Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Hydrothermal time analysis of watermelon (<Emphasis Type="Italic">Citrullus vulgaris</Emphasis> cv. ‘Crimson sweet’) seed germination

This study evaluated the ability of a hydrothermal time model (HTT) to describe the kinetics of watermelon (Citrullus vulgaris cv. ‘Crimson sweet’) seed germination under different temperatures (T) and water potentials (ψ) and also to determine the cardinal temperatures of watermelon. Results indicated that ψ influenced germination rate and germination percentage. For this seed lot, cardinal temperatures were 10 °C for T _b, 28.34 °C for T _o and 40.8 °C for T _c in the control (0 MPa) treatment. There was a decrease in hydrotime constant (θ _H) when T was increased to T _o and then remained constant at supra-optimal temperatures (30 MPah^?1). Also, at temperatures above T _o, ψ _b(50) values increased linearly with T. The k _T value (the slope of the relationship between ψ _b(50) and T exceeds T _o) of this seed lot was calculated as 0.076 MPa°Ch^?1. Results this study show that when the HTT model is applied, it can accurately describe ψ _b(g) and the course of germination around Ts (R ² = 0.82). Moreover, the ψ _b(50) was estimated to be ?0.96 MPa based on this model. Consequently, the germination response of watermelon for all Ts and ψs can be adequately described by the HTT model and enabling it to be used as a predictive tool in watermelon seed germination simulation models.     

A note on imitative behavior

In a preceding paper (Bull. Math. Biophysics,27, 175–185) the distribution function ofφ=? ₁-? ₂,—the difference of excitations in the two mutually inhibiting centers, has been derived in terms of the distribution functionsf ₁(? ₁) andf ₂(? ₂) of the two excitations. In the present note some properties of the distribution functionf(?) in terms of the propertiesf ₁(? ₁) andf ₂(? ₂) are derived.     

Genetically engineered cell lines for α1-antitrypsin expression

Objectives

To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination.

Results

α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT.

Conclusions

CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.

     

What can cell cycle and ultrastructure tell us about desiccation tolerance in Leucaena leucocephala germinating seeds?

Desiccation tolerance (DT) is the ability to tolerate dehydration to levels below 0.1 g(H₂O) g^?1(dry mass) and subsequent rehydration without lethal damage. Here, it is proposed that Leucaena leucocephala, a tree species, has potential to be model tolerant species in seed research. Using flow cytometry and transmission electron microscopy, cytological changes related to loss of DT in Leucaena primary roots were followed during germination. Leucaena seeds lost their DT at the end of germination and this coincided with an increase in cellular 4C DNA content. A negative correlation between the 8C DNA content and the capacity of germinating Leucaena seeds to tolerate desiccation was also observed. Apparently, the seeds of Leucaena underwent extra cycles of endoreduplication and accumulated a high content of DNA — an event not previously linked to DT. The ultrastructural damage imposed by drying overcame Leucaena primary root cell resilience and their ability to resume normal growth. Nuclear DNA content may be used as indicator of progress of germination and loss of DT in Leucaena.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Evaluation and application of constitutive promoters for cutinase production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.     

Cascading flowlines and layout modules: Practical strategies for machine duplication in facility layouts

The standard approach for design of a layout for a high-variety low-volume (HVLV) manufacturing facility has been to use either a from-to chart or a multi-product process chart to design a process layout or a cellular layout, respectively, for the facility. Considerable research has focused on making a go-no go decision to implement any one of these two traditional layouts as the preferred layout for an HVLV manufacturing facility. This paper introduces a variety of Hybrid Cellular Layouts (HCLs) which integrate the attributes of the traditional functional, cellular and flowline layouts. The mathematical models and methods for design of two HCLs—cascading flowline layout and modular layout—are discussed in detail. Unlike the standard models in the literature, the design of the cascading flowline layout introduces a novel string-to-graph aggregation and planar graph embedding method that allows machine duplication in the layout. Similarly, the design of the Modular layout introduces a substring clustering method instead of the standard method of cluster analysis to form part families using the complete routings of the parts. For each HCL, results from an industry project are presented to demonstrate the real-world viability of the concepts, methods and software developed to support the design of HCLs for high-variety low-volume manufacturing facilities.     

Inheritance of Traits Controlled by Odd Chromosomes Using Data on Transmission of Monosomic Addition S<Superscript>t</Superscript> Chromosome of the <Emphasis Type="Italic">Elymus Sibiricus</Emphasis> Genome

On the basis of the winter bread wheat cultivar Obryi, two independent disomic addition lines BC₁₂F_∞ with the chromosome of the E. sibiricus S^t genome are created. A practical algorithm for determining the probabilities of transmission of the odd chromosome separately through male and female gametes in selfpollination of hemizygous hybrids from the equation p²–(1 + f₁–f₄) × p + f₁ = 0 is proposed, where p is the probability of the formation of viable gametes with the considered chromosome and f₁ and f₄ are the empirical frequencies of the corresponding homozygotes with and without the trait. The probability of transmission of an alien univalent chromosome through pollen (p_♂) is associated with the frequency of its transmission through the egg cell (p_♀) in backcrosses and in self-pollination (1–f₄) by the equation p_♂ = 1–f₄/(1–p_♀). The calculated empirically dependent estimates of the probabilities of transmission of the added chromosome through the egg cell p_♀ = 18.7% and through pollen p_♂ = 4.3% correspond to the empirical frequencies obtained for backcrosses. The coefficients of the gamete selection V_♀ = 0.748 and V_♂ = 0.172 are calculated, and the expected segregation for the alien trait controlled by a dominant gene located in the added chromosome is determined—with the trait: without the trait is 0.222: 0.778 in F₂; 0.187: 0.813 in equational and 0.043: 0.957 in certational backcrosses.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Spreading and mechanisms of antimicrobial resistance in microorganisms,producing beta-lactamases. Molecular mechanisms of resistance to beta-lactam antibiotics of <Emphasis Type="Italic">Klebsiella</Emphasis> spp. strains,isolated in cases of nosocomial infections

Antibiotic sensitivity has been investigated in nosocomial bacterial Klebsiella spp. strains isolated from patients treated in 30 hospitals of 15 Russian regions. Among Klebsiella strains (n = 212) studied the following species were found: Klebsiella pneumoniae ss. pneumoniae—182 (85.8%), Klebsiella pneumoniae ss. ozaenae—1 (0.5%), Klebsiella oxytoca—29 (13.7%) strains. Their sensitivity to antibacterial preparations was estimated by the method of serial dilutions in microvolume (the microdilution method). Carbapenems (imipenem and meropenem) exhibited the highest antibacterial activity against the strains studied. Among third generation cephalosporins the lowest MIC (Minimum inhibitory concentration) were found in the inhibitor protected preparations: ceftazidime/clavulanic acid (MIC₅₀ of 0.25 μg/ml; MIC₉₀ of 64 μg/ml) and cefoperazone/sulbactam (MIC₅₀ of 16 μg/ml; MIC₉₀ of 64 μg/ml). Using the PCR method the detection of class A betalactamases genes (TEM, SHV, CTX) was carried out in 42 strains of Klebsiella pneumoniae ss. pneumoniae. TEM type beta-lactamases were found alone or in various combinations in 16 (38.1%) strains, SHV—in 29 (69%), and CTX—in 27 (64.3%). Combinations of 2 and 3 different resistance determinants were detected in 23.8 and 26.2% of strains, respectively. Screening of carbapenem-resistant Klebsiella strains for production of class B metallo-beta-lactamases did not reveal nosocomial strains with phenotypically documented production of these enzymes.     

Alginate biosynthesis by <Emphasis Type="Italic">Azotobacter</Emphasis> bacteria

The ability of representatives of various species of the bacterial genus Azotobacter (A. chroococcum 7B, A. chroococcum 12B, A. chroococcum 12BS, A. agile 12, A. indicum 8, A. vinelandii 17, and A. vinelandii 5B) to alginate synthesis has been studied. It has been shown that all tested bacterial strains have this ability to different extents. Capsular alginate comprises from 2.6 to 32% of the total amount of synthesized alginate in various bacterial species. Strains that are able to active synthesis of alginate have been selected; the effect of the medium composition on their biosynthesis has been studied. The optimal conditions for alginate synthesis by the A. chroococcum 12BS producer strain include the presence of mannitol (40 g/L), yeast extract (1%), and low concentration of phosphates (KH₂PO₄—0.008 g/L, K₂HPO₄—0.032 g/L) in the medium; alginate production under these conditions is 4.5 g/L. The effect of aeration on polymer biosynthesis has been revealed: an increase in aeration causes an increase in alginate synthesis, while its decrease promotes the synthesis of poly-3-hydroxybutirate. It has been shown by IR spectroscopy that alginates obtained under various conditions of cultivation contain different ratios of residues of mannuronic and guluronic acids (M/G from 70/30 to 80/20) in the polymer chain and also differ in the amount of acetyl groups (from 10 to 25%) in the polyme structure.     

Mechanism for phenanthridines synthesis by nitrogenation of 2-acetylbiphenyls in acidic solution: a DFT study

The mechanism of phenanthridines synthesis from the nitrogenation of 2-acetylbiphenyls (1) by TMSN₃ in TFA has been studied by DFT calculations. Results at the B3LYP/6-311G(d) level showed that: 1) reaction of TMSN₃/HN₃ with the protonated form of 1 (1H⁺), which generates the key intermediate C ^x+ by removal of TMSOH/H₂O, is the rate determining step, and TMSN₃ as the nitrogen source is certainly preferred over HN₃. 2) from C ^x+, the two pathways leading to 2 ^x H⁺ and 3 ^x H⁺ are both thermodynamically and kinetically feasible and competitive to each other. 3) The high barriers of the reverse reactions suggest that the ratio of the final products 2 ^x :3 ^x is determined by the branching ratio of reaction rates of C ^x+ to intermediates D ^x+ in pass_I and E ^x+ in pass_II.

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Graphical Abstract DFT results indicate that the replacement of -OH by -N₃ which generates C ^x+ controls the consumption rate of 1 ^x H⁺, and the ratio of C ^x+ transforms to D ^x+ and C ^x+ transforms to E ^x+ (k:k') determines the final ratio of products 2x:3x.

     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Measurements of the plasma density in the FTU tokamak by a pulsed time-of-flight X-wave refractometer

On-line control over the plasma density in tokamaks (especially, in long-term discharges) requires reliable measurements of the averaged plasma density. For this purpose, a new method of density measurements—a pulsed time-of-flight plasma refractometry—was developed and tested in the T-11M tokamak. This method allows one to determine the averaged density from the measured time delay of nanosecond microwave pulses propagating through the plasma. For an O-wave, the measured time delay is proportional to the line-averaged density and is independent of the density profile (f?f _p ) τ_o ≈ k _o \(\tfrac{1}{{f^2 }}\mathop \smallint \limits_l \) N(x dx. A similar formula is valid for an X-wave: τ_X = ≈ k _x \(\tfrac{{f^2 + f_c^2 }}{{(f^2 - f_c^2 )^2 }}\mathop \smallint \limits_l \) N(x)dx. Here, f is the frequency of the probing wave, f _p is the plasma frequency, l= 4 a is the path length for two-pass probing in the equatorial plane, a is the plasma minor radius, k _O and k _X are numerical factors, f _c is the electron-cyclotron frequency at the axis of the plasma column, and f _p ?f _c , f. Measurements of the time delay provide the same information as plasma interferometry, though they do no employ the effect of interference. When the conditions f _p ?f _c , f are not satisfied, the measured time delay depends on the shape of the density profile. In this case, in order to determine the average density regardless of the density profile, it is necessary to perform simultaneous measurements at several probing frequencies in order to determine the average density. In ITER (Bt ～ 5T), a spectral window between the lower and upper cutoff frequencies in the range of 50–100 GHz can be used for pulsed time-of-flight X-wave refractometry. This appreciably simplifies the diagnostics and eliminates the problem of the first mirror. In this paper, the first results obtained in the FTU tokamak with a prototype of the ITER pulsed time-of-flight refractometer are presented. The geometry and layout of experiments similar to the planned ITER experiments are described. The density measured by pulsed time-of-flight refractometry is shown to agree well with the results obtained in FTU with a two-frequency scanning IR interferometer. The results obtained are analyzed, and the future experiments are discussed.     

Spatial genetic structure in seed stands of <Emphasis Type="Italic">Pinus lumholtzii</Emphasis> B.L. Rob. &amp; Fernald in Durango,Mexico

Sad pine is one of the most prominent pine species in Mexico due to its conspicuous pendulous foliage and extreme habitat. However, scientific studies of the species are scarce, and genetic information on sad pine populations is lacking. This endemic tree species occurs naturally on the Sierra Madre Occidental where it covers a total area of about 1,600,000 ha. It typically grows with several species of Quercus and Pinus or in pure stands in uneven-aged forests. Pinus lumholtzii is naturally spatially fragmented, and genetic research on seed and pollen dispersal patterns and spatial genetic structure (SGS)—and the possible implications of these in terms of evolution, conservation and breeding management—is particularly important. Given the fragmented occurrence of the preferred soil type, the goal of this research was to use amplified fragment length polymorphism (AFLP) markers to identify potential differences in spatial genetic structure within and between five P. lumholtzii seed stands at fine and large scales. At the fine scale, we almost always observed non-significant autocorrelation, suggesting that the genetic variants of P. lumholtzii are randomly distributed in space within each sampled seed stand. At the larger scale, our findings provide strong support for the theory of isolation by distance that predicts the expected pattern of SGS at drift–dispersal equilibrium. We recommend a network of P. lumholtzii seed stands of maximum distances of 100 km among stands to prevent greater loss of local genetic variants and use the seeds for reforestations in a radius of maximal 50 km from their proveniences.     

Antimicrobial activity,cytotoxicity and chemical analysis of lemongrass essential oil (<Emphasis Type="Italic">Cymbopogon flexuosus</Emphasis>) and pure citral

The aim of this study was to determine the antimicrobial effects of lemongrass essential oil (C. flexuosus) and to determine cytotoxic effects of both test compounds on human dermal fibroblasts. Antimicrobial susceptibility screening was carried out using the disk diffusion method. Antimicrobial resistance was observed in four of five Acinetobacter baumannii strains with two strains confirmed as multi-drug-resistant (MDR). All the strains tested were susceptible to both lemongrass and citral with zones of inhibition varying between 17 to 80 mm. The mean minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of citral (mic—0.14 % and mbc—0.3 % v/v) was lower than that of Lemongrass (mic—0.65 % and mbc—1.1 % v/v) determined using the microtitre plate method. Cell viability using human dermal fibroblasts (HDF; 106-05a) was determined following exposure to both compounds and a control (Grapeseed oil) using the XTT assay and the IC₅₀ determined at 0.095 % (v/v) for citral and 0.126 % (v/v) for lemongrass. Grapeseed oil had no effect on cell viability. Live cell imaging was performed using the LumaScope 500 imaging equipment and changes in HDF cell morphology such as necrotic features and shrinkage were observed. The ability of lemongrass essential oil (EO) and citral to inhibit and kill MDR A. baumannii highlights its potential for use in the management of drug-resistant infections; however, in vitro cytotoxicity does suggest further tests are needed before in vivo or ex vivo human exposure.     

Fatty acid composition and <Emphasis Type="Italic">PlFADs</Emphasis> expression related to α-linolenic acid biosynthesis in herbaceous peony (<Emphasis Type="Italic">Paeonia lactiflora</Emphasis> Pall.)

As a new α-linolenic acid (ALA) resource, there has been little known about the relationship between expression levels of fatty acid desaturase (FAD) genes during seed development and fatty acid (FA) composition in herbaceous peony (Paeonia lactiflora Pall.). In this study, oil content and FA composition of nine cultivars were measured at four different stages during seed development. Moreover, five genes including PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 related to the ALA biosynthesis were isolated. Furthermore, the relative expression levels of these genes in seeds were investigated in two cultivars, that is, ‘PL1’ and ‘PL2’ with higher and lower ALA content, respectively. The results showed that oil content was from 17.03 ± 0.15 to 24.51 ± 0.15%; 15 kinds of FA were detected. However, the relative content of ALA was decreased during seed development. Although, the genes PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 were all expressed during the seed development, the expression levels varied significantly. Most of the genes were expressed strongly in the early stage but weakly in the late stage except PlFAD6. Moreover, expression level of gene PlFAD2-2 was the highest while that of PlFAD7 was lowest at S1. In addition, the relative expression level of genes in ‘PL1’ (high ALA content) was higher than those in ‘PL2’ (low ALA content) during the early stage of the seed development apart from gene PlFAD6. This study provides a reliable theoretical basis for improving the contents of ALA by means of genetic engineering in the herbaceous peony seed oil.