Engineering chimeric two-component system into <Emphasis Type="Italic">Escherichia coli</Emphasis> from <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.     

Fluorescence change of <Emphasis Type="Italic">Fusobacterium nucleatum</Emphasis> due to <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

The aim of this study was to measure changes in the fluorescence of Fusobacterium nucleatum interacting with Porphyromonas gingivalis for excitation with blue light at 405-nm. P. gingivalis was mono- and co-cultivated in close proximity with F. nucleatum. The fluorescence of the bacterial colonies was photographed using a QLF-D (Quantitative Light-induced Fluorescence-Digital) Biluminator camera system with a 405 nm light source and a specific filter. The red, green and blue intensities of fluorescence images were analyzed using the image analysis software. A fluorescence spectrometer was used to detect porphyrin synthesized by each bacterium. F. nucleatum, which emitted green fluorescence in single cultures, showed intense red fluorescence when it was grown in close proximity with P. gingivalis. F. nucleatum co-cultivated with P. gingivalis showed the same pattern of fluorescence peaks as for protoporphyrin IX in the red part of the spectrum. We conclude that the green fluorescence of F. nucleatum can change to red fluorescence in the presence of adjacent co-cultured with P. gingivalis, indicating that the fluorescence character of each bacterium might depend on the presence of other bacteria.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

The use of parameters of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction to evaluate the state of plants under anthropogenic load

The physiological state of the leaves of the small-leaved linden (Tilia cordata), silver birch (Betula pendula), and northern white cedar (Thuja occidentalis) under urban conditions was assessed via recording the kinetics of chlorophyll under fluorescence induction. Different sensitivities of the plants to adverse growing conditions were revealed. The most sensitive parameters of the fluorescence JIP test, viz., PI _ABS , F _V/F ₀, and F _V/F _M, were identified as indicators of the physiological state of the urban phytocoenosis. Recommendations for the application of the method for monitoring studies are presented.     

<Emphasis Type="BoldItalic">In vivo</Emphasis> conditions influence the coding of stimulus features by bursts of action potentials

The functional role of burst firing (i.e. the firing of packets of action potentials followed by quiescence) in sensory processing is still under debate. Should bursts be considered as unitary events that signal the presence of a particular feature in the sensory environment or is information about stimulus attributes contained within their temporal structure? We compared the coding of stimulus attributes by bursts in vivo and in vitro of electrosensory pyramidal neurons in weakly electric fish by computing correlations between burst and stimulus attributes. Our results show that, while these correlations were strong in magnitude and significant in vitro, they were actually much weaker in magnitude if at all significant in vivo. We used a mathematical model of pyramidal neuron activity in vivo and showed that such a model could reproduce the correlations seen in vitro, thereby suggesting that differences in burst coding were not due to differences in bursting seen in vivo and in vitro. We next tested whether variability in the baseline (i.e. without stimulation) activity of ELL pyramidal neurons could account for these differences. To do so, we injected noise into our model whose intensity was calibrated to mimic baseline activity variability as quantified by the coefficient of variation. We found that this noise caused significant decreases in the magnitude of correlations between burst and stimulus attributes and could account for differences between in vitro and in vivo conditions. We then tested this prediction experimentally by directly injecting noise in vitro through the recording electrode. Our results show that this caused a lowering in magnitude of the correlations between burst and stimulus attributes in vitro and gave rise to values that were quantitatively similar to those seen under in vivo conditions. While it is expected that noise in the form of baseline activity variability will lower correlations between burst and stimulus attributes, our results show that such variability can account for differences seen in vivo. Thus, the high variability seen under in vivo conditions has profound consequences on the coding of information by bursts in ELL pyramidal neurons. In particular, our results support the viewpoint that bursts serve as a detector of particular stimulus features but do not carry detailed information about such features in their structure.     

Trojan Horse Strategy for Non-invasive Interference of <Emphasis Type="Italic">Clock</Emphasis> Gene in the Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.     

Parasitism may alter functional response comparisons: a case study on the killer shrimp <Emphasis Type="Italic">Dikerogammarus villosus</Emphasis> and two non-invasive gammarids

The Ponto-Caspian freshwater amphipod Dikerogammarus villosus has colonized most of the water bodies of continental Europe where it causes strong structural alterations in recipient communities that can lead to changes in ecosystem-level processes, mainly because of a strong predatory behaviour. Most of the D. villosus populations from the invaded range have been found infected with the co-introduced microsporidian parasite Cucumispora dikerogammari, known to decrease the predation rate of its host. Infection might thus mitigate the ecological impact of D. villosus and we wanted to test this assumption using the comparative functional response approach. We compared the relationship between resource use and resource availability (i.e. the functional response, FR) of D. villosus, either with infected individuals or not, to that of two non-invasive gammarids: Gammarus pulex and Echinogammarus berilloni. With infected individuals included, D. villosus displayed a higher FR than the two non-invasive gammarids. Although this effect was not significant, C. dikerogammari infection tended to alter the FR of D. villosus with a slight decrease in attack rate and handling time, resulting in a less steep initial slope and a higher asymptote, respectively. Removing infected D. villosus from the dataset did not affect the FR comparison with G. pulex but suppressed the difference in FR with E. berilloni. Although we cannot exclude the role of sample size reduction in this effect, this suggests that C. dikerogammari infection might increase the predation pressure on local prey populations in case of species replacement between D. villosus and E. berilloni. From a more general perspective, our study illustrates how parasites may alter our capacity to predict invasive species impacts from FR comparisons.     

<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a PEO-Gellan Gum Semi-Interpenetrating Polymer Network for the Oral Delivery of Sulpiride

In this study, an optimized epichlorohydrin-crosslinked semi-interpenetrating polymer network xerogel matrix system (XePoMas) for the controlled delivery of sulpiride was prepared. The ability of XePoMas to sustain drug release was determined by in vitro and in vivo drug release experiments. Swelling of the xerogel over the 24-h experimental period ranged from 346 to 648%; swelling was observed to increase exponentially over the initial 8 h. In vitro drug release depicted a linear zero order drug release profile with an R ² value of 0.9956. The ability of the fabricated XePoMas to sustain drug release and enhance bioavailability of sulpiride in vivo was investigated by evaluating the plasma drug concentration over 24 h in the large pig model. The optimized XePoMas formulation was shown to increase intestinal absorption of sulpiride to a greater extent than the marketed product in vivo, with a C _max of 830.58 ng/mL after 15 h.     

Long non-coding RNA <Emphasis Type="Italic">HOTAIR</Emphasis> regulates proliferation and invasion via activating Notch signalling pathway in retinoblastoma

Retinoblastoma is the most frequently occurring tumour in the eyes in early childhood. Novel targets that are important for the diagnosis or treatment of retinoblastoma could be valuable in increasing the survival rate of patients affected by this disease. Long non-coding RNAs (lncRNAs) are a recently discovered type of RNAs with no protein-coding function; yet it has become increasingly clear that lncRNAs are responsible for important gene regulatory functions in various diseases. In this study, the expression of lncRNA HOTAIR was measured by qRT-PCR, and HOTAIR expression was found to be significantly upregulated in human retinoblastomas tissues as compared with that in paracancerous tissues. Knockdown of HOTAIR restricted the proliferation and invasion of the more invasive retinoblastoma Y79 cells, and led to G0/G1 arrest, possibly through inhibiting phospho-RB1, RB1 and CCNE. Furthermore, we found that the Notch signalling pathway was activated abnormally in retinoblastoma cell lines, while knockdown of HOTAIR attenuated the endogenous Notch signalling pathway in vitro and in vivo. In addition, knockdown of HOTAIR could inhibit the tumour progression in a xenograft model of retinoblastoma. In summary, our findings indicate that HOTAIR may play important roles in retinoblastoma progression via Notch pathway. HOTAIR has the potential to enhance the development of novel targeted diagnostic and therapeutic approaches for retinoblastoma.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Growth Response to Hormones by a New Rat Ovary Cell Line

SEVERAL endocrine cell lines established in recent years show a functional response to hormones in vitro¹ but, except for one mammary cell line², none of them exhibits the normal hormone requirement for growth in vivo. We have now isolated a rat ovarian cell line whose growth in vitro is markedly stimulated by bovine luteinizing hormone (LH-NIH-B7), a pituitary gonadotrophin and by dexamethasone, a synthetic glucocorticoid. This cell line provides the first permanent in vitro system for studying the growth stimulation of gonadal cells by hormones.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

Characterization of <Emphasis Type="Italic">SWEET</Emphasis> family members from loquat and their responses to exogenous induction

The plant SWEET family is a sugar transporter family that plays a significant role in plant development. Here, seven loquat SWEET family members were identified by RNA-seq. These were designated as EjSWEET1, EjSWEET2a, EjSWEET2b, EjSWEET2c, EjSWEET4, EjSWEET15, and EjSWEET17. Phylogenetic and predictive functional annotation analyses suggest that the loquat SWEETs are classified as having sucrose, glucose and fructose transportation features. The in vivo responses of loquat SWEETs to exogenous sugar or NaCl was investigated by applying high concentrations of sugar or salt to 7-month-old loquat seedlings cultured in a nutrient medium. The results showed that most loquat SWEET genes can respond to exogenous applications of sucrose, glucose, fructose and salt. The response of EjSWEET1 to exogenous fructose was faster than the others, indicating that EjSWEET1 is more sensitive to exogenous fructose compared with other loquat SWEETs. EjSWEET15 can be induced by sucrose, but is suppressed by glucose. This indicates its possible role in sucrose transporting. The response of loquat SWEETs to NaCl showed broadly similar patterns compared to sugars. However, after a longer time of NaCl treatment, most loquat SWEETs are upregulated, especially EjSWEET15. This indicates its long-term response to high salinity.     

Arabinose C Protein: Regulation of the Arabinose Operon <Emphasis Type="Italic">in vitro</Emphasis>

The detection of the gene ara C protein using a DNA-dependent in vitro protein synthesizing system represents the first isolation of an operon specific positive regulator with an in vivo role that has been genetically defined.     

Investigation of the photosynthetic activity of bark phelloderm of arboreous plants using the fluorescent method

Seasonal changes in the characteristics of chlorophyll fluorescence were studied in the bark of several species of trees originating in various climatic zones: Siberian cedar (Pinus sibirica), larch (Larix sibirica), eastern arborvitae (Thuja occidentalis), pendent white birch (Betula pendula), wild black cherry (Padus virginiana), horse chestnut (Aesculus hippocastanum), red oak (Quercus rubra), Manchurian catalpa (Catalpa bungei), linden (Tilia cordata), goat willow (Salix caprea), Amur cherry (Padus maackii), and apple Korichnaya (Malus domestrica B.). Tree bark has a sufficient amount of chlorophyll for measuring the parameters of chlorophyll fluorescence throughout the year. The relative yield of the variable fluorescence of chlorophyll (F _v/F _m) can be used to assess seasonal changes in the physiological state of various trees.     

Detection of Antigen-binding Cells by Combined Rosette Formation and Autoradiography

DETERMINATION of the frequency of chromosome aberrations in cultured blood lymphocytes may provide a means of measuring ionizing radiation doses, at least after whole body exposure. Much work has been done with human blood irradiated in vitro^1,2, but before these results can be applied to radiation exposure in vivo, the difference between in vitro and in vivo exposure must be shown to be quantitatively negligible.