Monophasic Medium Research Articles

Silencing TUBB3 Expression Destroys the Tegument and Flame Cells of Echinococcus multilocularis Protoscoleces.

Simple SummaryEchinococcus multilocularis is a fox and carnivore tapeworm with important zoonotic potential, as the larval stage can cause alveolar echinococcosis in both humans and livestock. An understanding of the molecular mechanisms of parasite growth and development is essential for disease diagnosis, management and control. This study investigates the potential role of E. multilocularis TUBB3 in protoscoleces through RNA interference in vitro with specific short interfering RNAs. Our results show that EmTUBB3 might contribute to tegument formation and protonephridial system flame cell integrity in E. multilocularis protoscoleces.Alveolar echinococcosis (AE), caused by infection with the larvae of Echinococcus multilocularis, is a neglected tropical disease and zoonosis that causes remarkable morbidity in humans and has economic importance in the livestock industry worldwide. The growth of this parasite resembles the invasion and proliferation of malignant tumours. Microtubules, especially the β-tubulin subunit in the exposed end, are the targets of many antitumour drugs. However, the role of TUBB3, which is the most studied isotype in solid tumours and is also a marker of biological aggressiveness associated with the modulation of tumour metastatic abilities in the growth and development of platyhelminths, is unknown. In this study, protoscoleces (PSCs) are cultivated in monophasic medium in vitro. Using electroporated short interfering RNA (siRNA), EmTUBB3 knockdown was performed with two EmTUBB3-specific siRNAs (siRNA-1 and siRNA-2). qRT–PCR was performed to detect the expression of TUBB3. PSCs viability and the evagination rate and number of body contractions were quantified under a light microscope. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the ultra-morphological changes of the parasites. After siRNA interference, the EmTUBB3 expression in E. multilocularis PSCs was significantly reduced. Reduced viability, a decreased evagination rate and a decreased number of body contractions were also documented. In particular, shrinkage and roughness of the tegument were observed. Ultrastructural changes included marked damage to flame cells, cracked cilia structures enclosed in the cell body and ruptured microtubule structures. EmTUBB3 possibly plays a crucial role in tegument and flame cell integrity in E. multilocularis PSCs. Novel drugs targeting this specific beta-tubulin isotype in E. multilocularis are potential methods for disease control and deserve further attention.

Comparison of two monophasic culture systems

Objective Comparison of a low lactate monophasic culture medium (medium 1) to a KSOM based monophasic culture medium (medium 2). Design Prospective sibling oocyte study. Materials and methods 115 patients undergoing ICSI between January and May 2019 were included in this study. A 60mm dish was prepared with 30µL drops covered with light mineral oil and pre-equilibrated at least 16 hours at 37°C 6.5%CO2/5%O2 to maintain a pH of 7.2 to 7.3. Both media were in the same dish. Oocytes were randomly distributed among the two culture-media immediately post-ICSI. Fertilization was evaluated at 17 ± 1-hour post-ICSI. 2PN zygotes with clearly visible pronuclei were cultured through day 6. Blastocyst quality was evaluated at days 5 and 6. Ploidy data was included if available. Results 115 patients with an average age of 37.4 years had 1,308 embryos cultured in media 1 (640) and media 2 (655). Day 5 and day 6 blastocysts yields were 34% and 29%, respectively for media 1 and 14% and 19%, respectively for media 2. Overall, blastocyst-quality was similar between both media 1(50.9%) and medium 2 (51.6%). A non-significant trend showed medium 1 to produce a higher proportion of good-quality blastocysts on Day 5 (67% vs 56% when compared to medium 2 (P=0.09, paired sample t-test). Conversely, medium 2 had more good quality blastocysts on Day 6, than medium 1 (difference of 8%, P=0.03, paired sample t-test). A sub-set of 27 patients (N=95) had PGT-A on D5 Blastocysts (50, medium 1 vs 45, medium 2). The proportion of euploid embryos was similar between the two media with both demonstrating 44% euploidy rate on day 5. Conclusions A wide variety of commercially available media have been formulated for the culture of human pre-implantation embryos. A meta-analysis concluded “there is insufficient evidence to support or refute the use of any specific culture medium1”. Previous reports indicated that a low lactate medium yielded significantly more euploid blastocysts when compared to a monophasic medium with higher lactate concentration2. Our data indicated a trend towards a higher number of day 5 euploid blastocysts in a low lactate medium than a medium with higher lactate. Even if the present study suffers from constraints of sample size and lack of a more representative demography, it does indicate robustness of low-lactate media in supporting embryo development and ploidy in a sibling split experimental design. Disclosures None

Chlorinated biphenyl degradation by wild yeasts pre-cultured in biphasic systems

Environmental biotechnology has developed as an offshoot from sanitary engineering, and only recently the biological component of the ecosystems had been recognized as relevant when bioremediation strategies must be chosen to solve environmental problems. Yeasts were isolated on 2,4-dichlorobiphenyl, 2,3',4- and 2,4',5-trichlorobiphenyl, poorly soluble compounds in water, as carbon sources. Debaryomyces castelli , Debaryomyces maramus and Dipodascus aggregatus composed the mixed culture and represented 72% of the isolates; their degradation potential were studied in biphasic and monophasic systems. The biphasic cultures were obtained with phenol as the organic phase and MSM as the aqueous ones, the monophasic medium only with MSM. Both cultures were supplied with 50, 100, 150 and 200 ppm DCB, TCB-3' and TCB-4' as substrate. The growth rates varied with the dispersion degree, agitation rates and cell adhesion to the organic phase. The water-phenolic system improved yeasts selection in pollutant presence with low water solubilities, indeed, the adaptation and degradation were more slowly in the monophasic aqueous medium. Bioremediation is based on the presence of efficient microbial populations and pollutant availability; the tested yeasts and the organic-water system assayed put forward the possibility that hydrophobic substrates could be mineralized in natural habitats by wild yeast consortium.

Exploiting benign ionic liquids to effectively synthesize chiral intermediate of NK-1 receptor antagonists catalysed by Trichoderma asperellum cells

This study aims to exploit an ionic liquid (IL)-containing medium for efficient production of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) from 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) catalysed by whole cells of Trichoderma asperellum ZJPH0810. A new type of quaternary ammonium-based ILs (QAILs) was investigated and compared with more conventional pyridinium or imidazolium-based ILs. [N1,1,1,1]+ comes up as a cation of ILs to the T. asperellum ZJPH0810 cell in the 3,5-BTAP bioreduction. After selection and optimization, the highest concentration of substrate and product for the bioreduction observed in a [N1,1,1,1][PF6]-distilled water reaction system were 75 mM and 65.4 mM, which is a 1.5-fold increase and 1.4-fold increase compared with the monophasic aqueous medium, respectively. Consequently, [N1,1,1,1][PF6] was considered to be a promising IL for T. asperellum ZJPH0810-catalysed reduction owing to its good biocatalytic reduction results and better cell biocompatibility.

Immobilization of purified β-glucuronidase on ZnO nanoparticles for efficient biotransformation of glycyrrhizin in ionic liquid/buffer biphasic system

Purified recombinant β-glucuronidase (PGUS-E) from Aspergillus oryzae Li-3 was immobilized on the zinc oxide nanoparticles (ZnO-NP) for glycyrrhizin (GL) biotransformation. The optimal loading efficiency of the PGUS-E on ZnO-NP was 6.52U/mg with an average of 85.83% immobilization yield. The adsorption of the PGUS-E on ZnO-NP was confirmed using scanning electron microscope (SEM) and fourier transform infrared (FTIR) spectroscope. The comparative catalytic efficiency of the immobilized PGUS-E was evaluated in the ionic liquids (ILs) media and buffer. The higher catalytic efficiency of the immobilized PGUS-E was recorded in the hydrophobic ionic liquid [Bmim]PF6 (20% volumetric ratio) compared with other ILs media and pure buffer. The temperature and pH profiles of the immobilized PGUS-E were also determined for ionic liquid (ILs) media and pure buffer. The higher operational stability of the immobilized PGUS-E was observed in the IL co-solvent medium than the pure buffer medium; and after 8 repeated uses an average 30.54% and 7.42% of its catalytic activity was respectively retained. The recovery rate of the IL medium ([Bmim]PF6) was as high as 76.11%. The measurement of the enzyme kinetic parameters and activation energy also explicitly display the superiority of the IL co-solvent media over the monophasic media.

Papain Catalyzed Oligomerization in Monophasic Aqueous Organic Media - Synthesis and Characterization of Neutral and Polar Amino Acid Oligomers

Synthesis of oligopeptides is generally carried out in aqueous or bi-phasic reaction media; use of monophasic reaction media is quite limited. In this study, homo-oligopeptides of lysine (Lys), glycine (Gly), methionine (Met) and tyrosine (Tyr) were synthesized through papain-catalyzed reaction in monophasic systems consisting of acetonitrile/ water. Reaction conditions were optimized for peptide bond formation and to minimize enzyme denaturation. Such media are especially attractive because they can minimize the secondary and reverse hydrolysis of the acyl complex and the oligopeptide, respectively. The synthesized oligopeptides were purified and characterized by reversed phase liquid chromatography (RPLC) and Electrospray ionization mass spectrometry (ESI-MS). The yields of oligopeptides were approximately 80% for all amino acids. The stereo specificity of papain in acetonitrile/water media was also investigated. The separated enantiomers of methionine were characterized using Chiral High-Pressure Liquid Chromatography (HPLC). The results indicate the L-specificity of papain is maintained in monophasic media.

Sequential versus Monophasic Media Impact Trial (SuMMIT): a paired randomized controlled trial comparing a sequential media system to a monophasic medium

To determine whether sequential or monophasic media is the more optimal formulation for blastocyst development and sustained implantation rates (SIR) in IVF. Paired randomized controlled trials. Academic. Infertile couples (N = 192) with female partner ≤42years old and normal ovarian reserve. Fertilized zygotes from each patient were randomly divided into two groups: [1] cultured in sequential media and [2] cultured in monophasic medium. Sequential media consisted of Quinn's Advantage Cleavage Medium (SAGE) followed by Blast Assist (Origio). The monophasic medium used was Continuous Single Culture (Irvine Scientific). Paired ETs were accomplished by transferring the best euploid blastocyst from each media group. DNA fingerprinting was used to link outcomes. The primary outcome measure was the proportion of blastocysts suitable for clinical use. Secondary outcome measures included timing of blastulation, aneuploidy rates, and SIR. Sustained implantation rate is defined as the number fetal heart beats at 8-9weeks of gestation, divided by the number of embryos transferred. A total of 192 patients had their 2PN embryos (N = 2,257) randomized to each culture system. Sequential media had higher blastulation rate than monophasic medium (55.2% vs. 46.9%). No differences were found in the day of blastulation or aneuploidy rate. Of the 168 patients who had euploid blastocysts suitable for transfer, 126 completed a paired ET. Among the double ETs, there was no difference in implantation between groups. This is the first randomized controlled trial to examine paired euploid transfers of sibling zygotes cultured in sequential versus monophasic media. This study demonstrates that the usable blastocyst rate is greatest after culture in the sequential media tested in comparison with the monophasic formulation selected for study. However, no difference exists in timing of blastulation, aneuploidy, or SIR. Whether these observations are generalizable to other media systems remains to be determined. NCT01917240.

Toward Biorelevant Dissolution: Application of a Biphasic Dissolution Model as a Discriminating Tool for HPMC Matrices Containing a Model BCS Class II Drug

The potential of a biphasic dissolution system to assist with the analysis of controlled-release (CR), Biopharmaceutics Classification System (BCS) Class II pharmaceutical products has been investigated. Use of a biphasic dissolution medium (aqueous/octanol) provided sink conditions and afforded complete dissolution of nifedipine formulated in a CR matrix tablet while maintaining the dosage form in an aqueous environment. This was not possible in a monophasic (aqueousonly) dissolution medium. Consequently, the biphasic model allowed the discrimination of three formulations containing different HPMC loadings while the monophasic medium did not. The performance of the formulations in conventional dissolution media incorporating the inorganic salt dibasic sodium phosphate, the surfactant sodium dodecyl sulfate (SDS), and ethanol as solubility modifiers was assessed. The addition of the salt and surfactant failed to produce complete discrimination in a predictive manner. The use of a hydroalcoholic medium comprising water and ethanol enabled the statistical discrimination of the three formulations but not as effectively as the biphasic medium.

Comparison of Mono and Biphasic Culture Media in Isolating Bacteria from Blood and evaluation Bu-Ali Hospital Lab Quality

Blood Culture is an important diagnostic method in infectious diseases and has positive results in 30%- 50% and even to 80% of cases due to sample volume. In Bu-Ali Hospital, Tehran, it decreases to 2-3%. In this survey, quality of hospital lab and difference between Mono and Biphasic culture media in isolating bacteria from blood of patients suggestive of sepsis were evaluated.106 (48 F + 58 M) newly admitted patients with impression of sepsis as SIRS (Systemic Inflammatory Response Syndrome) (36˚C >OT>38.3˚C , tachycardia more than 100/min, leukocytosis with shift to left or leucopenia) with infectious source were sampled for culture (5ml blood 3 times in 1 Biphasic and 2 Monophasic media) in the infectious ward. One Monophasic Media in hospital lab and the two other (1 Monophasic + 1 Biphasic Media) in Reference Laboratory of Iran, Research Center were handled. Media were quality-controlled at beginning and in the middle of study by NCCLS (National Committee for Clinical Lab Standard) with ATCC (American Type Culture Collection) samples. Sampling, transfer, and handling were all in standard conditions usually used in hospital. Results were compared by Fisher Exact Test. Clinical diagnosis were bacterial in 84 (79%), and nonbacterial in 22 (21%) patients at admission. 57 (54%) patients had not used antibiotics in the past 72 hours. In Monophasic Media of hospital lab 2 (1.9%) positive cultures (S. epidermidis) one with history of Erythromycin use were reported. In both Monophasic and Biphasic Media in reference lab 3 (2.8%) positive cultures (2 S.epidermidis, 1 E. coli) were reported equally, one with history of Erythromycin use. Growth Index in both Monophasic and Biphasic Media were standard in quality control. Qualities of Mono and Biphasic Media in growing bacteria were alike and Biphasic Media had no superiority to Monophasic Media in routine bacterial isolation. Positive culture in both labs had no significant statistical difference. So, negative results are not due to media and laboratorial fields, and it is needed to educate and evaluate two other fields: Sampling and Transferring. Also, we may have more positive cultures by increasing blood samples from 5 to 20 ml which can be compared in next studies.

Herpetomonas spp. isolated from tomato fruits ( Lycopersicon esculentum) in southern Spain

A flagellate of the family Trypanosomatidae was isolated from fruits of Lycopersicon esculentum (tomato) in southeastern Spain. The isolate was successfully adapted to in vitro culture in monophasic media. The morphology showed the kinetoplast to be positioned towards the middle of the body, and the typical opistomastigote form characteristic of members of the genus Herpetomonas. Amplification of the mini-exon gene was negative, whilst for the 5S ribosomal rRNA gene the result was positive. The DNA sequence was obtained and its alignment with other trypasomatids, obtained using the BLAST algorithm, suggested it was closely related to Herpetomonas samuelpessoai.

Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in Bis-(2-ethylhexyl) phthalate

The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.

Poly(4-tert-butylstyrene) as a Soluble Polymer Support in Homogeneous Catalysis

[reaction: see text] Use of soluble poly(4-tert-butylstyrene) (PtBS) as a support in synthesis is demonstrated. These soluble polymers supported catalysts that were used in a monophasic medium. Subsequent separation of the catalysts after reaction was effected either by cooling- or by water-induced liquid/liquid-phase separation. Specific catalysts studied included both phosphine and DMAP nucleophilic catalysts. Low loadings of an azo dye quantified the efficiency of separation and recovery of these catalysts through multiple reaction cycles.

Synthesis of enriched solutions of chloramine starting from hypochlorite at high chlorometric degree

The synthesis of high-grade chloramine was studied starting from sodium hypochlorite solutions containing 25% in NaOCl. The reagents at 100 chlorometric degrees are relatively stable and lose approximately 1.4° by 24 h at 17°C. They present a temperature of crystallization of 14.2°C (precipitation of NaOCl·5H 2O) but it is possible to use them at −20°C in undercooling state. To obtain quantitative yields, it is necessary to neutralize the totality of hydroxide ions generated by the reaction and to operate below −10°C in order to limit oxidation of chloramine by the hypochlorite ions. The addition of an acceptor soluble salt is the most appropriate solution because it regenerates ammonia as soon as it is consumed and stabilizes chloramine by maintaining the pH around 10. The ammoniacal combinations NH 4NO 3–NH 4Cl–NH 3–H 2O and NH 4NO 3–NH 3–H 2O are compatible and allow to operate in monophasic medium until −30°C. The yields are close to 95% with a titre in NH 2Cl of 2.3 M (11.4%). Experiments carried out in continuous rating lead to a monochloramine solution of 2 M.

Emulsion electrosynthesis in the presence of power ultrasound Biphasic Kolbe coupling processes at platinum and boron-doped diamond electrodes

The electrochemical oxidation of aliphatic carboxylic acids, hexanoic, heptanoic, and lauric acid, under biphasic conditions is studied as a model system for ultrasound enhanced Kolbe electrosynthesis processes. Power ultrasound is used to generate an in situ emulsified medium and to remove reaction products continuously from the electrode surface. A clean and highly efficient process at platinum electrodes with formation of ‘one electron’ products only occurs in marked contrast to processes in monophasic media. For hexanoic acid the Kolbe dimer product R–R is formed in up to 75% yield with 45% current efficiency at 0.18 A cm −2 current density and in the presence of 190 W cm −2 ultrasound. The mechanism is explained in terms of a dynamically modified electrode surface, at which hydrophobic products are immediately ‘trapped’ via partitioning into a non-polar organic phase and transported away into the emulsion system. Kolbe electrosynthesis is undertaken both at platinum electrodes and at free-standing polycrystalline boron-doped diamond electrodes, in order to minimize the surface erosion effect induced by power ultrasound. The type and yield of products obtained from the biphasic Kolbe electrolysis process at diamond electrodes are essentially identical to those observed at platinum and based on this observation, the presence of a biphasic reaction layer at the electrode surface is postulated to govern the process.

In vitro taurocholate-induced segmentation and clustering of Mesocestoides vogae (syn. corti) tetrathyridia (Cestoda)--inhibition byh cestocidal drugs.

Mesocestoides vogae (syn. M. corti) tetrathyridia were cultured in the presence of sodium taurocholate, for the purpose of exploring the suitability of this organism for the in vitro assay of cestocidal drugs. Parasite clustering and segmentation were observed as taurocholate-dependent effects in biphasic and monophasic media, respectively. Interestingly, representative members of two major classes of known cestocidal agents (namely, albendazole and praziquantel) blocked these effects. Furthermore, it was possible to determine a specific concentration of the drugs that inhibited clustering and segmentation (minimum inhibitory concentration). In contrast, no inhibition was obtained in the presence of anthelmintics without cestocidal activity. These observations open the way for further studies focused at understanding how the activity of the drugs is involved in the suppression of the taurocholate-induced effects.

Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium

N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial-scale production. A formyl group (F-) was found to be a more desirable protecting group for the N-terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F-AspPheOMe (N-formyl-L-aspartyl-L-phe- nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F-L-Asp and L-PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F-AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid–liquid extraction was thus applied to the F-AspPheOMe synthesis. We succeeded in synthesizing F-AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 57–65, 2000.

Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium

N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial-scale production. A formyl group (F-) was found to be a more desirable protecting group for the N-terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F-AspPheOMe (N-formyl-L-aspartyl-L-phe- nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F-L-Asp and L-PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F-AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid-liquid extraction was thus applied to the F-AspPheOMe synthesis. We succeeded in synthesizing F-AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium.

The diagnosis of acute aortic diseases with multiplanar-detector CT using the spiral technic

Acute diseases of the central arteries require an immediate investigation. An efficient, fast and reliable diagnosis is necessary because of the high mortality, if the patient remains untreated. These requirements are perfectly fulfilled by the new CT-techniques. Suspected aortic diseases were examined with a new multi-slice helical CT. The thoracic or the abdominal aorta as well as the entire vascular tree from the supra-aortic branches to the inguinal arteries were investigated with different CT protocols. The slice-thickness and the scan mode were changed while the total examination time was kept constant for the first two groups. In the third group a monophasic examination was compared to a biphasic one. In the diagnosis of acute aortic diseases multi-slice helical CT proved to be a fast and reliable method with all scan protocols. The objective measurements of contrast homogeneity and image quality were comparable in the first two groups. The monophasic contrast medium injection protocol was superior to the biphasic administration mode. Multi-slice helical CT appears to be a very effective approach for the diagnosis of acute aortic diseases and seems to be the new gold standard.

Experimental Study of the Oxidation of Gaseous Monomethylhydrazine in a Strictly Monophasic Medium

Intermediate and final products of the oxidation of monomethylhydrazine by dioxygen are studied using an original device which allows analysis of the gaseous medium under strictly monophasic conditions.

Experimental Study of the Oxidation of Gaseous Monomethylhydrazine in a Strictly Monophasic Medium

Intermediate and final products of the oxidation of monomethylhydrazine by dioxygen are studied using an original device which allows analysis of the gaseous medium under strictly monophasic conditions.