24-h Culture Research Articles

Purification, characterization and cloning of a chitinase from Stenotrophomonas rhizophila G22.

In the presented research the extracellular chitinase of Stenotrophomonas rhizophila G22 was biochemically and molecularly characterized. The studied enzyme was purified from a 72-h bacterial culture about 14 times, with a recovery of 63%. The molecular weight of the purified protein was estimated at 50kDa by SDS-PAGE. The enzyme showed high activity against colloidal chitin. Significantly lower activities were observed with native chitin powder and chitosan. Adsorption of the enzyme to colloidal chitin and to powdered chitin at the level of 75% and 37%, respectively, was observed after 30min of reaction. Optimum temperature and pH were 37°C and 5.9, respectively. The enzyme demonstrated higher activity against nitrophenyl-β d N, N', N″-triacetylchitotriose and approx. 5 times lower activity for 4-nitrophenyl-N, N'-diacetyl-β-d-chitobiose. The enzyme is an endochitinase, which is confirmed by the K m and V max values determined in the studies. S. rhizophila G22 endochitinase was inhibited in the presence of cysteine-specific inhibitors, which indicates the role of cysteine moieties in the mechanism of catalysis or in stabilisation of the enzyme molecule. Also Ca2+ and Mn2+ ions may stabilise the protein's spatial structure. SDS and ions: Fe2+, Cu2+, Co2+, Zn2+ inhibited the activity of enzyme. A full-length (2109bp) gene coding chitinase from S. rhizophila G22 was obtained. Four domains typical for glycoside hydrolase family 18 (GH 18) chitinases were identified: catalytic Gly_18, chitin-binding-ChtBD3, type-III fibronectin-FN3 and polycystic kidney disease domain-PKD domain.

Kinetics on aggregate behaviors of human induced pluripotent stem cells in static suspension and rotating flow cultures

Understanding of kinetics on aggregate behaviors of human induced pluripotent stem cells (hiPSCs) is critical knowledge for culture design because aggregate behaviors are considered to affect cell growth. In this study, we elucidated kinetics on aggregate behaviors of two types of hiPSCs (253G1 and 201B7 lines) to clarify the influence of aggregate behaviors on cell growth by comparing aggregate morphology, size of cell aggregates, and kinetic parameters in 72h culture under static and floating conditions, which were realized by multi-dimple plate and rotating wall vessel, respectively. In the case of 253G1 line under floating condition, aggregate number decreased and size increased drastically during culture time, t= 0-24h due to coalescence between cell aggregates. The apparent specific growth rate decreased after t= 24h although cell number and aggregate size gradually increased under static condition. In the case of 201B7 line under floating condition, cell and aggregate number, and aggregate size kept constant levels during t= 24-72h due to collapse of cell aggregates by stripping of single cells from aggregate, suggesting that specific death rate increased after t= 24h despite constant levels of apparent specific growth rate and aggregate number under static condition. Our kinetic analysis concluded that excessive increase of aggregate size due to coalescence and cell death due to collapse critically affected growth of hiPSCs in suspension culture.

Synthesis and cellular affinity of a water-soluble sulfated diselenide compound as a H2O2-responsive ionic cross-linker

Because various stimuli can affect living organisms, substances that response to stimuli, such as H2O2, have been studied for biomaterials. To synthesize a biocompatible H2O2-responsive ionic cross-linker, we synthesized a water-soluble H2O2-responsive diselenide compounds containing both sulfate end groups (diSe-SO4). The synthesis of diSe-SO4 was evaluated by 1H NMR and FT-IR. H2O2 responsiveness was evaluated by UV–vis spectra, FT-IR, and XPS. The optical density at 303nm due to diselenide in diSe-SO4 was decreased in the H2O2 concentration increased. FT-IR and XPS results showed new peaks at 880cm and 3500cm−1, and at 63.0eV, respectively, due to oxidized Se, which indicated the cleavage of the diselenide bond in diSe-SO4 as the H2O2 concentration increased. The ionic binding efficiency of diSe-SO4 to cationic materials was confirmed by measuring the zeta-potential difference (from –15.6mV to 10.3mV) before and after mixing with chitosan. MRC-5 cell viability and apoptosis in the presence of diSe-SO4 was evaluated using a CCK-8 assay and FACS, respectively. A high cell viability exceeding 95% was observed at 500μMdiSe-SO4 over 48h of culture. In conclusion, the synthesized water-soluble diSe-SO4 provides a H2O2-responsive ionic cross-linking material with a high cell affinity.

42 Vitrification of invitro-produced feline embryos

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of invitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n=32), vitrification with EG (n=31), or control (n=47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5mL of 1M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1min. Next, embryos were moved to 0.5mL of 0.5M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3min. Finally, embryos were transferred to 0.5mL of FOCMH for 5min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24h after warming (84%) than embryos vitrified in PrOH (59%; P<0.05). The continued embryonic growth of viable embryos after culture for 48h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P>0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.

183 Effects of epidermal growth factor and progesterone on invitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that invitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n=400) were aspirated from antral follicles (1-3mm) and cultured for 48h in control medium (TCM-199 plus 5.0mgmL−1 LH, 0.5mgmL−1 FSH, and 5% fetal bovine serum) alone or with EGF (10ngmL−1), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20h in TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, and 10μM cilostamide. Then, COCs were matured invitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P<0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48h of culture for COCs in all groups (P<0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P<0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P<0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P<0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P<0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration invitro.

28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles

BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

Characterization of gallium resistance induced in a Pseudomonas aeruginosa cystic fibrosis isolate.

The repurposing of gallium nitrate as an antibacterial, a drug used previously for the treatment of hypercalcemia, is a plausible alternative to combat infections by Pseudomonas aeruginosa, since it has antipseudomonal properties in vitro and in vivo in animal models and in human lung infections. Furthermore, gallium nitrate tolerance in clinical isolates is very rare. Nevertheless, studies on the reference strains PA14 and PAO1 show that resistance against gallium nitrate is achieved by decreasing gallium intracellular levels by increasing the production of pyocyanin. In this work, we induced resistance in a cystic fibrosis P. aeruginosa isolate and explored its resistance mechanisms. This isolated strain, INP-58M, was not a pyocyanin producer, and its pyoverdine levels remained unchanged upon gallium addition. However, it showed higher activities of NADPH-producing enzymes and the antioxidant enzyme SOD when gallium was added, which suggests a better antioxidant response. Remarkably, gallium intracellular levels in the resistant isolate were higher than those of the parental strain at 20h but lower after 24h of culture, suggesting that this strain is capable of gallium efflux.

Optimization of C. crescentus β-Xylosidases and Expression of xynB1–5 Genes in Response to Agro-Industrial Waste

The effect of the response surface methodology was applied to the production of Caulobacter crescentus (strain NA1000) β-xylosidases using corn cob. The components of the medium that presented the greatest influence on the enzyme production were chosen for optimization including the concentration of the corn cob residue and temperature variation. Optimal concentrations were determined by a central composite rotational design and a combination of 3.5% (w/v) corn cob concentration and 27 °C temperature was found to be optimal. When C. crescentus was cultivated using the optimized conditions, a maximum activity of 393.36 U/mL of β-xylosidases was achieved in 24-h cultures with a yield of 95% in real test conditions compared to the predicted one. In parallel, there was an increase of 3.6 times in the production of intracellular xylanases when compared to cultures without statistical application. In the C. crescentus genome, 5 genes that encode β-xylosidases are present. In order to evaluate which of them would be induced in the optimized conditions, the quantitative expression (qPCR) of the xynB1–xynB5 genes was evaluated in the presence of 1 or 3.5% corn cob (w/v) and surprisingly, all showed a constitutive expression in relation to the control. Assays of Western Blot performed with a polyclonal antiserum against C. crescentus β-xylosidase II in the optimized condition also did not show mass variation of the referred protein. These data strongly suggest that post-transcriptional controls are operating in the induced condition to increase the activity of C. crescentus β-xylosidases I-V, but β-xylosidase II. To our knowledge, this is the first time that these data are reported in literature for a bacterial system.

NOTCH2 Contributes to Venetoclax Resistance in Chronic Lymphocytic Leukemia

BACKGROUND: Chronic lymphocytic leukemia (CLL) has recently experienced an unprecedented revolution thanks to the discovery of crucial pathogenetic mechanisms. Despite considerable therapeutic advancements, the eradication of the disease is not complete and resistance and transformation may occur. Venetoclax is a small-molecule BH3 mimetic that competes for binding in the hydrophobic groove of Bcl-2, a key protein involved in CLL cells survival. Venetoclax was shown to have an excellent antitumor activity in patients with relapsed CLL including those with high risk features. However, venetoclax monotherapy shows an overall response rate of 79% and complete response rates of 16% and leave some open questions mainly related to intrinsic features of CLL patients that may guide a pattern of resistance. A recent work shows that durable response to venetoclax based therapy is more likely in patients having minimal adenopathy, mutated IGHV gene, wild type TP53 and Notch1 genes. AIM OF THE WORK: We hypothesize that specific intrinsic features of CLL cells may contribute to drive possible mechanisms of resistance to venetoclax (ABT-199) treatment. METHODS: Notch2 expression was monitored by western blot in purified CLL cells. Modulation of Notch2 expression in vitro was performed by using siRNA strategy. ABT-199 was used at doses of 0.1 nM or 1 nM. RESULTS: Notch signaling is relevant in CLL pathogenesis. We detected a peculiar high expression of Notch2 in a subgroup of CLL patients carrying trisomy 12. The high expression of Notch2 correlated with high levels of Mcl-1, CD23 and Hes1. Interfering with Notch2 expression in trisomy 12 CLL patients, by siRNA silencing, was able to affect leukemic cells viability, reducing CD23 and Mcl-1 expression. Since Mcl-1 is involved in ABT-199 resistance, we wondered if ABT-199 may have a different effect in CLL cells isolated from patients carrying trisomy 12 comparing to no trisomy 12 cases. After 24h of culture in complete medium, trisomy 12 CLL cells showed a gain in survival rate of 10% during treatment with both 0.1 nM or 1 nM in comparison to no trisomy 12 cases. This advantage in viability reflected the maintenance of Notch2 and Mcl-1 expression in trisomy 12 both at 0.1 nM and 1 nM of ABT-199 compared to control. Conversely, a reduction of Notch2 and consequently Mcl-1 levels were observed in no trisomy 12 cases at both doses of ABT-199. We did not detect any variation in Bcl-2 levels. We wondered if Notch2 expression may reduce the response to ABT-199 in trisomy 12 CLL. To demonstrate this hypothesis, we interfered with Notch2 by silencing strategy. Treatment with Notch2 siRNA decreased the expression of Notch2 and Mcl-1 in combination with ABT-199 as shown in Figure 1A. We also found that Notch2 down-regulation cooperated with ABT-199 decreasing CLL cells viability (Figure 1B). CONCLUSIONS: Collectively, our results show a novel mechanism that may compromise the clinical response to venetoclax in CLL patients. Although the excellent mechanism of action of venetoclax, Bcl-XL and Mcl-1, two major anti-apoptotic proteins of Bcl-2 family not inhibited by venetoclax, are key determinants of both acquired and intrinsic resistance to treatment. The possibility to identify patients with more pronounced expression of Mcl-1 may help to optimize the treatment. The expression of Notch2 identify a subset of CLL patients, mainly harboring trisomy 12 aberration, that through the maintenance of high levels of Mcl-1, may be involved in a reduced response to ABT-199. Disclosures Luppi: Gilead Sci., MSD, Pfizer, Novartis, Abbvie, Sanofi, Daiichi Sankyo, Jazz Pharmaceuticals: Honoraria. Marasca:Janssen and Gilead Sci, Abbvie, Roche and Shire: Honoraria, Research Funding.

Complete reutilisation of mixed mackerel and brown seaweed wastewater as a high-quality biofertiliser in open-flow lettuce hydroponics

Abstract Biodegradation using Bacillus species can convert mixed fishery wastewater into biofertiliser in a clean manner. Herein, fishery wastewater from mackerel and brown seaweed was tested, and the ratio most suitable for biodegradation was 10:1. During the 72-h biodegradation at the optimum mixing ratio, 36.4% hydrolysis occurred due to stable protease, alginate lyase and laminarinase activities, which chemical oxygen demand decreased by 69.1% and total nitrogen by 62.0%. The highest antioxidant activities toward 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals were 84.9% and 93.1% respectively at 24 h, and the total amino acid content at 72 h was 7715.7 μg mL-1. As a biofertiliser, the 72-h culture broth also met the requirements for standard content of nitrogen (N), phosphorus (P), potassium (K) and heavy metals, and the number of viable cells. In open-flow hydroponics using the produced biofertiliser, 1-month-old lettuce plants exhibited significantly enhanced growth rate compared with controls, with high chlorophyll and carotenoid content and high antioxidant activity. Moreover, there was no permeation of pathogens in the circular biofertiliser solution. These results indicate the potential of the biofertiliser for the production of high-quality lettuce, and demonstrate the complete reutilisation of mixed fishery wastewater.

Diversity of soil microbial community identified by Biolog method and the associated soil characteristics on reclaimed Scirpus mariqueter wetlands

We examined how various soil characteristics are associated with Scirpus mariqueter growth and spatial heterogeneity in Shanghai Nanhui Dongtan wetlands and addressed a major knowledge gap regarding the effects of reclamation on microbial communities in the soil. Biolog was used to examine soil carbon resources, diversity, abundance, and community structure of S. mariqueter soil microbes after a 72-h culture. Tidal elevation influenced soil microbes, which used carbon resources at higher rates, exhibited more community diversity and had greater species richness in areas with dense S. mariqueter cover than in bare tidal flats (P < 0.05). Microbial functional diversity also differed significantly across the study region in a manner that reflected S. mariqueter spatial heterogeneity (P < 0.05). In terms of soil characteristics, microbial community diversity was positively correlated with soil salinity, organic carbon, and total phosphorus (P < 0.05), whereas negatively correlated with soil particle size (P < 0.05). The results of this study provide insight into plant–soil interactions of disturbed S. mariqueter wetland ecosystems through clarifying factors that influence soil microbes.

Antimicrobial Activity of Supernatant of Lactobacillus plantarum against Pathogenic Microorganisms.

We studied ntimicrobial activity of L. plantarum strain against different pathogens: Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus. It was shown that supernatant of 48-h L. plantarum culture in liquid nutrient medium exhibits inhibitory activity against gram-positive and gram-negative pathogenic microorganisms. Supernatant of 24-h culture exhibited lower activity, while supernatant of 72-h culture produced no inhibitory effect. Boiling and proteinase K treatment did not affect activity of the preparation, i.e. antimicrobial activity of the supernatant was not associated with protein or peptide component. These data were confirmed by the results observed after ultrafiltration of the preparation: the growth of E. coli, P. aeruginosa, and S. aureus was inhibited by the low-molecular-weight fraction, but not high-molecular-weight fraction of the supernatant. On the other hand, the high-molecular-weight fraction suppressed the growth of streptococcus by 3 times. We hypothesized that L. plantarum supernatant obtained in our experiments contained at least two antimicrobial components with different molecular weights.

Estrogen suppresses bcl2 expression through dnmt1 in Japanese eel ovarian follicle.

Japanese eels are commercially valuable species in Asian aquaculture. This study evaluated whether salmon pituitary extract (SPE) or 17β-estradiol (E2) treatment can induce cytotoxic activity in eel ovarian follicles. Follicular cells died after exposure SPE for 24-h culture in an in vitro culture. Moreover, the E2 treatment also significantly reduced follicular cell counts. These results reveal that the inhibition of follicular cell numbers by SPE may occur through SPE-induced steroidogenesis. Results of a quantitative PCR analysis indicated that adding E2 to the culture decreased bcl2 and increased dnmt1 mRNA expression in Japanese eel follicular cells after 24 h. The results of a promoter assay revealed that E2 significantly increase dnmt1 promoter activity through estrogen receptor-binding site. An in silico analysis predicted several putative transcription factors targeting the bcl2 gene promoter region. Methylation of the bcl2 promoter accounted for the downregulation of bcl2 by E2-mediated dnmt1. The DNA methylation level of the bcl2 gene was significantly higher in E2-treated follicular cells than that in the control group. Finally, the E2-induced hypermethylation pattern of the bcl2 promoter and the reduction in follicular cell numbers were suppressed by adding an MTase inhibitor. Our findings demonstrate that estrogen has a negative effect on the reproductive system of female eels by regulating an epigenetic mechanism during artificial maturation.

Hyaluronic acid is required for palatal shelf movement and its interaction with the tongue during palatal shelf elevation

Palatal shelf elevation is an essential morphogenetic process that results from palatal shelf movement caused by an intrinsic elevating force. The nature of the elevating force remains unclear, but the accumulation of hyaluronic acid (HA) in the extracellular matrix (ECM) of the palatal shelves may play a pivotal role in developing the elevating force. In mammals, HA is synthesized by hyaluronic acid synthases (HAS) that are encoded by three genes (Has1-3). Here, we used the Wnt1-Cre driver to conditionally disrupt hyaluronic acid synthase 2 (Has2) in cranial neural crest cell lineages. All Has2 conditional knockout (cko) mice had cleft palate due to failed shelf elevation during palate development. The HA content was significantly reduced in the craniofacial mesenchyme of Has2 cko mutants. Reduced HA content affected the ECM space and shelf expansion to result in a reduced shelf area and an increased mesenchymal cell density in the palatal shelves of Has2 cko mutants. We examined palatal shelf movement by removal of the tongue and mandible from unfixed E13.5 and early E14.5 embryonic heads. Reduced shelf expansion in Has2 cko mutants altered palatal shelf movement in the medial direction resulting in a larger gap between the palatal shelves than that of littermate controls. We further examined palatal shelf movement in the intact oral cavity by culturing explants containing the maxilla, palate, mandible and tongue (MPMT explants). The palatal shelves elevated alongside morphological changes in the tongue after 24-h culture in MPMT explants of early E14.5 wild type embryos. On the contrary, shelf elevation failed to occur in MPMT explants of age-matched Has2 cko mutants because the tongue obstructs palatal shelf movement, suggesting that reduced shelf expansion could be essential for the palatal shelves to interact with the tongue and overcome tongue obstruction during shelf elevation. Has2 cko mutants also showed micrognathia due to reduced HA content in the mandibular mesenchyme including Meckel’s cartilage. Through 3D imaging and morphometric analysis, we demonstrate that mandibular growth results in a significant increase in the vertical dimension of the common oral-nasal cavity that facilitates palatal shelf movement and its interaction with the tongue during shelf elevation.

Caffeinated beverages contribute to a more efficient inflammatory response: Evidence from human and earthworm immune cells

The role of caffeinated beverages on efficiency of acute inflammatory responses is not yet fully understood. This study analyzed the effect of five hot water extracts, coffee (CO), black/green tea (BT/GT), yerba mate (YM), and guarana (GU) on inflammatory modulation of non-activated human peripheral blood mononuclear cells (PBMCs), yeast-activated human neutrophils, and granulocytic coelomocytes from Eisenia fetida earthworm. Based on preliminary tests, a concentration of 10 μg/mL was chosen for subsequent assays, as at this concentration, the extracts exhibited antioxidant, genoprotective, and non-cytotoxic properties. Immunoassays using 24-h PBMC supernatant showed that all extracts decreased levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ), and increased levels of the anti-inflammatory cytokine, IL-10. Further, these extracts induced overexpression of cytokine genes in 24-h cultures. These results suggest that an increase in the levels of mRNAs and/or inactive cytokines in the cytoplasm improves the “immune cytokine response. Analysis of the yeast encapsulation processes, and production of human neutrophils and coelomocyte extracellular DNA traps suggests that extracts also improve the immune response in humans and earthworms. However, for E. fetida, the intensity of these results varied from extract. Overall, our results suggest that caffeinated beverages may improve an organism's efficiency against acute inflammatory processes.

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

10. DIFFERENCES IN MRNA EXPRESSION OF SELENOPROTEINS IN DOMESTIC CAT OOCYTES AND CUMULUS CELLS DURING IN VITRO MATURATION

Introduction Reactive oxygen species (ROS) is one of the major factors that has a direct negative impact on oocyte quality. The presence of antioxidant enzymes including selenoproteins across different mammalian species suggests that defence mechanisms are conserved and are likely to be important for the final steps of in vitro oocyte maturation. Domestic cat serves as a good animal model for future research in conservation breeding of endangered feline species, but also it is a valuable model for studying developmental pathways that can be affected by in vitro culture and maturation of oocytes for many mammalian species including human. Competence of in vitro development of cat oocytes is low in comparison with other model organisms. A major factor affecting the viability of cells during in vitro culture is an increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). The objective of this study was to investigate the relative mRNA expression pattern of three genes - Sep15 (selenoprotein 15), Gpx1 (glutathione peroxidase 1) and Sephs2 (selenophosphate synthetase 2), each involved in protection against free radicals, between oocyte and the surrounding cumulus cells during in vitro maturation. Material and methods Ovaries from healthy, feral, 8-12 months old female domestic cats (n=10), were obtained by ovariectomy in local veterinary clinic. After 48h of culture in maturation media oocytes were collected at different stages (GV n= 40, MII n=75) and pooled in groups. Total RNA was isolated and level of mRNA expression was tested by qPCR method. Results Results revealed that all three transcripts were detected, independently of the maturation stage. Nevertheless, significantly higher level of Gpx1 and Sephs2 expression was observed in oocytes, compared with their surrounding cumulus cells. Particularly higher level of Gpx1 mRNA expression was observed in MII oocytes (P 0.05). Conclusions In conclusion, our results indicate that Gpx1 and Sephs2 can be involved in in vitro maturation of oocytes and may be a part of anti-oxidant pathways induced by oxidative stress. Further studies should be conducted on the oxidative response pathways in relation to developmental potential of oocytes matured in vitro and conservation of the mechanism in human. This study was financed by the NCN, research project no. 2012/07B/NZ9/02196 and KNOW Leading National Research Centre-Scientific Consortium Healthy Animal - Safe Food No. 05-1/KNOW2/2015

First Report of Pseudomonas viridiflava Causing Bacterial Blight on Globe Artichoke in Turkey

The production of globe artichoke (Cynara scolymus L.), which is a major part of Mediterranean cuisine and packs an abundance of nutritional value, has been increasing over recent decades in Turkey. In the spring of 2018, globe artichoke (cv. Sakiz) plants with blight symptoms on outer bracts, dark brown sunken lesion on inner sides, red-brown areas on margins, and small and malformed buds were found in artichoke-growing areas in the Karaburun district of Izmir, Turkey. In some areas, disease incidence was up to 5% of the plants. Several buds in harvest stage exhibiting symptoms were collected from 30-ha artichoke production fields that have heavy soil texture and humid weather conditions and processed to identify the causal agent on 15 artichoke samples. Symptomatic outer bracts showing characteristics of bacterial blight symptoms as well as spines were surface sterilized with 1% NaOCl, rinsed in sterilized distilled water (SDW), and cultured on King’s medium B (KB) for 24 h at 28°C. After incubation, a predominant bacterium was isolated from all samples. Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. Round, flat, and blue-green translucent colonies fluorescing under 360-nm UV light when grown on KB medium were observed in all samples taken. The isolates caused soft rot of potato tubers, induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. Acid was produced from mannitol, erythritol glycerol, D-sorbitol, D-mannose, and inositol but not from L-tyrosine, D-cellobiose, D-arabinose, and D-trehalose, all of which are typical characteristics of Pseudomonas viridiflava (Lelliott et al. 1966; Schaad et al. 2001). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using the universal bacterial primers 8F and 1492R (Galkiewicz and Kellogg 2008) and gyrase subunit B (gyrB) gene using primers gyrB-Rps/gyrB-Fps (Sawada et al. 1999). The sequences were submitted to GenBank under accession numbers MH763694 for 16S and MK546431 for gyrB. BLASTn analysis of the 1,042-bp amplicon for 16S and 545-bp amplicon for gyrB indicated that it had 99% similarity with P. viridiflava strain 4G1113 (accession no. MG972916) and 100% similarity with P. viridiflava ATCC 13223 strain (accession no. KY612187), respectively. Pathogenicity tests were conducted twice on outer bracts and main stems of artichoke cultivar Sakiz by inoculating 20 µl of bacterial suspension of a 24-h culture in KB medium (1.9 × 10⁹ CFU/ml in SDW). The inoculated plants were placed in a transparent box for 7 days and kept at 25°C and 70% relative humidity. The bract leaves inoculated with only SDW did not develop symptoms; however, the bracts treated with isolated bacterial suspension developed blight symptoms similar to those observed in the field, 7 days postinoculation. The stems inoculated with the isolate also exhibited soft rot symptoms, and the same bacterium was consistently reisolated from both bracts and stems but not from parts of negative control plants. Reisolated bacteria exhibited the same biochemical profile with our isolate, thereby fulfilling Koch’s postulates. P. viridiflava causing a bacterial blight of artichoke bracts was previously reported in Greece (Sarris et al. 2012). To our knowledge, it is the first report of this bacterium causing bacterial blight on leaves and soft rot on stems of globe artichoke in Turkey. Given the availability of climatic conditions, the potential of disease spreading can cause a serious reduction in yield and marketability of this vegetable, which has increasing popularity owing to having high nutritional value, leading to substantial economic losses.

Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.