in the cell fraction and the cultured medium.

Effect of Incorporation of Zeolite Containing Silver-Zinc Nanoparticles into Mineral Trioxide Aggregate on Odontogenic Activity of Human Dental Pulp Stem Cells.

Statement of the Problem: The stimulation of odontogenic activity is considered an essential property for biomaterials used in vital pulp therapy.Purpose: The present study aimed to evaluate the effect of the incorporation of zeolite containing silver-zinc nanoparticles (Ze-Ag-Zn) into Angelous mineral trioxide aggregate (AMTA) on the odontogenic activity of human dental pulp stem cells (HDPSCs).Materials and Method: In this in vitro study, HDPSCs were treated with 2% wt of synthesized Ze-Ag-Zn particles+AMTA, AMTA and Ze-Ag-Zn disks. The negative control cells did not receive any treatment. Then, cell viability was measured using the MTT assay after 7 and 14 days of the treatment course. The alkaline phosphatase (ALP) activity and calcium ion level were also measured in the supernatant culture media using auto-analyzer kits. The obtained data were analyzed using one-way ANOVA and Student t-test where appropriate. The level of the statistical significance was set at p< 0.05.Results: The results indicated that HDPSCs treated with AMTA and AMTA+Ze-Ag-Zn particles did not show any significant cell death compared with the control cells after 14 days of the treatment course while the ALP activity and calcium ion levels were significantly (p< 0.05) elevated. Also, the addition of AMTA particles to the cell culture media resulted in increased ALP activity and calcium ion level compared with HDPSCs treated with AMTA+ Ze-Ag-Zn particles on day 7 of the treatment course (p< 0.05).Conclusion: It seems that the incorporation of Ze-Ag-Zn particles into AMTA did not have any significant positive effect on the biomineralization properties of AMTA.

Isolation, characterization, and evaluation of a high-siderophore-yielding bacterium from heavy metal-contaminated soil.

Heavy metal-resistant siderophore-producing bacteria (SPB) with plant growth-promoting traits can assist in phytoremediation of heavy metal-contaminated soil. We isolated siderophore-producing bacteria from Pb and Zn mine soil in Shangyu, Zhejiang, China. The isolate with the highest siderophore production, strain SX9, was identified as Burkholderia sp. Burkholderia sp. SX9 produced catecholate-type siderophore, with the highest production at a pH range of 6.0 to 8.0, a temperature range of 20 to 30 °C and NaCl concentration below 2%. Siderophore production was highest without Fe3+ and became gradually lower with increasing Fe3+ concentration. Minimal inhibitory concentrations (MIC) of Pb2+, Zn2+, Cu2+, and Cd2+ were 4000, 22000, 5000, and 2000 μmol L-1, respectively. The strain had a strong metal solubilization ability: the contents of Cu2+, Zn2+, and Cd2+ in the supernatant were 47.4%, 133.0%, and 35.4% higher, respectively, in strain SX9-inoculated cultures than in the not inoculated controls. The siderophore produced by strain SX9 could combine with Fe3+, Zn2+, and Cd2+ with good effectiveness. The plant growth-promoting traits of the strain included indole acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and phosphate solubilization capability. Compared to the uninoculated growth medium and SX9 culture supernatant, the germination rate of Lolium perenne seeds was higher when inoculated with strain SX9 culture. In the experiment of seed germination, adding bacterial culture or supernatant could alleviate the toxicity of heavy metals to L. perenne seed germination. Under Cu2+ and Zn2+ stress, strain SX9 promoted the germination rate. Taken together, Burkholderia sp. SX9 had properties beneficial in the microbial enhancement of phytoremediation of soil contaminated with heavy metals.

Cytokine-Related Effect of Buccal-Delivered Collagen Peptide Incorporated in Mucoadhesive Films to Improve Female Skin Conditions

Recently, interest in collagen products has increased in the industries However, collagen products that are taken orally have the problem of being degraded by digestive enzymes. Therefore, a collagen peptide buccal delivery film (C-BDF) was developed to enhance the absorption without destruction and a clinical trial was conducted. A C-BDF was developed as a double layer and the permeation of collagen peptide (CP) through swine mucosa was investigated. This clinical study was performed on 43 healthy women, who were divided into either a control (n = 21) or test group (n = 22), over the course of 4 weeks. Skin assessments analyzed the hydration, elasticity, and roughness. In addition, the production of peroxynitrite and IL-1α in RAW 264.7 cells in supernatant media was conducted. A total of 1 kDa of CP in BDF showed significantly stronger permeation through swine mucosa compared to 3 kDa of CP in BDF. The C-BDF significantly enhanced skin hydration, elasticity, and roughness, and it removed wrinkles with no side effects after 2 weeks of intake. In addition, the production of peroxynitrite and IL-1α after the treatment with CP was significantly increased. Therefore, this study showed that collagen peptides could be completely absorbed into mucosa via a buccal delivery system and homeopathic effects might occur.

4-Octyl itaconate modified demineralized bone matrix scaffold improves bone repair by regulating early inflammation

Macrophages are involved in the early inflammation during tissue repair and play a distinctive role in maintaining the stability of the tissue microenvironment. However, it is still very difficult to accurately regulate the phenotypic transformation of macrophages during bone repair. The current study innovatively used 4-octyl itaconate (OI), a cell-permeable derivative of an endogenous anti-inflammatory metabolite itaconate (IA), to regulate the polarization of macrophages and enhance the quality of bone repair. Chitosan (CS) was selected as a bridge in coating OI on demineralized bone matrix (DBM) scaffold to generate three-dimensional (3D) bone integration scaffold (OI/CS/DBM scaffold). Whether this metabolite derivative can exert the same anti-inflammatory effect as the metabolite itself was validated both in vitro and in vivo. In vitro cellular results showed that both OI and OI/CS/DBM scaffold could promote the formation of anti-inflammatory phenotype of macrophages, and the osteogenic differentiation of mesenchymal stem cells (MSCs) was indirectly enhanced by macrophage supernatant medium. In vivo studies of a rat skull defect model further proved that the OI/CS/DBM scaffold could improve the bone integration. In this study, the OI/CS/DBM scaffold was successfully fabricated as a vehicle for delivering OI, which was proved to have the ability to promote bone integration by regulating early inflammation. The present results may provide a novel method to improve bone repair with metabolites derivative to regulate the inflammation.

Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases.Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at −20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing.Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at −20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group.Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at −20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

Functional analyses of three different mutations in the AVP-NPII gene causing familial neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI), a rare disorder, which is clinically characterized by polyuria and polydipsia, results from mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The aim of this study was to perform functional analyses of three different mutations (p.G45C, 207_209delGGC, and p.G88V) defined in the AVP-NPII gene of patients diagnosed with FNDI, which are not included in the literature. For functional analysis studies, the relevant mutations were created using PCR-based site-directed mutagenesis and restriction fragment replacement strategy and expressed in Neuro2A cells. AVP secretion into the cell culture medium was determined by radioimmunoassay (RIA) analysis. Fluorescence imaging studies were conducted to determine the differences in the intracellular trafficking of wild-type (WT) and mutant AVP-NPII precursors. Molecular dynamics (MD) simulations were performed to determine the changing of the conformational properties of domains for both WT and 207-209delGGC mutant structures and dynamics behavior of residues. Reduced levels of AVP in the supernatant culture medium of p.G45C and p.G88V transfected cells compared to 207_209delGGC and WT cells were found. Fluorescence imaging studies showed that a substantial portion of the mutant p.G45C and p.G88V AVP-NPII precursors appeared to be located in the endoplasmic reticulum (ER), whereas 207_209delGGC and WT AVP-NPII precursors were distributed throughout the cytoplasm. The mutations p.G45C and p.G88V cause a failure in the intracellular trafficking of mutant AVP-NPII precursors. However, 207_209delGGC mutation does not result in impaired cellular trafficking, probably due to not having any significant effect in processes such as the proper folding, gain of three-dimensional structure, or processing. These results will provide valuable information for understanding the influence of mutations on the function of the AVP precursor hormone and cellular trafficking. Therefore, this study will contribute to elucidate the mechanisms of the molecular pathology of AVP-NPII mutations.

HOXC13-AS Induced Extracellular Matrix Loss via Targeting miR-497-5p/ADAMTS5 in Intervertebral Disc.

Background/Aims: LncRNAs are a new modulator in the development of intervertebral disc degeneration. However, the functional role and mechanism of HOXC13-AS in intervertebral disc degeneration remain unclear. Methods: qRT-PCR analysis was performed to measure the relative expression levels of HOXC13-AS and miR-497-5p, and the levels of IL-1β, IL-6, and TNF-α in the medium supernatant were analyzed by ELISA. The related mechanism between HOXC13-AS and miR-497-5p was detected by luciferase assays. Results: The results revealed that TNF-α and IL-1β induced HOXC13-AS expression in NP cells. HOXC13-AS was overexpressed in IDD specimens compared to control specimens, and higher expression of HOXC13-AS was correlated with high Pfirrmann scores. Ectopic expression of HOXC13-AS promoted MMP-3 and ADAMTS4 and inhibited aggrecan and collagen II expression in NP cells. Furthermore, overexpression of HOXC13-AS increased the expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Our results demonstrated that TNF-α and IL-1β induced ADAMTS5 expression and suppressed miR-497-5p expression. miR-497-5p was downregulated in IDD specimens compared to control specimens, and the lower expression of miR-497-5p was correlated with high Pfirrmann scores. The miR-497-5p level was negatively proportional to HOXC13-AS expression in IDD specimens. Luciferase analysis data indicated that ADAMTS5 was a direct target gene of miR-497-5p. HOXC13-AS induced inflammatory cytokine expression and ECM degradation by modulating miR-497-5p/ADAMTS5. Conclusion: HOXC13-AS may be a treatment target for IDD.

Immune regulatory effect of tacrolimus in patients with severe aplastic anemia and in murine model

Objective: To investigate the immunomodulatory effect of tacrolimus in severe aplastic anemia (SAA). Methods: Patients diagnosed with SAA at the Department of Hematology, General Hospital of Tianjin Medical University from June 2015 to January 2018 were enrolled. CD8+T cells were sorted by immunomagnetic separation from peripheral blood of SAA patients. MTT method was used to detect the proliferation of CD8+T cells. The SAA mouse model was established by total body irradiation (TBI) and donor lymphocyte infusion (DLI). There were 10 normal controls without pretreatment, 10 rats in TBI group, 15 rats received TBI and DLI. The expression of perforin and granzyme in CD8+T cells and the ratio of CD4+/CD8+cells in peripheral circulation were measured by flow cytometry. The level of interferon-γ (IFN-γ) in medium supernatant of cultured CD8+T cells was tested with enzyme-linked immunosorbent assay (ELISA). SAA mouse model was established to study the recovery of hemogram and survival time after treatment. Results: A total of 16 SAA patients were enrolled, and there were 10 males and 6 females, with a median age of 35 (22-49) years. Tacrolimus inhibited the proliferation of CD8+T cells when IL-2 concentration was 20.0,200.0 and 2 000.0 U/ml (P<0.05). The expression of perforin in CD8+T cells of SAA patients treated with tacrolimus was significantly lower than that in blank control group and IL-2 group [(2.25±0.76)%, (6.70±0.82)% vs (9.10±1.90)%,all P<0.05]. The level of IFN-γ in CD8+T cells group after applying tacrolimus was significantly lower than that in the blank control group (P<0.05). After 10 days of administration, the peripheral blood hemoglobin (Hb), white blood cell (WBC) and platelet (PLT) counts of SAA mice in tacrolimus group were all higher than those in SAA group (all P<0.05). The expression of perforin in CD8+T cells in tacrolimus group was significantly lower than that in SAA group [(18. 39±6.65) vs (29. 99±9.83),P<0.05]. The median survival time of SAA group was 18.6 days, and the 90 day survival rate was 0. The median survival time of tacrolimus group was 44.6 days, and the 90 day survival rate was 80%. The survival time of SAA mice in tacrolimus group was significantly longer than that in SAA group (P<0.05). Conclusion: The immunomodulatory effect of tacrolimus in SAA is similar to CsA. It has an immunosupressive effect on CD8+T lymphocyte.

Regulatory T Cells Promote the Stemness of Acute Myeloid Leukemia Cells through IL10 Cytokine Related Signaling Pathway

Regulatory T cells (Tregs) could maintain the stemness property and inhibit the differentiation of hematopoietic stem/progenitor cells (HSPCs) in normal hematopoietic system. Recent studies have shown that Tregs, as an important component of acute myeloid leukemia (AML) microenvironment, helped AML cells to evade immunoesurveillance. However their function in regulating the stemness property of AML cells remains elusive.In this study, we analyzed the proportion of Tregs and leukemic stem cells (LSCs) in 68 patients' bone marrow, and found that the proportion of LSCs in Tregs high group (Tregs ≥ 9%) was higher than that in Tregs low group (Tregs < 9%) (Figure A). So, we hypothesized that Tregs up-regulated the stemness property of AML cells. Therefore, we treated Kasumi-1 and SKNO-1 with the supernatant of Tregs culture medium (condition medium, CM) which was isolated from donors, and verified that the proportion of side population (SP) cells was increased (Figure B) and the efficiency of sphere formation was enhanced (Figure C) by CM treatment. Further, Tregs promoted the initiation of AML and shortened the life span of mice in AML1-ETO mice model (Figure D).As is well known that, Tregs could secrete IL10 cytokine to establish a suppressive microenvironment. Therefore, we analyzed the expression level of IL10 in 35 patients' bone marrow, and found that the expression of IL10 in Tregs high group was higher than that in Tregs low group (Figure E), meanwhile, the expression of IL10 in LSCs high group (LSCs ≥ 5%) was higher than that in LSCs low group (LSCs < 5%). Thus, we treated Kasumi-1 and SKNO-1 with human recombinant IL10 cytokine (rhIL10). rhIL10 treatment increased proportion of SP, enhanced the efficiency of sphere formation and induced both activating phosphorylation of PI3K and AKT at S473 with similar kinetics in a dose-dependent (with concentrations ranging from 1 to 100 nM) manner. Given the ability of AKT to increase the expression of OCT4 and nanog, we analyzed changes in the expression of OCT4 and nanog. As expected, expression levels of OCT4 and nanog increased after treatment with IL10.To investigate whether IL10 is responsible for stemness property induction, we blocked PI3K/AKT signaling pathway by IL10R neutralizing antibody (IL10R Ab) and PI3K/AKT inhibitor- LY294002 following CM treatment. In contrast to the control (CM treatment only), LY294002 and IL10R Ab could greatly reduce the proportion of SP, lessen the efficiency of sphere formation, and decrease the expression of OCT4 and nanog.Consequently, our study revealed that Tregs increased the proportion of LSCs in AML through IL10/PI3K/AKT/OCT4/nanog signaling pathway and indicated that targeting on the communication between Tregs and AML cells might be a promising strategy in AML therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bio-Preservative Potential of Microorganisms Isolated from Red Grape against Food Contaminant Fungi.

Fungal spoilage is one of the main reasons of economic losses in the food industry, especially in the wine sector. Consequently, the search for safer and new preservation techniques has gained importance in recent years. The objective of this study was to investigate the antifungal and anti-mycotoxigenic activity from 28 microorganisms (MO) isolated from red grape. The antifungal activity of a cell free supernatant of fermented medium by the isolated MO (CFS) was tested with the agar diffusion method and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) assay. Additionally, different antifungal compounds from the CFS were identified and quantified (organic acids, phenolic compounds, and volatile organic compounds). Finally, the most active CFS were tested as red grape bio-preservative agents. Results evidenced that CFS fermented by the strain UTA 6 had the highest antifungal activity, above all isolates, and produced a wide pool of antifungal compounds. The use of UTA 6 CFS as bio-preservative agent showed a reduction of 0.4 and 0.6 log10 spores per gram of fruit in grapes contaminated by A. flavus and B. cinerea, respectively. Moreover, UTA 6 CFS treatment reduced the occurrence of aflatoxin B1 and fumonisin (B2, B3, and B4) production in grapes contaminated by 28–100%.

Bioleaching of Gold from Silicate Ore by Macrococcus caseolyticus and Acinetobacter calcoaceticus: Effect of Medium, Amino Acids and Growth Supernatant

The aims of this work were to study the gold leaching by the isolated bacteria from silicate ore. Three strains were isolated and identified as Macrococcus caseolyticus, Acinetobacter calcoaceticus, and Bacillus sp. MBEA40. However, only M. caseolyticus and A. calcoaceticus were capable of gold bioleaching. In order to examine only the effect of microorganisms involved in the gold bioleaching process, minimal medium and ethanol mineral salt medium without amino acids were used for culturing M. caseolyticus and A. calcoaceticus, respectively. The result showed that the growth supernatant (in the absence of microorganisms) of both strains might be more suitable to leaching gold from ore than leaching by microorganisms (in the presence of microorganisms) directly. This might be due to the fact that there is no interference of gold absorption and metal toxicity in microorganisms in the long-term operation. The result also confirmed that amino acids/peptides/proteins produced by microorganisms might be involved in gold bioleaching, as shown in the high-performance liquid chromatography (HPLC) results. The Fourier transform infrared spectroscopy (FTIR) study also found that amine groups and carboxylic groups played important roles in gold bioleaching by M. caseolyticus and A. calcoaceticus. In addition, the bioleaching process had significantly higher gold leaching than mixed pure amino acids due to the growth supernatant containing mixed amino acids/peptides/proteins and other compounds. Therefore, the growth supernatant of M. caseolyticus and A. calcoaceticus can be applied in gold bioleaching under neutral pH conditions, which is considered to be a safe, not corrosive, and environmentally friendly leaching process. This study is also needed further study in order to increase the percentage of gold bioleaching and decrease times.

Metformin alters therapeutic effects in the BALB/c tumor therapy model

BackgroundDespite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin.MethodsCytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting.ResultsInitial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells.ConclusionIn conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.Graphical abstract

Pulsatilla Decoction Combined with 5-Fluorouracil Triggers Immunogenic Cell Death in Colorectal Cancer Cells.

Background: Our research is designed to explore the role of 5-FU and Pulsatilla decoction (PD) through modulation of Immunogenic cell death (ICD) for the co-treatment of Colorectal cancer (CRC). Materials and Methods: Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assays. Cell apoptosis was assessed using flow cytometry. Phosphorylation of STAT3 and expression of Mcl-1 and Bcl-xl were measured by Western blot assays. The levels of ATP and HMGB1 in the supernatants of the culture medium were analyzed by ATP assays and the HMGB1 enzyme linked immunosorbent assay kit. The cell surface levels of CRT were measured by immunofluorescence assays. The tumor growth was analyzed in mice. Results: PD increased 5-FU-induced ICD in CRC cells, as demonstrated by the extracellular levels of adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), and the surface levels of calreticulin (CRT). Our mechanism study showed that PD promoted 5-FU-induced ICD by inactivating signal transducer and activator of transcription 3 (STAT3). Furthermore, the co-treatment of 5-FU and PD further promoted 5-FU-induced CRT expression and T cell infiltration in vivo. Tumorigenicity analysis revealed that 5-FU combined with PD notably reduced tumor growth. Conclusion: This study indicated that PD enhances 5-FU-induced ICD and anti-tumor effect in CRC by inactivating STAT3. The combined application of 5-FU with PD may improve the anti-tumor activity of 5-FU in CRC.

Treatment optimization of patients with genital endometriosis

The aim of the research: to optimize the treatment of patients with combined genital pathology, including internal endometriosis (adenomyosis) and inflammatory diseases. Materials and methods: prospective study has been conducted on 160 women with adenomyosis. There were 24 (15 %) patients with the I degree of adenomyosis spreading, 72 (45.0 %) women with the II degree, 33 (20.6 %) patients with the III degree, and 31 (19.4 %) woman with the IV degree of adenomyosis spreading. Microbial flora analysis included bacterioscopic, bacteriological research methods with determination of sensitivity to antibiotics, and PCR method. The concentration of cytokines in the culture medium (supernatant) was determined by the enzyme immunoassay. Results: the obtained data from the study indicate a high percentage of the combination of adenomyosis with chronic inflammatory diseases of the pelvic organs. An immuno-inflammatory reaction preceding adenomyosis is accompanied by the violation of the local cytokine balance. In turn, the increased activity of cytokines and the presence of infectious agents can participate in the relapse of endometriosis. Conclusion: considering the immuno-inflammatory reaction, accompanied by the violation of the local cytokine balance in the development of adenomyosis. The study substantiates the necessity of using antimicrobial therapy in patients with combined genital pathology, including adenomyosis and inflammatory diseases

The culture microenvironment of juvenile idiopathic arthritis synovial fibroblasts is favorable for endochondral bone formation through BMP4 and repressed by chondrocytes

BackgroundWe examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF).MethodsPrimary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA.ResultsIn culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFβ superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2.ConclusionsJFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.