Modification Of Medium Research Articles

Enhanced Production of Herpes Simplex Virus 1 Amplicon Vectors by Gene Modification and Optimization of Packaging Cell Growth Medium

Herpes simplex virus 1 (HSV-1)-derived amplicon vectors are unique in their ability to accommodate large DNA molecules allowing whole genomic loci to be included with all of their regulatory elements. Additional advantages of these amplicons include their minimal toxicity and ability to persist as episomes, with negligible risk of insertional mutagenesis, being particularly well-suited for gene therapy of neurological disorders due to their outstanding ability to deliver genes into neurons and other neural cells. However, extensive gene therapy application has been hindered by difficulties in vector production. This work improved HSV-1 amplicons production by genetic modification of the packaging cell line and optimization of the culture medium. A stably-transfected Vero 2-2 cell line overexpressing the anti-apoptotic Bcl-2 protein was generated, exhibiting an increased resistance to apoptosis, prolonged culture duration, and a significant improvement in viral vector production. Additionally, supplementation of the growth medium with antioxidants, polyamines, amino acids, and reduced glutathione further increased the yield of packaged amplicon vectors. With these modifications, HSV-1 amplicons could be isolated from culture supernatants instead of cell lysates, leading to vector preparations with higher titer and purity and paving the way for generation of stable cell lines that are capable of continuous herpesviral vector production.

Metaxalone Suppresses Production of Inflammatory Cytokines Associated with Painful Conditions in Mouse Macrophages RAW264.7 Cells in Vitro: Synergistic Effect with β-caryophyllene.

Mouse macrophage RAW264.7 cells were cultured in Dulbecco's Modification of Eagle's Medium containing 10% fetal bovine serum in the presence of metaxalone. Cell growth was assayed by counting the number of cells attached to culture dishes. Inflammatory cytokines released into the culture medium were analyzed with the ELISA kit. Metaxalone (1-100 μM) was found to decrease the number of macrophages by inhibiting the proliferation and stimulating the death of RAW264.7 cells in vitro. The combination of metaxalone (0.1 or 1 μM) and β-caryophyllene (10 or 50 μM), which alone did not have a significant effect on the cell number, caused potential effects on the growth and death of RAW264.7 cells. Mechanistically, molecular levels of mitogenactivated protein kinase were decreased by treatment with metaxalone or β- caryophyllene, and each effect was enhanced by their combination. Furthermore, levels of caspase-3 were increased by metaxalone or β-caryophyllene and enhanced by their combination. Notably, productions of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 or prostaglandin E2, which were enhanced by lipopolysaccharide (LPS), were repressed by culturing with metaxalone. Levels of cyclooxygenase (COX)-1, COX-2 and nuclear factor kappa B, which were increased by LPS treatment, were reduced by metaxalone. Metaxalone was found to suppress the activity of inflammatory macrophages in vitro.

Modeling Normal and Dysbiotic Subgingival Microbiomes: Effect of Nutrients

Screening for microbiome modulators requires availability of a high-throughput in vitro model that replicates subgingival dysbiosis and normobiosis, with a tool to measure microbial dysbiosis. Here, we tested various formulations to grow health- and periodontitis-associated subgingival microbiomes in parallel, and we describe a new subgingival dysbiosis index. Subgingival plaque samples pooled from 5 healthy subjects and, separately, 5 subjects with periodontitis were used to inoculate a Calgary Biofilm Device containing saliva-conditioned, hydroxyapatite-coated pegs. Microbiomes were grown for 7 d on either nutrient-rich media—including a modification of SHI medium, brain-heart infusion (BHI) supplemented with hemin and vitamin K, and a blend of SHI and BHI, each at 3 sucrose concentrations (0%, 0.05% and 0.1%)—or nutrient-limited media (saliva with 5%, 10%, or 20% inactivated human serum). The microbiomes were assessed for biomass, viability, and 16S rRNA profiles. In addition to richness and diversity, a dysbiosis index was calculated as the ratio of the sum of relative abundances of disease-associated species to that of health-associated species. The supplemented BHI and blend of SHI and BHI resulted in the highest biomass, whereas saliva-serum maximized viability. Distinct groups of bacteria were enriched in the different media. Regardless of medium type, the periodontitis-derived microbiomes showed higher species richness and alpha diversity and clustered with their inoculum separate from the health-derived microbiomes. Microbiomes grown in saliva-serum showed the highest species richness and the highest similarity to the clinical inocula in both health and disease. However, inclusion of serum reduced alpha diversity and increased dysbiosis in healthy microbiomes in a dose-dependent manner, mainly due to overenrichment of Porphyromonas species. The modification of SHI stood second in terms of species richness and diversity but resulted in low biomass and viability and significantly worsened dysbiosis in the periodontitis-derived microbiomes. Overall, saliva with 5% human serum was optimal for replicating subgingival microbiomes from health and disease.

Effect of Flattening Cracked Medium on Positioning Accuracy of a Linear Magnetic Encoder

Linear magnetic recording medium is a crucial and necessary component in a linear magnetic position sensing system. During the manufacturing of the magnetic medium, shape defects would often be observed due to various manufacturing processes. As a result, shape defects in the magnetic medium would alter the recording accuracy during position measurement of the magnetic reader. Thus, a flattening process is introduced for flattening of curved magnetic medium. Previous studies have demonstrated its effectiveness in flattening of magnetic medium. In this article, a further step is taken to examine the crack formation in the microstructure of the magnetic medium during flattening process to ensure the quality and recording accuracy of the magnetic medium. The experimental results have demonstrated that crack propagation would continue to grow under the influence of flattening process if crack initiation is found prior to the flattening process. Nonetheless, the measured linear position accuracy showed that the accuracy improved after the flattening process despite crack propagation occurring on the mechanical quality of the medium. In other words, the results have demonstrated that the effect of mechanical shape defects on linear position accuracy is more important than that of crack formation on the magnetic medium. Through understanding the crack formation of the magnetic medium, the accuracy of the recording medium can be improved by modification of the magnetic medium and flattening conditions.

Studies on peroxidase production and detection of Sporotrichum thermophile-like catalase-peroxidase gene in a Bacillus species isolated from Hogsback forest reserve, South Africa

This study sought to determine the process conditions for optimum peroxidase production by a Bacillus species (Bacillus sp. FALADE-1-KX640922) isolated from Hogsback forest reserve in South Africa and characterize the peroxidase gene in the bacteria. We optimized peroxidase production by manipulating the environmental and nutritional parameters under submerged fermentation. Subsequently, the gene encoding heme-peroxidase was determined through nested polymerase chain reaction and Sanger DNA sequencing. The studied bacteria had maximum peroxidase production at pH 8, 30 °C and 150 rpm. The addition of guaiacol to lignin fermentation medium enhanced peroxidase production by over 100 % in the studied bacteria. However, the other lignin monomers (veratryl alcohol, vanillin, vanillic acid and ferulic acid) repressed the enzyme activity. Modification of the fermentation medium with ammonium sulphate gave the maximum peroxidase yield (8.87 U mL−1). Under the predetermined culture conditions, Bacillus sp. FALADE-1 expressed maximum specific peroxidase activity at 48 h (8.32 U mg−1). Interestingly, a search of the sequenced gene in PeroxiBase showed 100% similarity to Sporotrichum thermophile catalase-peroxidase gene (katG), as well, the deduced protein sequence clustered with bacterial catalase-peroxidases and had a molecular weight of about 11.45 kDa with 7.01 as the estimated isoelectric point. Subsequently, the nucleotide sequence was deposited in the National Center for Biotechnology Information (NCBI) repository with the accession number MF407314. In conclusion, Bacillus sp. FALADE-1 is a promising candidate for improved peroxidase production.

Doxorubicin-loaded PLGA nanoparticles for the chemotherapy of glioblastoma: Towards the pharmaceutical development.

Brain delivery of drugs by nanoparticles is a promising strategy that could open up new possibilities for the chemotherapy of brain tumors. As demonstrated in previous studies, the loading of doxorubicin in poly(lactide-co-glycolide) nanoparticles coated with poloxamer 188 (Dox-PLGA) enabled the brain delivery of this cytostatic that normally cannot penetrate across the blood-brain barrier in free form. The Dox-PLGA nanoparticles produced a very considerable anti-tumor effect against the intracranial 101.8 glioblastoma in rats, thus representing a promising candidate for the chemotherapy of brain tumors that warrants clinical evaluation. The objective of the present study, therefore, was the optimization of the Dox-PLGA formulation and the development of a pilot scale manufacturing process. Optimization of the preparation procedure involved the alteration of the technological parameters such as replacement of the particle stabilizer PVA 30-70 kDa with a presumably safer low molecular mass PVA 9-10 kDa as well as the modification of the external emulsion medium and the homogenization conditions. The optimized procedure enabled an increase of the encapsulation efficiency from 66% to >90% and reduction of the nanoparticle size from 250 nm to 110 nm thus enabling the sterilization by membrane filtration. The pilot scale process was characterized by an excellent reproducibility with very low inter-batch variations. The in vitro hematotoxicity of the nanoparticles was negligible at therapeutically relevant concentrations. The anti-tumor efficacy of the optimized formulation and the ability of the nanoparticles to penetrate into the intracranial tumor and normal brain tissue were confirmed by in vivo experiments.

Possible replacement of cytokinins and auxins in culture in vitro

Aim. To perform an analysis of the effectiveness of our proposed modification of the classical MS medium in which auxin and cytokinin are replaced by derivatives of the class of tetrahydrothio-fendioxide and pyridine. Methods. In this study, the work with cell culture in vitro, in particular aseptic sprouting of seeds, microclonal reproduction, callusogenesis and initiation of various types of morphogenesis was carried out according to known techniques. Two varieties of potatoes (Slovyanka, Lugovskaya) and two varieties of tomato (Lagidny, Bobrytsky) were used to induce somaclonal variability. Results. The ability of preparations of tetrahydrothiophenedioxide and pyridine derivatives to perform the function of auxins and cytokinins in the MS medium in the callusogenesis of Solanacea cultures in vitro was established. Conclusions. It has been shown that with the use of the new generation drug (tetrahydrothiophenedioxide-pyridine), as a substitute for auxin and cytokinin in the MS medium, the growth of callus tissue is even increased. The production of the pilot batch of this substitute proved to be much cheaper than synthesis or the purchase of phytohormones.
 Keywords: modification of MS medium, phytohormones substitute, tetrahydro-thiophenedioxide-pyridine, potato, tomato.

Selection of bacteria inducing calcium carbonate precipitation for self-healing concrete application

A modification of bacterial medium using calcium lactate pentahydrate was developed for calcium carbonate precipitation. A total of five strains of bacteria were cultivated on the solution medium containing nutrient broth and calcium lactate pentahydrate. In this study, the variation of 3.2 mM and 16.2 mM of calcium lactate pentahydrate was used to obtain the optimum condition for bacterial growth. The results showed that isolated strains CPB 1, CPB 3, and CPB 5 with medium containing nutrient broth and 3.2 mM calcium lactate pentahydrate gave the optimum growth, pH and Eh, thus being favourable for the process of calcium carbonate precipitation. Hence, this will be useful for self-healing concrete.

Genetic transformation of einkorn (Triticum monococcum L. ssp. monococcum L.), a diploid cultivated wheat species

BackgroundDomesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~ 5.7 GB), low ploidy (2n = 2x = 14, AmAm) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn.ResultsIn this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T1-T2 populations of transgenic einkorn that are resistant to herbicides were selected.ConclusionTo the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

STUDY OF THE INFLUENCE OF THE COMPOSITION OF CULTURAL MEDIUM ON THE BASIS OF A WHITE CABBAGE ON DEVELOPMENT OF LEUCONOSTOC MESENTEROIDES AT THE PRE-FERMENTATION STAGE

The aim of the research was to study the regularity of the influence of the culture medium (substrate) on the development of microflora on the pre -fermentation stage of a model medium made from white cabbage «Parus», using strains of lactic acid microorganisms Leuconostoc mesenteroides VKPM B-8818. The main task in the research process was to perform a stepby-step mathematical processing of experimental data and analyze them, to obtain functional dependencies adequately approximating the experimental data for the base (BMS) and modified (MMC) model media. Analysis of the experimental data showed that, depending on the type (composition) of the medium, the same species of microorganisms exhibit different dynamics of titer growth. In connection with this, an algorithm was developed to determine the optimal duration of pre-fermentation – «stop points». The results of the research showed that modification of the model medium with the addition of table salt and ascorbic acid to it promotes the formation of positive dynamics of the comparison indicator. This dynamics has three extreme extremes, but only extremes are of practical significance, which are in the interval of the monotonic decrease of the titer. One of the conditions for successful development of the starting culture at the main stage of fermentation is a relatively small amount of the first culture's titer at the end of the preliminary fermentation stage in order to exclude competition. Consequently, the position of the "stoppoint" corresponds to the period after the last peak of the comparison indicator. These studies on the regularity of the effect of the pre-cultivation of gram-positive microorganisms on the activity of lactic acid microorganisms in the process of fermentation are relevant, since the whole process and the production of high-quality products depend on this approach in full.

Improving the sintering performance of blends containing Canadian specularite concentrate by modifying the binding medium

Canadian specularite concentrate (CSC) possesses high total iron grade and low impurity content. However, due to the poor granulating performance and weak reactivity of CSC at high temperature, the proportion of CSC used in sintering blends is restricted. In this research, the effects of fine limonite, slake lime, and bentonite particles on the granulation performance of blends containing a high ratio of CSC were studied through granulation test. Based on the test results, the effects of modification of the binding medium on the sintering performance of blends containing a high ratio of CSC were revealed by the sintering pot test. Both the granulation property and sintering performance of blends with a high proportion of CSC were improved by modifying the binding medium.

THE INFLUENCE OF THE COMPOSITION OF THE CULTURE MEDIUM ON THE DEVELOPMENT OF LEUCONOSTOC LACTIS PRE-FERMENTATION

The regularity of the influence of the culture medium (substrate) on the development of microflora at the stage of preliminary fermentation of the model medium on the basis of white cabbage varieties "Parus" was studied. During the research, strains of lactic acid microorganisms Leuconostoc lactis were used. Step-by-step mathematical processing of the experimental data was carried out. Functional dependencies are obtained that most adequately approximate experimental data for modified (MMC) and basic (BMS) model media. Analysis of the experimental data showed that, depending on the type (composition) of the medium, the same species of microorganisms exhibit different dynamics of titer growth. In connection with this, an algorithm was developed to determine the optimal duration of pre-fermentation – «stop points». As a result of the research, it can be seen that the modification of the model medium with the addition of table salt and ascorbic acid to it promotes the formation of positive dynamics of the comparison indicator. This dynamics has three extremes, but only extremes are of practical significance, which were in the interval of the monotonic decrease of the titer. For successful development of the starting culture of the stage of the main fermentation, one of the conditions is a relatively small amount of the titer of the first culture at the end of the preliminary fermentation step to exclude competition. Thus, the position of the «stop-point» position corresponds to the period after the last peak of the comparison indicator. The investigated regularity of the effect of the preliminary cultivation of gram-positive microorganisms on the activity of lactic acid microorganisms in the process of fermentation is topical, since the whole process and the production of high-quality products fully depend on this approach.

Modification of growth medium of mixed-culture species of microalgae isolated from southern java coastal region

Globally, there is growing interest in microalgae as production organisms. Microalgae contain lipids (oil), proteins and carbohydrates (sugars), and, especially marine algae have been used as food and feed for centuries. Recently, production cost reduction related to the supply of growth nutrients is necessary to make it profitable. Therefore, utilization of molasses, a byproduct of sugar production, as the natural carbon, macronutrients, and micronutrients sources can be helpful. The analysis showed that the content of sucrose, glucose, fructose, potassium, zinc, and magnesium was 68.4% w/w, 18.5% w/w, and 13.1% w/w, 5.5% w/w, 3.91 ppm, and 1,370 ppm respectively. This work aimed to determine the effect of molasses addition to the physio-chemical properties of multi-culture species of microalgae isolated from southern Java coastal region in Indonesia grown under mixotrophic culture. The cultivation in this work used medium which was self-formulated by the authors consisting of NaNO3 (5 mL/L), H3BO3 (1 mL/L), EDTA (1 mL/L), N2H2PO4 (5 mL/L), FeSO4 (1 mL/L), MgSO4 (1 mL/L), NaCl (1 mL/L), micronutrients (1 mL/L), vitamin B1 (1 mL/L), and vitamin B12 (1 mL/L) in 500 mL of water. The medium will be treated to have molasses concentration of 0.05% v/v, 0.15% v/v, 0.25% v/v, 0.35% v/v, and 0.45% v/v.

Pedestal formation of all-semiconductor gratings through GaSb oxidation for mid-IR plasmonics

Mid-IR localized surface plasmon resonances (LSPR) have been demonstrated in nano-ribbons of highly Si-doped InAsSb alloys on GaSb substrates. We show that the slow, steady and selective oxidation of GaSb in water leads to an all-semiconductor mid-IR pedestal configuration consisting of highly doped InAsSb plasmonic resonators on top of GaSb pedestals embedded in an amorphous oxide layer. The homogeneity of the pedestal structure is imaged with an attenuated-total reflection Fourier transform infrared (ATR-FTIR) microscope by measuring around the plasmonic excitation at the plasma wavelength of 5.5 µm. As the plasmonic properties are influenced by modifications of the surrounding medium, we show for all-semiconductor gratings with defined doping level and defined grating geometry, that the GaSb oxidation process allows post-fabrication targeting of mid-IR plasmonic resonances to cover the mid-IR range from 5 to 20 µm. Additionally, the pedestal formation reduces the refractive index mismatch between the two interfaces of the plasmonic resonators which allows the exploitation of a second plasmonic peak and which favors plasmonic field enhancement at the top (air-) side of the structure relevant for enhanced molecular vibration spectroscopy.

Sheep Isolated Secondary Follicles Are Able to Produce Metaphase II Oocytes After Vitrification and Long-Term In Vitro Growth.

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss)

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera)

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190h) and its mean cell diameter (14.5±0.7μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

Bacterial endotoxin-induced endothelial cell injury and calcium overload associated with Toll-like receptor and calcium signal

To explore the effect of toll-like receptor 4 (TLR4), myeloid differentiation protein-2 (MD2), and stromal interaction molecular 1 (STIM1) for regulating human vascular endothelial calcium overload injury and inflammatory reaction induced by bacterial endotoxin (LPS). Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco's modification of Eagle's medium (DMEM). (1) The levels of TLR4, MD2 and nuclear factor-κB (NF-κB) were detected by reverse transcriotion-polymerase chain reaction (RT-PCR) before and 0.5, 1, 6, 12, 24 hours after LPS stimulation. (2) Intracellular calcium peak level was detected by confocal following probe fluo-3 AM loading in HUVEC cells induced with LPS and transfected by psiSTIM or psiTLR. (3) MD2, STIM1 or NF-κB protein level was detected by immunoprecipitation (IP) and immuno-blotting in HUVEC cells which were transfected by TLR4 inhibited expression (psiTLR) for 12 hours and followed by LPS stimulation for 6 hours. (4) HUVEC cells were randomly divided into 6 groups: control group, LPS group, PDTC 0.1 mg/L group, PDTC 1 mg/L group, psiTLR 1 h group and psiTLR 12 h group. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme linked immunosorbent assay (ELISA) in supernatant. The mRNA levels of STIM1 and NF-κB were detected by RT-PCR. (1) The mRNA levels of TLR4, MD2, and NF-κB gradually increased after LPS induction and peaked at 6 hours (2-ΔΔCt: 23.52±2.88, 17.43±3.43, 18.13±2.99, respectively), which were statistically significant before the stimulation with LPS (2-ΔΔCt: 7.02±2.81, 5.19±3.22, 8.11±1.42, all P < 0.05). (2) Extracellular calcium influx in LPS group was increased significantly higher than control group (nmol/L: 108.13±22.33 vs. 41.57±13.19, P < 0.01). Extracellular calcium influx in psiSTIM+LPS group (nmol/L: 62.61±14.12 vs. 108.13±22.33, P < 0.05) and psiTLR+LPS group (nmol/L: 50.78±8.05 vs. 109.43±20.21, P < 0.01) were both suppressed as compared with LPS group. While extracellular calcium peak level in psiTLR+psiSTIM+LPS group further decreased (nmol/L: 39.31±6.42 vs. 109.43±20.21, P < 0.01). (3) MD2 protein but not STIM1 or NF-κB can be detected in anti-TLR4 precipitates in control (ctrl-) by immunoprecipitation. MD2 protein level increased in anti-TLR4 precipitates in LPS group (ctrl+) and was suppressed in TLR4 inhibiting group (psiTLR). (4) The levels of TNF-α in PDTC 1 mg/L group were significantly lower than those of LPS group (ng/L: 0.60±0.24 vs. 1.77±0.66, P < 0.01). The levels of IL-6 in PDTC 0.1 mg/L, 1 mg/L group and psiTLR 12 h group decreased significantly lower than that of LPS group (ng/L: 232.10±63.54, 134.32±37.23, 284.23±56.14 vs. 510.22±89.23, all P < 0.05). Compared to LPS group, the mRNA levels of NF-κB and STIM1 were obviously inhibited in PDTC 1 mg/L group and psiTLR 12 h group [NF-κB mRNA (2-ΔΔCt): 17.22±2.35, 13.24±3.54 vs. 30.16±2.06; STIM1 mRNA (2-ΔΔCt): 12.57±2.43, 12.21±2.46 vs. 25.12±2.02, all P < 0.05]. TLR4, MD2, NF-κB signal and SOC calcium channel STIM1 mediate LPS induced-calcium influx and inflammatory mediators level in HUVEC cells. Extracellular calcium overload and inflammatory response by endotoxin induction can be effectively inhibited by down-regulation of TLR4, NF-κB and/or STIM1.

Laser modification of liquid nutrient medium and antibiotic solutions with the assessment by the bacteria growth dynamics

The processes of laser photoinduced modification of nutrient media and antibiotic solutions are considered. It is shown experimentally that the growth of colonies number is slower on the irradiated medium. The impact of laser radiation on the antibiotic activity is also studied experimentally. It is found that the laser-induced photomodification can affect the antimicrobial activity of the medicinal preparations.

Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

Tomato (Solanum lycopersicum) has rapidly become a valuable model species for a variety of studies including functional genomics. A high-throughput method to obtain transgenic lines sooner than standard methods would greatly advance gene function studies. The goal of this study was to optimize our current transformation method by investigating medium components that would result in a decreased time for recovery of transgenics. For this study, 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings were infected with the Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium with indole-3-acetic acid (IAA) at concentrations of either 0.05 or 0.1 mg/l decreased the recovery time for transgenic lines by 6 weeks as compared to our standard medium that contains zeatin as the only plant growth regulator. We observed 50 and 54 % transformation efficiency on plant regeneration medium containing 0.05 and 0.1 mg/l IAA, respectively. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in earlier root development than medium that did not contain IAA. Addition of IAA to the plant regeneration and rooting media did not have any negative effects on plant development. Recovery of transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time and resources needed to complete investigations of gene function.