Agrobacterium-Mediated Gene Transfer in Plants and Biosafety Considerations

Agrobacterium, the natures?? genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world??s population.     

Strategies for genetic manipulation of adult stem cell-derived organoids

Organoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology.Subject terms: Mechanisms of disease, Genetic engineering, Mutagenesis, Experimental models of disease     

Modeling in biological chemistry. From biochemical kinetics to systems biology

A brief review on biochemical kinetics in the twentieth century mainly concerned with enzyme kinetics and cooperative processes is presented. Molecular biology and, in particular, structural biology provided the basis for modeling biological phenomena at the molecular level. Structure was recognized as the ultimate and only level at which biological processes find an explanation that is satisfactory for chemists and physicists. A new epoch in biology was initiated by successful extensions of the molecular approach from individual molecules and reactions to the cellular and organismic level. Starting with sequencing of whole genomes in the 1980s more and more techniques became available that are suitable for upscaling from molecules to cells. A series of research programs was initiated: genomics dealing with sequencing the DNA of whole organisms, proteomics considering all proteins of a cell and their interactions, metabolomics studying all metabolic reactions of a cell or an organism, and functional genomics or systems biology aiming at an exploration of the dynamics of complete biological entities. At the same time computational facilities have experienced an unexpected development in speed of calculations and storing devices. At present computer simulations of whole cells at molecular resolution are within reach. The challenge for the theorist in biology is to develop methods for handling the enormously complex networks of gene regulation and metabolism in such a way that biological questions can be addressed. This goal cannot be achieved by dynamical systems theory alone. What is needed is a joint effort from different mathematical disciplines supported by empirical knowledge and tools from discrete mathematics to informatics. Two sections with selected examples from our own laboratory dealing with structural bioinformatics of RNA and with a dynamical systems approach to gene regulation are added.     

Functional characterisation of metal(loid) processes in planta through the integration of synchrotron techniques and plant molecular biology

Functional characterisation of the genes regulating metal(loid) homeostasis in plants is a major focus for phytoremediation, crop biofortification and food security research. Recent advances in X-ray focussing optics and fluorescence detection have greatly improved the potential to use synchrotron techniques in plant science research. With use of methods such as micro X-ray fluorescence mapping, micro computed tomography and micro X-ray absorption near edge spectroscopy, metal(loids) can be imaged in vivo in hydrated plant tissues at submicron resolution, and laterally resolved metal(loid) speciation can also be determined under physiologically relevant conditions. This article focuses on the benefits of combining molecular biology and synchrotron-based techniques. By using molecular techniques to probe the location of gene expression and protein production in combination with laterally resolved synchrotron techniques, one can effectively and efficiently assign functional information to specific genes. A review of the state of the art in this field is presented, together with examples as to how synchrotron-based methods can be combined with molecular techniques to facilitate functional characterisation of genes in planta. The article concludes with a summary of the technical challenges still remaining for synchrotron-based hard X-ray plant science research, particularly those relating to subcellular level research.     

Synthesis of bis[benzyl‐N′‐hydrazinecarbodithioato‐κ2 N′,S]nickel(II) complex as a novel lead molecule for cancer treatment

A novel bis[benzyl‐N′‐hydrazinecarbodithioato‐κ² N′,S]nickel(II) complex was synthesized and characterized by means of various physical, chemical, and spectroscopic techniques. The X‐ray single crystal diffraction analysis indicated two independent close comparable bis‐chelated square planar complexes of trans‐configuration, where S‐benzyl dithiocarbazate (SBDTC) ligand is coordinated via N,S‐donor set. The complex is able to inhibit Ehrlich ascites carcinoma (EAC) cell proliferation by 51.81% and 75.75%, with 0.3 and 50 mg kg^?1 (dose adjusted) dose, respectively, administered intraperitoneally for five successive days in mice model. Apoptotic cell morphological changes were examined using optical and fluorescence microscopy techniques. Expression pattern of apoptosis regulatory genes in EAC cells treated with the synthesized nickel(II) complex for five consecutive days showed an increased expression of P⁵³, Bax, Cas‐8, Cas‐9, Cas‐3, Cyt‐c, and TNF‐α proapoptotic genes and decreased expression of antiapoptotic Bcl‐2 gene. The Ni(II) complex and Bleomycin (standard drug) were used in molecular docking coupled with molecular dynamics simulation studies with the aim to support the experimental results and to investigate the apoptotic effect towards the targeting apoptotic genes. Both experimental and computational studies reveal that the nickel(II) complex inhibits EAC cells growth successfully, suggesting a potential compound for cancer treatment.     

NMR and Molecular Genetics as Tools for Investigating Protein Interactions in Membrane Systems

In our laboratory, we have applied the tools of nuclear magnetic resonance (NMR) spectroscopy and molecular genetics to investigate the structural and dynamic properties of membrane-associated proteins and their interactions with membrane components. There are two general classes of membrane proteins, i.e., intrinsic and peripheral ones. For the intrinsic membrane proteins, we have chosen the membranebound D-lactate dehydrogenase (D-LDH) of Escherichia coli as a model to study protein-lipid interactions in membranes. D-LDH is a respiratory enzyme of molecularweight 65, 000 containing flavin adenine dinucleotide (FAD) as a cofactor. The activity of purified D-LDH is enhanced up to 100-fold by lipids and detergents. The gene for D-LDH has been sequenced, and production of the enzyme amplified up to 300-times normal levels. We have biosynthetically incorporated 5-fluorotryptophan (5F-Trp) into D-LDH and studied the five Trp residues by ¹⁹F-NMR spectroscopy. In order to gain additional information using ¹⁹F-NMR, site-specific, oligonucleotide-directed mutagenesis has been used to insert a sixth Trp into D-LDH at various positions throughout the 571-amino acid chain. These mutant D-LDHs are being characterized biochemically and through NMR. For peripheral membrane proteins, we have chosen two periplasmic binding proteins, histidine-binding protein J (J protein) of Salmonella Typhimurium and glutamine-binding protein (GlnBP) of E. coli as models to investigate the structure-function relationship in periplasmic binding protein-mediated active transport systems. These two proteins both have molecular weights of approximately 25, 000. By using mutant J proteins and GlnBPs and site-specific, oligonucleotide-directed mutagenesis techniques, we have assigned several resonances to specific amino acid residues. We are investigating the relationship between ligand-induced conformational changes in these two proteins and their roles in the active transport of ligand across the cell membrane. We have found that a combination of isotopic labeling, biochemistry, molecular biology, and NMR is a very useful approach to investigate various interactions of membrane-associated protein systems.     

Transient Expression of Minimum Linear Gene Cassettes in Onion Epidermal Cells Via Direct Transformation

A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5′-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca²⁺, surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 °C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.     

Advances and Developments in Strategies to Improve Strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and Processes to Obtain the Lignocellulosic Ethanol−A Review

The conversion of biomass into ethanol using fast, cheap, and efficient methodologies to disintegrate and hydrolyse the lignocellulosic biomass is the major challenge of the production of the second-generation ethanol. This revision describes the most relevant advances on the conversion process of lignocellulose materials into ethanol, development of new xylose-fermenting strains of Saccharomyces cerevisiae using classical and modern genetic tools and strategies, elucidation of the expression of some complex industrial phenotypes, tolerance mechanisms of S. cerevisiae to lignocellulosic inhibitors, monitoring and strategies to improve fermentation processes. In the last decade, numerous engineered pentose-fermenting yeasts have been developed using molecular biology tools. The increase in the tolerance of S. cerevisiae to inhibitors is still an important issue to be exploited. As the industrial systems of ethanol production operate under non-sterile conditions, microbial subpopulations are generated, depending on the operational conditions and the levels of contaminants. Among the most critical requirements for production of the second-generation ethanol is the reduction in the levels of toxic by-products of the lignocellulosic hydrolysates and the production of low-cost and efficient cellulosic enzymes. A number of procedures have been established for the conversion of lignocellulosic materials into ethanol, but none of them are completely satisfactory when process time, costs, and efficiency are considered.     

Single-Crystal X-Ray Diffraction Studies of Oligonucleotides and Oligonucleotide–Drug Complexes

Nucleic acids constitute the library of genetic information for all living organisms. They also play a regulatory role in many biological events concerned with the utilization of genetic information. The double-helical model of DNA, proposed by Watson and Crick in 1953, suggested the structural basis for its biological role, but this insight into nucleic acid structures seems to have generated as many questions as it has provided answers. Experimental studies, in particular fiber diffraction work, yielded a wealth of information on the conformational flexibility of nucleic acids and on the importance of interactions with water and cations. Major advances in synthetic organic chemistry, with implications for molecular biology, propelled nucleic acid research forward in the late 1970s. The availability of milligram quantities of synthetic oligonucleotides of defined sequence and high purity paved the way for detailed and accurate structural analysis using single-crystal X-ray diffraction methods and, in more recent times, NMR spectroscopy. This article is a detailed survey of the structural results generated by crystallographic techniques as applied to DNA, RNA, and nucleic acid–drug complexes over the period 1979–1990. The appendix lists important definitions used in the characterization of oligonucleotide structures.     

Universal calibration in GPC: A study of polystyrene,poly-α-methylstyrene,and polypropylene

The theoretical justification for using M[_η], or a similar quantity, as a universal calibration parameter in GPC is reviewed. The equation based on this parameter is applied to transform the primary calibration curve, obtained by means of polystyrene samples, into calibration curves for poly-α-methylstyrene, polypropylene, and linear polyethylene. The Mark–Houwink equations for these polymers, as they are used in the transformation, are discussed. The resulting GPC calibration curves are compared with molecular weights and peak elution volumes of fractionated poly-α-methylstyrene and polypropylene. The same comparison is made with samples of polypropylene and polyethylene having very broad molecular weight distributions. The agreement lies within experimental error.     

GENETIC AND MOLECULAR ANALYSES OF UV RADIATION-INDUCED MUTATIONS IN THE FEM-3 GENE OF CAENORHABDITIS ELEGANS

The utility of a new target gene (fem-3) is described for investigating the molecular nature of mutagenesis in the nematode Caenorhabditis elegans. As a principal attribute, this system allows for the selection, maintenance and molecular analysis of any type of mutation that disrupts the gene, including deletions. In this study, 86 mutant strains were isolated, of which 79 proved to have mutations in fem-3. Twenty of these originally tested as homozygous inviable. Homozygous inviability was expected, as Stewart and coworkers had previously observed that, unlike in other organisms, most UV radiation-induced mutations in C. elegans are chromosomal rearrangements of deficiencies (Mutat. Res. 249 , 37–54, 1991). However, additional data, including Southern blot analyses on 48 of the strains, indicated that most of the UV radiation-induced fem-3 mutations were not deficiencies, as originally inferred from their homozygous inviability. Instead, the lethals were most likely “coincident mutations” in linked, essential genes that were concomitantly induced. As such, they were lost owing to genetic recombination during stock maintenance. As in mammalian cells, yeast and bacteria, the frequency of coincident mutations was much higher than would be predicted by chance.     

Isotachophoresis for rapid transformation of Escherichia coli

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2–3 × 10⁵) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 μm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (–16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.     

Darwin and Molecular Biology

Darwin's “idea of thehe century”, the principle of selection, is as important in the age of molecular biology as it was a hundred years ago. As a natural law it is open to rigorous physical proof, if certain prerequisites are met, and to quantitative experimental test—in vitro and in vivo—under defined laboratory conditions.     

Modern Trends in the In Vitro Production and Use of Callus,Suspension Cells and Root Cultures of Medicinal Plants

This paper studies modern methods of producing and using callus, suspension cells and root cultures of medicinal plants in vitro. A new solution for natural product production is the use of an alternative source of renewable, environmentally friendly raw materials: callus, suspension and root cultures of higher plants in vitro. The possibility of using hairy root cultures as producers of various biologically active substances is studied. It is proven that the application of the genetic engineering achievements that combine in vitro tissue culture and molecular biology methods was groundbreaking in terms of the intensification of the extraction process of compounds significant for the medical industry. It is established that of all the callus processing methods, suspension and root cultures in vitro, the Agrobacterium method is the most widely used in practice. The use of agrobacteria has advantages over the biolistic method since it increases the proportion of stable transformation events, can deliver large DNA segments and does not require special ballistic devices. As a result of the research, the most effective strains of agrobacteria are identified.     

Proton transport through hydrated chitosan‐based polymer membranes under electric fields

Proton transport is essential in many areas of chemistry and biology and is especially important in the fields of proton exchange membrane fuel cells and biocompatible, protonic semiconductors. These devices make use of membranes to control the flow of protons for either the generation of energy or to more closely couple electronics and biology. In the present study, we make use of ab initio molecular dynamics simulations, including the effect of applied electric fields, to gain atomistic insight into the intrinsic conductivity of chitosan‐based polymers and demonstrate that chitosan does not act as a significant source of friction for the transport of protons while increasing the number of free ions. Published 2017.^? J. Polym. Sci., Part B: Polym. Phys. 2017 , 55 , 1103–1109     

Combinatorial Synthesis of Small Organic Molecules

Combinatorial synthesis has developed within a few years from a laboratory curiosity to a method that is taken seriously in drug research. Rapid progress in molecular biology and the resulting ability to determine the activity of new substances extremely efficiently have led to a change in paradigm for the synthesis of test compounds: in addition to the conventional procedure of synthesizing one substance after another, new methods allowing simultaneous creation of many structurally defined substances are becoming increasingly important. A characteristic of combinatorial synthesis is that a reaction is performed with many synthetic building blocks at once—in parallel or in a mixture— rather than with just one building block. All possible combinations are formed in each step, so that a large number of products, a so-called library, is obtained from only a few reactants. Several methods have been developed for combinatorial synthesis of small organic molecules, based on research into peptide library synthesis: single substances are produced by highly automated parallel syntheses, and special techniques enable targeted synthesis of mixtures with defined components. Many structures can be obtained by combinatorial synthesis, and the size of the libraries created ranges from a few individual compounds to many thousand substances in mixtures. This article gives an overview of the combinatorial syntheses of small organic molecules reported to date, performed both in solution and on a solid support. In addition, different techniques for identification of active compounds in mixtures are presented, together with ways to automate syntheses and process the large amounts of data produced. An overview of pionering companies active in this area is also given. The final outlook attempts to predict the future development of this exponentially growing area and the influence of this new thinking in other areas of chemistry.     

Fast detection of Piscirickettsia salmonis in Salmo salar serum through MALDI‐TOF‐MS profiling

Piscirickettsia salmonis is a pathogenic bacteria known as the aetiological agent of the salmonid rickettsial syndrome and causes a high mortality in farmed salmonid fishes. Detection of P. salmonis in farmed fishes is based mainly on molecular biology and immunohistochemistry techniques. These techniques are in most of the cases expensive and time consuming. In the search of new alternatives to detect the presence of P. salmonis in salmonid fishes, this work proposed the use of MALDI‐TOF‐MS to compare serum protein profiles from Salmo salar fish, including experimentally infected and non‐infected fishes using principal component analysis (PCA). Samples were obtained from a controlled bioassay where S. salar was challenged with P. salmonis in a cohabitation model and classified according to the presence or absence of the bacteria by real time PCR analysis. MALDI spectra of the fish serum samples showed differences in its serum protein composition. These differences were corroborated with PCA analysis. The results demonstrated that the use of both MALDI‐TOF‐MS and PCA represents a useful tool to discriminate the fish status through the analysis of salmonid serum samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA

Some RNA classes require folding into the proper higher‐order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA‐binding ligand, naphthyridine carbamate tetramer with Z‐stilbene linker (Z‐NCTS). Binding of Z‐NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z‐NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z‐NCTS‐responsive genetic switch using the HHR sequence inserted into the 3′ untranslated region as a cis‐acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z‐NCTS and was functional in mammalian cells.     

Silica Nanoparticles-mediated Stable Genetic Transformation in Nicotiana tabacum

Nanoparticles as gene carriers become popular in the mammalian cells, whereas the application of them in plant cells is still very limited. Herein lies a report on silica nanoparticles(SiNPs) modified with positively charged poly-L-lysine(PLL) successfully delivering plasmid-encoded β-glucuronidase(GUS) gene into tobacco with the help of gene gun. The stable transgenic tobacco plants mediated by SiNPs can be obtained. Furthermore, we revealed the quantity of gene and types of receptor materials could affect the expression efficiency. In comparison to conventional gold particles-mediated transformation, the silica nanoparticles-mediated stable genetic transformation enhances transformation efficiency, potentially overcoming transgenic silencing. Our results demonstrate the great potential of SiNPs as gene carrier in plant genetic transformation and prove a novel approach for plant genetic decoration.     

QSAR and Mechanistic studies on the genotoxic compounds including environmental effects

The effects of a proximate condensed environment as the solvent and cellular structured patterns (biopolymers, membranes, etc.) play an important role in determination of the courses of molecular processes in biology. We present here the background of methods developed for such an environmental effects estimation combining the continuum and discrete models. Their applications within theoretical studies into the mechanisms of carcinogenic action of alkylating N-nitrosocompounds are shown. The results given cover four different areas, namely the quantitative structure-activity relationship, mechanistic studies into their metabolic activation reactions, interactions of the ultimate carcinogens with DNA, and finally their genetic consequences.