24-h Culture Research Articles

A tool for evaluating novel osteoarthritis therapies using multivariate analyses of human cartilage-synovium explant co-culture

There is a need to incorporate multiple tissues into in vitro OA models to evaluate novel therapeutics. This approach is limited by inherent donor variability. We present an optimized research tool: a human OA cartilage-synovium explant co-culture model (OA-EXM) that employs donor-matched lower and upper limit response controls combined with statistical approaches to address variability. Multiple rapid read-outs allow for evaluation of therapeutics while cataloguing cartilage-synovium interactions. 48-h human explant cultures were sourced from OA knee arthroplasties. An OA-like cartilage-synovium co-culture baseline was established relative to donor-matched upper limit supraphysiological pro-inflammatory cytokine and lower limit OA cartilage or synovium alone controls. 100nM dexamethasone treatment validated possible "rescue effects" within the OA-EXM dual tissue environment. Gene expression, proteoglycan loss, MMP activity, and soluble protein concentrations were analyzed using blocking and clustering methods. The OA-EXM demonstrates the value of the co-culture approach as the addition of OA synovium increases OA cartilage proteoglycan loss and expression of MMP1, MMP3, MMP13, CXCL8, CCL2, IL6, and PTGS2, but not to the extent of supraphysiological stimulation. Conversely, OA cartilage does not affect gene expression or MMP activity of OA synovium. Dexamethasone shows dual treatment effects on synovium (pro-resolving macrophage upregulation, protease downregulation) and cartilage (pro-inflammatory, catabolic, and anabolic downregulation), and decreases soluble CCL2 levels in co-culture, thereby validating OA-EXM utility. The OA-EXM is representative of late-stage OA pathology, captures dual interactions between cartilage and synovium, and combined with statistical strategies provides a rapid, sensitive research tool for evaluating OA therapeutics.

Cytokine TNF-α and its receptors TNFRI and TNFRII play a key role in the in vitro proliferative response of BLV infected animals.

Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48h. PBMC from BLV-infected animals showed spontaneous proliferation after 48h in culture but did not show changes in proliferation rates after 48h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.

Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

BackgroundH. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer.ResultsA 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.ConclusionsA VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

Hierarchically Assembled Type I Collagen Fibres as Biomimetic Building Blocks of Biomedical Membranes.

Wet spinning is an established fibre manufacturing route to realise collagen fibres with preserved triple helix architecture and cell acceptability for applications in biomedical membranes. However, resulting fibres still need to be chemically modified post-spinning to ensure material integrity in physiological media, with inherent risks of alteration of fibre morphology and with limited opportunities to induce fibrillogenesis following collagen fixation in the crosslinked state. To overcome this challenge, we hypothesised that a photoactive type I collagen precursor bearing either single or multiple monomers could be employed to accomplish hierarchically assembled fibres with improved processability, macroscopic properties and nanoscale organisation via sequential wet spinning and UV-curing. In-house-extracted type I rat tail collagen functionalised with both 4-vinylbenzyl chloride (4VBC) and methacrylate residues generated a full hydrogel network following solubilisation in a photoactive aqueous solution and UV exposure, whereby ~85 wt.% of material was retained following 75-day hydrolytic incubation. Wide-angle X-ray diffraction confirmed the presence of typical collagen patterns, whilst an averaged compression modulus and swelling ratio of more than 290 kPa and 1500 wt.% was recorded in the UV-cured hydrogel networks. Photoactive type I collagen precursors were subsequently wet spun into fibres, displaying the typical dichroic features of collagen and regular fibre morphology. Varying tensile modulus (E = 5 ± 1 − 11 ± 4 MPa) and swelling ratio (SR = 1880 ± 200 − 3350 ± 500 wt.%) were measured following post-spinning UV curing and equilibration with phosphate-buffered saline (PBS). Most importantly, 72-h incubation of the wet spun fibres in PBS successfully induced renaturation of collagen-like fibrils, which were fixed following UV-induced network formation. The whole process proved to be well tolerated by cells, as indicated by a spread-like cell morphology following a 48-h culture of L929 mouse fibroblasts on the extracts of UV-cured fibres.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

BackgroundCultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations).MethodsTime-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs.ResultsTo minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs.ConclusionsThe bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Abstract 2888: Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture

Abstract The cytotoxicity and cytostatic effects of imipridone family drugs ONC201 and its analog TR57 (Madera, USA) in variety of cultured breast cancer cell line (estrogen dependent BT474) was manifested through induction of ISR (Integrated Stress Response), UPRER and UPRMT. The major consequences of drug treatment are inhibition of proliferation and mitochondrial structural and functional damage(s). Our preliminary data demonstrated that 24 h exposure of BT474 leads to dysregulation and suppression in mitochondrial Ca2+ uptake and Ca2+ capacity (Odinokova et al., 2021). In the present work, we extend our study to include the effect of these drugs on the rate of migration intact and treated cells in culture. Treatment of BT474 cells in culture for 24h resulted in decreased motility of cells from 17 ± 2 µm/h in the absence of the drugs to 5 ± 1 µm/h and to 3±1 µm/h in cells treated with 10µM of ONC201 or 50nM of TR57 (n=5). This suppression was fully reversible and removal of drugs for 120h resulted in time dependent restoration of the initial motility of BT474 cells, and TR57 caused larger inhibitory effect on motility as compared with ONC201. Buffering of extracellular Ca2+ with 5 mM EGTA or intracellular Ca2+ with 10 µM BAPTA, 30 min suppressed motility of cells (19 ± 2 µm/h vs 6 ± 1 µm/h). Gd3+, selective blocker of store operated Ca2+ entrance (SOCE) pathway at concentration of 10 µM suppressed the motility of BT474 cells, and rate of migration changed for from 16 ± 2 µm/h in control to 7± 2 µm/h, in Gd3+ treated cells. In line with our preliminary data, taken together these data indicate that ONC201 and/or TR57 induced dysfunction in mitochondrial Ca2+ homeostasis, which translates into the suppression of the migration of human breast cancer cells in culture. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Yana Evstratova, Margarita Kobyakova, Irina Odinokova, Alexander Stolyarov, Artyom Mishukov, Lee M. Graves, Ekhson Holmuhamedov. Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2888.

Albumin used in human IVF contain different levels of lipids and modify embryo and fetal growth in a mouse model.

Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown. Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography. Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05). Indicates the importance of a balance between different types/amount of lipids, and an "optimal ratio" required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF.

Genomic Effects of Arginine on Cell Behavior of Conceptus Trophectoderm in Ungulates

Abstract Hatched ungulate (e.g., pigs, sheep and other ruminants) blastocysts undergo dramatic morphological transitions from spherical to tubular to filamentous forms to conceptuses (embryo/fetus and associated extraembryonic membranes) before implantation. L-Arginine (Arg), a conditionally essential amino acid, is required for this process to activate the mTOR cell signaling pathway to induce proliferation of both porcine and ovine conceptus trophectoderm cells. However, the genomic effects of arginine on trophectoderm cells is unknown. RNA-seq was used for a comparative transcriptome analysis of porcine and ovine trophectoderm cells to further understand effects of Arg on regulation of metabolism in trophectoderm cells. An established porcine trophectoderm (pTr) cell line isolated from D12 porcine conceptuses, as well as an established ovine trophectoderm (oTr) cell line isolated from D15 ovine conceptuses were used to determine response to Arg at the physiological concentration of 0.2 mM in a 48-h culture. In pTr cells, a total of 2,723 differentially expressed genes (DEG; 1,482 up and 1,241 down) were identified in response to Arg. In oTr cells, a total of 5,369 DEG (2,819 up and 2,550 down) were detected. Comparison analyses showed that the Arg-treated pTr and oTr transcriptomes share 873 common DEG (273 up and 342 down). Canonical pathway analyses identified the top enriched pathways in both pTr and oTr cells, including activation of actin cytoskeleton signaling, adrenomedullin signaling, and IGF-1 signaling; and inhibition of cell cycle G2/M checkpoint regulation, and p53 signaling. In response to Arg, pathways associated with cholesterol biosynthesis, and estrogen-mediated S-phase entry were exclusively activated in the pTr cells; whereas interferon signaling, ephrin receptor signaling, and integrin signaling were specifically activated in the oTr cells. Results from this study advance understanding of mechanisms responsible for elongation of ovine and porcine conceptuses and enable the rational design of future experiments.

Bisphenol B stimulates Leydig cell proliferation but inhibits maturation in late pubertal rats

Bisphenol B (BPB) has been used as a substitute for bisphenol A (BPA) in plastic materials. Whether BPB disrupts the male reproductive system remains unknown. Here, we report the effect of BPB on Leydig cell maturation in late puberty. Male Sprague-Dawley (35 days old) rats were gavaged with BPB at 0, 10, 100, and 200 mg/kg/day for 21 days. BPB significantly reduced body and epididymis weight at 200 mg/kg. BPB markedly decreased serum testosterone levels at 100 and 200 mg/kg and serum luteinizing hormone and follicle-stimulating hormone levels at 200 mg/kg. BPB significantly increased Leydig cell number at 100 and 200 mg/kg, while down-regulating the expression of Leydig cell genes (Cyp11a1 and Hsd3b1) at ≥100 mg/kg and up-regulating the expression of Sertoli cell genes (Pdgfra, Fshr, Sox9) and cell cycle regulators (Pcna, Ccnb1, Cdk2, and Cdk4) at 10–200 mg/kg. BPB markedly increased the phosphorylation of AKT1, AKT2, and ERK1/2 at 200 mg/kg. BPB increased the proliferation of rat immature Leydig cells via promoting the S/M2 phase shift at 100 and 1000 nM after 24-h culture in vitro. In conclusion, BPB disrupts Leydig cell maturation in late puberty by increasing Leydig cell number while inhibiting its maturation.

Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells.

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte–macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

Fabrication of three dimensional bioactive Sr2+ substituted apatite scaffolds by gel-casting technique for hard tissue regeneration.

This study aimed to fabricate three-dimensional (3D) bioactive Sr2+ -substituted apatite (Sr-HAP) scaffolds prepared by gel-casting with polymer sponge infiltration technique. 3D Sr-HAP scaffolds were prepared as engineering constructs with interconnected porous structure with a pore size of 200-600μm ranging from a 10×10×6mm size was designed. The characterization of X-ray diffraction, field emission scanning electron microscopy, and energy dispersion spectroscopy was utilized in order to evaluate the crystalline phase, structure, and morphology in the interconnected porous of the synthesized Sr-HAP scaffold. The bioactive and biocompatible of the resultant Sr-HAP scaffolds were analyzed by using simulated body fluid solution. Furthermore, the cytotoxicity and proliferation of MG-63 cell lines on the scaffolds were examined in 24h culture. Furthermore, in vivo experiments demonstrated that the tibia bone defect with 4mm diameter in rabbits was successfully healed by Sr-HAP porous scaffold after 45days implantation. The histological images indicated the improved cell proliferation and new bone formation occurred in the porous scaffold treated group. The results indicated that the fabricated Sr-HAP scaffold is a promising capacity to infuse bone regeneration and promote in vivo tissue repair.

Bacteriocinogenic Potential of Bacillus amyloliquefaciens Isolated from Kimchi, a Traditional Korean Fermented Cabbage.

Bacteriocin production is considered a favorable property for various beneficial cultures. In addition to their potential as biopreservatives, bacteriocins are also promising alternatives for the control of multidrug-resistant pathogens and the inhibition of some viruses and cancer cells. The objective of this study was to screen and characterize a bacteriocin-producing strain with the aim of its future application for control of Listeria monocytogenes, an important food-borne pathogen. A total of 22 potentially bacteriocinogenic strains active against L. monocytogenes ATCC15313 were isolated from locally produced kimchi through a three-level approach. Pure cultures were obtained according to good microbiological practices and differentiated through RAPD-PCR using the primers OPL01, OPL09, and OPL11. Altogether, 5 strains were selected for further study. Specific focus was given to strain ST05DL based on its specific inhibitory activity against L. monocytogenes ATCC15313, while not affecting different strains belonging to the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella, most of which are beneficial microorganisms. The strain ST05DL was identified as Bacillus amyloliquefaciens based on its sugar fermentation profile obtained through API50CHB analysis and 16S rRNA partial sequencing. The antimicrobial compound produced by B. amyloliquefaciens ST05DL was found to be sensitive to pepsin and α-chymotrypsin, evidence of its proteinaceous nature. The presence of skim milk, NaCl, Tween 80, glycerol, and SDS did not affect the antimicrobial activity. The addition of 20% cell-free supernatant (CFS) obtained from a 24-h culture of B. amyloliquefaciens ST05DL to an exponentially growing culture of L. monocytogenes ATCC15313 successfully inhibited the test microorganisms during the monitored 10-h incubation. Optimal bacteriocin production by B. amyloliquefaciens ST05DL was observed during the stationary phase at 12 h (800 AU/mL) and remained stable for the next 15 h. The ratio between live and dead cells during this period was 74.37% and 25.66%, respectively, as determined by flow cytometry. The presence of the virulence genes hblA, hblB, hblC, nheA, nheB, and nheC was not detected in the total DNA of B. amyloliquefaciens ST05DL, and the strain was resistant only to ampicillin out of 10 tested antibiotics. Future evaluation of expressed bacteriocin/s by B. amyloliquefaciens ST05DL (amino acid sequence, molecular mass, cytotoxicity, detailed mode of action, etc.), will be the next step in the characterization and its potential application as biopreservative and/or pharmaceutical product.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1β, TGF-β, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1β that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-β (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-β expression and down-regulation of IFN-γ and IL-1β expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.

Long lasting protective effects of early l-arginine treatment on endothelium in an in vitro study

Excess nutrient supply, such as high fat and high glucose intake, promotes oxidative stress and advanced glycation end products accumulation. Oxidative stress and AGE accumulation cause pathological elevation of arginase activity and pro-inflammatory signaling implicated in endothelial dysfunction. Several studies showed positive effects of l-arginine supplementation in endothelial function but little is currently known about the role of l-arginine as prevention of endothelial dysfunction caused by excessive nutrient supply (overfeeding). Our aim was to evaluate a possible protective effect of l-arginine on endothelial dysfunction caused by excessive nutrient supply (overfeeding), using human endothelial cells line in an invitro study. Endothelial EA.hy926cells were pre-treated with 1.72mM of l-arginine for 24h and afterwards subjected to nutritional stress (high lipid, high insulin and high glucose concentrations) for further 24h. After treatment discontinuation, the cells were kept in culture for 48h, in physiological condition, to evaluate the effects of treatments after normalization. Excess nutrient supply in EA.hy926cell line showed an increase of oxidative and nitrosative stress, a rise of AGEs production, high arginase activity, leading the cells to acidosis and to cell death. l-arginine pretreatment protects the cells by reducing apoptosis, acidosis, oxidative and nitrosative stress, arginase activity and AGE accumulation. l-arginine pretreatment reduces AGEs generation and accumulation by regulating STAB1 and RAGE gene expression levels. STAB1, acting as receptor scavenger of AGEs, interferes with AGE-RAGE binding and thus prevents activation of intracellular signaling pathways leading to cell damage. Moreover the reduction of oxidative stress promotes a decrease of excessive activation of arginase involved in endothelial dysfunction. The effects of pretreatment with l-arginine last even in the absence of stimuli and despite after treatment discontinuation. An early l-arginine treatment is able to prevent oxidative stress and AGEs accumulation caused by overfeeding in human endothelial cell line by regulating STAB1/RAGE gene expression and by reducing excess arginase activity. The positive effects of l-arginine pretreatment continue even after treatment discontinuation in normal conditions.

Characterization of stem cells from human ovarian follicular fluid; a potential source of autologous stem cell for cell-based therapy.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluidexhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.

Nicosulfuron inhibits atrazine biodegradation by Arthrobacter sp. DNS10:Influencing mechanisms insight from bacteria viability, gene transcription and reactive oxygen species production.

Nicosulfuron is a sulfonylurea family herbicide which is commonly applied together with the triazine herbicide atrazine in agricultural practice. However, whether nicosulfuron can influence the biodegradation of atrazine is unclear. Therefore, the influence of nicosulfuron on atrazine removal as well as on cell viability and transcription of atrazine chlorohydrolase gene (trzN) in Arthrobacter sp. DNS10 was investigated in this study. Our results demonstrated that 76.0% of atrazine was degraded in the absence of nicosulfuron after 48h of culture, whereas 63.9, 49.1 and 42.6% was degraded in the presence of 1, 5, and 10mg/L of nicosulfuron, respectively. Nicosulfuron also induced an increase in the level of intracellular reactive oxygen species (ROS), thereby damaging the cell membrane integrity and inhibiting the growth of the strain DNS10. Flow cytometry analysis revealed that the cell viability of strain DNS10 decreased with an increase in nicosulfuron concentration. The transcription of trzN in strain DNS10 exposed to the three described levels of nicosulfuron was 0.99, 0.72 and 0.52 times, respectively, that without nicosulfuron. In brief, nicosulfuron could inhibit atrazine removal efficiency by strain DNS10 by inducing the over-production of ROS which ultimately enhances the population of membrane-damaged cells, as well as reducing cell viability and trzN transcription. The outcomes of the present study provide new insights into the mechanism of nicosulfuron inhibition on atrazine biodegradation by strain DNS10.

Novel Amides Derivative with Antimicrobial Activity of Piper betle var. nigra Leaves from Indonesia.

Piper betle var. nigra is a tropical plant closely related to the common piper. P. betle has also been dubbed a promising source of natural antioxidants in herbal health products, antibacterial, antifungal, antimalarial, cytotoxic activity against the cancer cell lines K562 and HL-60, and antileishmanial. The aim of this study to observation Antimicrobial activity and isolation of chemical compound. The antimicrobial activity of P. betle extract was performed by well diffusion method against two oral pathogenic bacteria (Streptococcus mutans and Streptococcus sanguinis) and opportunistic pathogenic yeast (Candida albicans). The inoculum (bacterial and yeast suspension) was prepared from a 24-h culture on NB for bacterial suspension and on TSB for yeast suspension. Extraction and isolation using various method of chromatography. Isolated compounds were characterized by spectroscopic means. Our study showed antimicrobial activity from crude ethanol extract of leaves P. betle L. var. nigra against two oral pathogenic bacteria and opportunistic pathogenic yeast with concentration 0.5% and 1%. The first report of two new amides derivatives, piperenamide A (1) and piperenamide B (2) in P. betle L. var. nigra.

Corrective effects of original bioflavonoid complex in the cyclophosphamide-induced immunity disorders

Our aim was to evaluate immunomodulatory properties of an original bioflavonoid complex in experimental immune disturbances induced by cyclophosphamide (Cy). We have studied morphometric indexes of thymus and spleen, as well as blood leukocyte counts, cell proliferative activity in lymphoid organs, delayed hypersensitivity responses to T cell-dependent antigen, along with differentiation activity of bone marrow stem cells in experimental animals during Cy-induced immune suppression after a course of bioflavonoid treatment. Suspension of the bioflafonoid complex was introduced to the male mice (СВАхC57Bl/6)F1 aged 12- 14 weeks at a daily dose of 2 mg/animal (80 mg/kg), per os, using gastric catheter, over 14 days. Cytostatic immunosuppression was produced by a single intraperitoneal Cy injection. Proliferative activity of spleen and thymic cells was determined by standard method with Н3 -thymidine incorporation in the 72-h cell culture. Cellular immune response was assayed by the degree of delayed-type hypersensitivity development in response to sheep erythrocytes. The number of hematopoietic progenitors was evaluated by culturing bone marrow cells in methylcellulose-based medium. The experiments have shown mitigation of immunosuppressive effects induced by Cy, in the course of bioflavonoid complex treatment, with respect to absolute and relative mass of lymphoid organs and leukocyte numbers in peripheral blood. Moreover, we have demonstrated decreased effects of Cy treatment upon the spontaneous activity of spleen cells, mitogen-induced thymocyte and splenocyte proliferation, intensivity of delayed-type hypersensitivity response that reached the values of intact animals. Following the course of bioflavonoids, we have revealed an increase in early hematopoietic progenitors. Alleviation of Cy-induced suppressive effects upon cellular immune response, proliferation rates of immune cells, as well as stimulation of hematopoietic stem cell functions suggest a sufficient capacity of the original bioflavonoid complex for modulation of immunity and hematopoiesis, thus presenting experimental proofs for its potential usage as an adjuvant treatment of the patients with malignant diseases.

A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.