PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 10. PRODUCTION OF BIOLOGICALLY ACTIVE SUBSTANCES BY GENETIC AND CELLULAR ENGINEERING METHODS

10.1 Recombinant DNA

Introduction/32.html">Genetic Engineering, or Recombinant DNA technology, is based on the in vitro construction of DNA fragments followed by the Introduction of new (recombinant) genetic structures into a living Cell and their expression. Genetic engineering techniques are used to study The Structure of Organism genes and to develop Gene Therapy and Molecular Diagnostics Methods. Recombinant DNA technology is employed to create new antibiotic producers that yield antimicrobial agents with modified properties. Furthermore, genetic engineering makes it possible to obtain Blood products from Transgenic Animals AND to produce human Proteins through the cultivation of recombinant microorganism strains (Table 20).

Class="center">Table 20. Recombinant proteins: commercial products and those undergoing clinical trials

10.2 Methods of In Vitro GENETIC ENGINEERING OF Microorganisms

A typical genetic engineering experiment consists of the following stages: 1) Obtaining DNA Fragments; 2) constructing in vitro recombinant DNA molecules consisting of the fragments obtained in The First stage and vectors, which are small structures that replicate autonomously in the recipient cell (Plasmids, phages, Viruses); 3) introducing the recombinant DNAs into the recipient cell; 4) selecting clones carrying the desired recombinant DNA (Fig. 62).

Fig. 62. Diagram of a typical genetic engineering experiment: Ampr — ampicillin resistance, a genetic marker of the plasmid.

10.3 Sources of DNA for Cloning

There are three sources of DNA molecules used in genetic engineering: fragments of genetic material from various organisms; double-stranded DNAs obtained on The basis of single-stranded DNA complementary to mRNA (cDNA); and DNA obtained by chemical-enzymatic synthesis. Obtaining cDNA is necessary for expressing human protein genes in Bacteria: eukaryotic genes contain introns, whereas bacterial cells lack a splicing mechanism. Therefore, special techniques are applied to obtain DNA consisting of nucleotide sequences that correspond to exons.

For cDNA synthesis, mature mRNA (devoid of introns) is used as a template, which is pre-polyadenylated (Fig. 63). Next, it is annealed with oligo-dT, which serves as a primer when copying the strand with Reverse Transcriptase. Annealing is The process of DNA reassociation involving The formation of Hydrogen Bonds between Base Pairs. Following reverse transcriptase Treatment, the mRNA is removed by alkaline Hydrolysis. The resulting cDNA has a hairpin structure at its 3' end, which can act as a primer for synthesizing the second DNA strand using DNA polymerase. To remove the oligo-dT and the single-stranded loop, S1 nuclease, which cleaves single-stranded DNA, is used.

Fig. 63. Scheme of double-stranded cDNA synthesis on mRNA.

10.4 Restriction Endonucleases

Restriction Enzymes are an essential tool in gene manipulation. These are specific endonucleases that form an integral part of the restriction-modification system of Prokaryotic Cells. This system is associated with protecting cells from the penetration of foreign DNA. The modification system methylates The Cell's own DNA immediately after Replication. Bacteria hydrolyze foreign DNA penetrating the cell using restriction enzymes. These endonucleases bind to DNA at specific sites (recognition sites) and cleave it into fragments (restriction fragments). Restriction enzymes do not destroy the host's own DNA because its recognition sites are methylated. Restriction-modification systems have been found in all studied bacteria and in some Yeasts.

There are three main classes of restriction enzymes. All of them recognize strictly defined nucleotide sequences on double-stranded DNA. However, class I restriction enzymes break DNA molecules at random points, whereas classes II and III cleave them at strictly defined points within recognition sites or at a fixed distance from them. Class I and III enzymes have a complex subunit structure and exhibit Two Types of activities: methylating and endonuclease activities. Class II enzymes consist of two separate proteins: a restricting endonuclease and a modifying methylase. For these reasons, exclusively class II restriction enzymes are used in genetic engineering.

There is a generally accepted nomenclature according to which restriction enzymes and methylases are designated by the letters R and M, respectively. The name of an enzyme is made up of the first letter of the genus and the first two letters of the species of bacteria from which it was isolated, for example, Bacillus subtilis — Bsu, Escherichia coli — Eco. If necessary, a typical strain characteristic is provided, for example, Hinc — an enzyme from Haemophilus influenzae, serotype C. If a specific bacterial strain has several restriction systems, an additional numerical designation is given. If the enzyme is encoded by plasmid or phage genes, the name of the extrachromosomal element is added to the enzyme name: EcoRI, EcoPI are E. coli enzymes encoded by plasmid R1 and phage P1, respectively.

Table 21 presents the Characteristics of Some restriction enzymes used in genetic engineering. Breaks in DNA strands can occur along the Symmetry axis, resulting in fragments with blunt ends (e.g., restriction enzyme Ball), or at some distance from the axis, resulting in fragments with single-stranded sticky 5' (EcoRI restriction enzymes) or 3' (PstI restriction enzymes) ends.

Table 21. Restriction enzymes used in genetic engineering.

Currently, more than 500 class II restriction enzymes have been isolated; however, among them, there are enzymes that recognize identical sequences in DNA. Such groups are called isoschizomers. A distinction is made between true isoschizomers, when enzymes recognize the same nucleotide sequence and cleave DNA at the exact same points, and false isoschizomers, when enzymes recognize the same site on the DNA but make breaks at different points within that same site.

Assuming that recognition sites are distributed randomly along the DNA strand, the target for enzymes recognizing a four-nucleotide site should occur on average once every 256 bp (base pairs), and for enzymes recognizing only six NUCLEOTIDES, once every 4096 bp. Obviously, if a restriction site turns out to be located within a gene, restriction enzyme treatment will lead to its inactivation. The probability of this event is very high when using frequent-cutter restriction enzymes and substantial when using rare-cutter ones (recognizing hexanucleotides). Therefore, to obtain an intact gene, treatment is carried out alternately with several rare-cutter restriction enzymes, or the partial Digestion technique is applied—i.e., treatment under conditions where Cleavage occurs at only one site out of several possible ones.

Restriction endonucleases, which generate fragments with self-complementary sticky ends, are of particular value as they are highly effective in constructing recombinant molecules.

10.5 Methods for Rejoining DNA Fragments

To join DNA fragments produced by restriction enzyme digestion, annealing is first performed to form hydrogen bonds between the complementary bases of the sticky ends, followed by treatment with DNA ligase. The ligase catalyzes the formation of a phosphodiester bond between adjacent nucleotides. The presence of sticky ends is not strictly required for joining DNA fragments with DNA ligase; certain enzymes can ligate entirely blunt-ended fragments, though this reaction only proceeds efficiently at high concentrations of DNA and ligase [27].

Combining donor and vector DNA via annealing and ligation yields not only hybrid molecules but also recircularized original vectors, which complicates subsequent cloning Procedures. To overcome this, specialized methods have been developed to direct the process primarily toward the formation of hybrid molecules. One approach involves digesting the vector DNA with multiple restriction enzymes to minimize the likelihood of self-assembly. Another effective method entails removing the terminal phosphate groups from linear vector DNA using alkaline phosphatase. In this case, ligase-mediated ring closure of the vector is possible only in the presence of donor DNA fragments bearing intact 5'-phosphate groups (Fig. 64).

Fig. 64. Diagram illustrating The Use of linker molecules for constructing hybrid DNA [27].

Identical, mutually complementary ends of two DNA molecules to be joined can be generated by hydrolyzing both molecules with the same restriction enzyme. However, it is frequently necessary to clone DNA fragments obtained via digestion with one restriction enzyme into a vector containing a cleavage site for a different enzyme. To achieve this, a method has been developed that allows virtually any DNA fragments to be recombined. It relies on the use of linkers—short, synthetic, double-stranded oligonucleotides containing recognition sites for a specific restriction enzyme. These linkers are attached to the ends of the DNA molecule to be cloned using ligase, and subsequently cleaved with the restriction enzyme to generate sticky ends. The vector molecule is digested with the same enzyme. Subsequent annealing and ligation yield recombinant DNA molecules. A linker molecule may contain more than one restriction enzyme recognition site, in which case it is referred to as a polylinker or adapter. The use of such molecules makes the restriction-ligation method of DNA recombination universal, as the starting fragments can be obtained through A wide variety of approaches.

The homopolymer tailing method (connector method) for joining fragments relies on the ability of terminal deoxynucleotidyl transferase to add nucleotide sequences onto the 3'-OH ends of DNA fragments. A single-stranded polynucleotide, such as poly-A (dA), is added to the ends of one DNA fragment, while a complementary homopolymer, such as poly-T (dT), is added to the ends of the other fragment. The modified fragments are then mixed and annealed. Any resulting gaps are repaired by treatment with DNA polymerase and ligase, yielding covalently closed circular molecules.

10.6 Vectors

Vectors are DNA molecules capable of carrying foreign DNA into a recipient cell and ensuring its expression. Specific genes are inserted into vector molecules using enzymes, and the resulting molecular hybrids are termed chimeras or recombinant DNA molecules. These are introduced into recipient cells, leading to the segregation of the molecules; each isolated clone contains an individual recombinant DNA molecule. This Procedure is known as the Cloning of recombinant DNA molecules (Gene cloning).

To persist stably within a cell, a vector must be a replicon (capable of autonomous replication). Furthermore, it must possess one or more selectable markers to phenotypically identify its presence in the recipient cell. To facilitate recombination with donor DNA, the vector molecule must contain restriction enzyme cleavage sites located within regions non-essential for vector replication. Additionally, It is important that the vector can accommodate donor DNA inserts of varying molecular weights and yield a high copy number per cell, which simplifies the Isolation and Purification of the recombinant DNA. Vector systems for E. coli as a recipient are the most highly developed and include the following types: plasmids, bacteriophage λ, cosmids, phagemids, and bacteriophage M13.

Vectors have also been developed for other microorganisms, including industrially important species (such as Bacillus, Pseudomonas, and Streptomyces). Dual-replicon (shuttle) hybrid vectors are particularly convenient; their replication systems originate from plasmids with different hosts, enabling them to replicate in various cells, such as E. coli, yeast, or animal cells. This allows all preliminary cloning steps to be performed in E. coli before transferring the recombinant DNA to the target organism.

10.6.1 Plasmids

Plasmids are extrachromosomal genetic elements that typically exist as closed, circular, supercoiled DNA molecules (Fig. 62). Small plasmids ranging from 2 to 10 kb in size are most frequently employed as vectors. A plasmid contains a specific origin of replication (ori).

As markers, plasmids often carry genes that confer Antibiotic Resistance to bacteria. Inserting a foreign (donor) gene into a marker gene leads to the inactivation of the latter. This makes it possible to distinguish transformed cells that have taken up the vector plasmid (losing antibiotic resistance) from cells that have acquired the recombinant molecule (retaining resistance). This approach is known as insertional inactivation of a marker.

Vectors have been developed that allow for the direct Selection of clones carrying hybrid molecules. These utilize genes that either cause cell death under specific conditions or encode the synthesis of an enzyme responsible for colony coloration on a selective medium. Cloning foreign DNA into such a gene results in its inactivation, which is readily observable phenotypically.

A limitation of many plasmids is the reduction in recombinant yield as the Molecular Weight of the insert increases. Consequently, cloning DNA fragments larger than 10 kb in plasmids is inefficient.

Plasmids are introduced into bacterial cells via transformation, a process in which typically only 1 out of every 1,000 to 10,000 DNA molecules is successfully taken up.

10.6.2 Bacteriophage λ-Based Vectors

The Genome of phage λ consists of a double-stranded DNA molecule of 48.5 kb, packaged into the phage HEAD as a linear molecule with 12 bp sticky ends. Within the host cell (E. coli), these sticky ends join, and the DNA circularizes. The feasibility of creating phage λ-based vectors stems from the fact that

the genes in the central region are non-essential for lytic development and can be replaced with foreign DNA fragments. Because recombinant DNA can be packaged into capsids in vitro, it is introduced into the host cell via infection (Transduction), which is significantly more efficient than transformation (approximately every tenth DNA molecule becomes infectious). The capsid packages DNA within a defined size range—no larger than 53 kb and no smaller than 38 kb—imposing upper and lower size limits on the DNA fragments that can be cloned using phage vectors.

Phage λ-based vectors are well-suited for constructing gene libraries (genomic banks), but less optimal for delicate manipulations of DNA fragments. For detailed analysis and modification, DNA fragments are typically subcloned into plasmids.

10.6.3 Cosmids

Cosmids are plasmids that incorporate the cos-site (complementary sticky ends) of phage λ DNA. The presence of the cos-site enables in vitro packaging of DNA into phage heads, allowing them to be introduced into cells via infection rather than transformation. Cosmid vectors can accommodate foreign DNA fragments ranging from 33 to 49 kb in size. Because they possess the highest cloning capacity among vectors, cosmids are specifically designed for cloning large eukaryotic DNA fragments and constructing Genomic Libraries.

10.6.4 Phagemids

Phasmids are hybrids between phages and plasmids that are capable of replicating both as a phage and as a plasmid. In terms of cloning capacity, phasmids are comparable to vectors based on phage X and are significantly inferior to cosmids. However, their regulated ability to propagate via either the phage or plasmid pathway offers several advantages, the most important of which is the relative simplicity of complementation analysis and DNA fragment reorganization. Phasmids make it possible to bypass the subcloning of genes from phage vectors into Plasmid Vectors.

10.6.5 Vectors Based on Bacteriophages Containing Single-Stranded DNA

The best vectors of this type have been developed on the basis of phage M13. The phage particle contains a single-stranded DNA molecule approximately 6,500 nucleotides long. Upon entering the host cell, this DNA is converted into a double-stranded replicative form (RF), which is isolated from the cells and used as a vector. Between 100 and 200 copies of RF DNA accumulate within an infected cell. Thereafter, synthesis becomes asymmetric, producing only a single DNA strand that is incorporated into the mature phage particle. Phage M13 does not lyse the host cell, but merely slows its growth. Mature phage particles are continuously secreted into the medium, and their titer can reach 10¹² per 1 ml.

The main advantage of phage M13 as a cloning vector is that the bacteriophage particles released by the cell contain single-stranded DNA homologous to one of the two complementary strands of the cloned DNA. Such DNA can be directly used for determining The nucleotide sequence of DNA (sequencing).

10.6.6 Recipient Cells

A host cell is the biological environment in which a recombinant DNA molecule can function. E. coli is frequently employed as a recipient for recombinant DNA; it is a well-characterized microorganism widely used in various fields of genetics and molecular biology. However, E. coli is not an ideal producer for biotechnological purposes for several reasons. Since E. coli is a natural component of the human normal microbiota, there is a risk of personnel infection with recombinant strains exhibiting properties undesirable for humans. In addition, E. coli is susceptible to phages, produces pyrogens, and does not secrete Biosynthesis products into the culture medium, which complicates their purification. Therefore, other microorganisms show considerable promise, including bacilli (Bac. subtilis, Bac. stearothermophilus, Bac. brevis), streptomycetes, and yeasts (Saccharomyces cerevisiae). Mammalian cell cultures are also utilized, as they yield products analogous to natural ones, which is advantageous for the Production of Human protein Pharmaceuticals. Nevertheless, the widespread application of such recipients is hindered by the challenges of their cultivation (slow growth rate, high cost of nutrient media, and the risk of bacterial and viral contamination).

10.7 Introduction of DNA Molecules into Cells

A cloning system consists of two core components: a vector and recipient cells. Recipient cells are used to isolate the desired type of recombinant molecules from a mixture, subsequently identify the cloned genes, and obtain the genes or their products. Permissive cells, defined as cells that support the replication of recombinant DNA, are typically used as recipients.

The method used to introduce DNA into cells is determined by The Nature of the vector. Plasmid vectors are introduced via transformation, whereas phage vectors are introduced via transfection or transduction. DNA transfer via conjugation is also possible.

Transfection is the process by which phage DNA enters bacterial cells, subsequently leading to The production of viral progeny. Viral

clones obtained As a result of transfection are termed transfectants. They are isolated from plaques after plating recipient cells onto a lawn of a culture sensitive to the given phage.

Transformation is a process of genetic recombination in which foreign DNA enters a recipient cell. Genetically transformed cells are referred to as transformants. Cells capable of adsorbing and taking up DNA are termed competent. The proportion of competent cells within a population depends on the species of the microorganism, its cultivation conditions, growth stage, and other factors. Competent cells are characterized by altered cell envelope properties, including a reduced surface charge and increased sensitivity to osmotic Shock. The latter is due to the partial exposure of cytoplasmic membrane regions in competent cells, which are directly involved in DNA adsorption and uptake. A special role in these processes is played by mesosomes and transformasomes—derivatives of the cytoplasmic membrane that facilitate The transport of DNA into the Cytoplasm.

Competence can be enhanced or ARTIFICIALLY INDUCED IN microorganisms lacking natural competence through specialized cell treatments. A widely used method involves inducing competence in E. coli using Calcium Ions. The cells are incubated in the presence of 50 mM Ca²⁺ at 0°C, followed by a brief heat shock at 37 or 42°C. Under these conditions, a state of general competence is established, enabling transformation and transfection. Co-treatment with Ca²⁺ ions alongside Mg²⁺, Mn²⁺, and Rb⁺ is also effective for inducing competence in many Gram-negative and Gram-positive bacteria. Yeast and filamentous fungal cells become competent following treatment with lithium salts. Physical Methods of competence induction include deep freezing (-196°C) followed by thawing (+42°C), as well as electroporation. The principle behind the latter method is that a brief exposure (5–20 ms) of The cell membrane to a high-intensity electric field (1–15 kV/cm) leads to the formation of pores (electroporation breakdown) large enough for DNA to enter the cell.

The use of protoplasts and spheroplasts is highly effective for transformation and transfection.

10.8 Methods for Identifying Clones Containing Recombinant Molecules

In most molecular cloning experiments, the action of restriction enzymes yields a complex mixture of DNA fragments. Special techniques exist to select for clones containing recombinant DNA molecules. For example, when using phasmids, exclusively phage particles are produced, into whose heads recombinant molecules are packaged. When using plasmids, clones containing recombinant DNAs are selected based on the inactivation of one of the vector markers, and so forth. The next, more challenging task is to find the clone carrying the gene of interest to the researcher among the recombinants.

Screening methods for recombinant clones can be based on altering the cellular phenotype through the newly synthesized product of the recombinant gene, or on The properties of the product itself. One such approach is the complementation test, which is used when the cloned gene complements Mutations in the host cell's genome, such as shifting it from an auxotrophic to a prototrophic state. In this case, the hybrid can be detected simply by selection on a selective medium.

If the gene product—namely, the protein—is produced in sufficient quantities, the desired clone can be isolated using immunological methods. The method of direct radioimmunoassay screening for colonies involves the following steps: cell colonies are lysed on the Agar surface and then blotted onto a polyvinyl membrane onto which Antibodies against the target protein have been adsorbed. Next, this membrane is treated with antibodies radiolabeled with I¹²⁵. This forms a protein-antigen complex with two antibody molecules: one attached to the membrane and the other labeled with iodine. Complex formation is detected by autoradiography. This method is highly sensitive and yields a positive result even when only one or a few protein molecules are present in the cell.

If Gene Expression does not occur in recipient cells, clones can be identified based on primary DNA Structure or the CHARACTERISTICS OF THE protein synthesized in a suitable system (such as frog oocytes or cell-free extracts). Testing of primary DNA structure is performed via Hybridization with labeled mRNA.

Using hybridization selection, the cloned DNA is denatured by melting, immobilized on a solid surface, and hybridized with mRNA. The resulting DNA-RNA duplex is heated to release the mRNA, which is then added to a cell-free protein-synthesizing system or injected into frog oocytes for Translation. Translation products are subsequently identified by their biological activity or through immunological assays.

If the Primary Structure of the gene or its encoded protein is known, synthetic oligonucleotides—probes complementary to the target gene—can be used for clone screening. These probes carry a radioactive label that enables their detection upon binding to the target DNA fragment.

10.9 Cell Engineering

Cell engineering is a vital branch of modern biotechnology, dealing with the manipulation of plant, animal, human, and microbial cell cultures. Cultures of higher organism cells can be utilized to produce Vaccines, Monoclonal Antibodies, other immunological preparations, and growth regulators for breeding new plant varieties. Obtaining protoplasts makes it possible to construct genetically novel objects via cell hybridization or to introduce foreign genetic material into them.

Protoplasts are structures formed after the complete removal of The Cell wall. Incomplete removal of the cell wall results in the formation of spheroplasts. Both protoplasts and spheroplasts are employed in cell engineering; however, in certain cases, experiments involving spheroplasts prove less efficient than those using protoplasts.

Protoplast transformation is a versatile method for introducing DNA molecules into the cells of bacteria, actinomycetes, yeasts, and Fungi.

Protoplast fusion makes it possible to obtain genetic recombinants in microorganisms that never cross naturally. This approach allows the creation of hybrid forms of microorganisms of major importance to the microbiological industry, laying the groundwork for their genetic study and expanding breeding possibilities. The protoplast fusion technique enables the combination within a single genome of productivity-enhancing mutations derived from different breeding lines—including those that are difficult or even impossible to induce in the same cell—while also eliminating deleterious mutations.

As a mechanism of genetic exchange, protoplast fusion differs from conjugation, transduction, and transformation (where only a portion of the donor DNA enters the recipient cell) by combining entire genomes along with all cytoplasmic Components of the parental cells. Furthermore, more than two protoplasts from different strains can participate in a single fusion event, immediately yielding recombinants that carry the traits of all parents.

Several methods are used to obtain microbial protoplasts (protoplastization). Some are based on the inhibition of cell wall synthesis. For bacteria, agents that disrupt murein formation are employed, such as penicillin, fosfomycin, and high concentrations of Amino Acids like Glycine, Methionine, and Threonine; for yeasts, 2-deoxy-D-glucose (a glucose analogue that prevents Cell wall formation) is used.

The second group of methods involves the enzymatic lysis of the cell wall. Lysozyme, which hydrolyzes murein, is used to obtain bacterial protoplasts, often in combination with Other Enzymes (proteases, lipases) and EDTA (ethylenediaminetetraacetic acid). For the protoplastization of filamentous fungi and yeasts, lytic enzymes from actinomycetes, fungi, or edible snail digestive juice (helicase)—which contains several dozen different enzymes—are utilized. Mixtures consisting of helicase, cellulase, chitinase, pectinase, and other enzymes are also applied.

Alternatively, cells previously grown on a medium containing a cell wall synthesis inhibitor can be subjected to enzymatic treatment.

Protoplasts are osmotically fragile structures. Therefore, all procedures involving protoplasts must be carried out in hypertonic solutions containing osmotic stabilizers at concentrations of 0.2–0.5 mol. Suitable osmotic stabilizers include mineral salts (KCl, NaCl, NH₄ Cl, NaNO₃), salts of organic acids (sodium succinate), polyols (mannitol, sorbitol), and CARBOHYDRATES (sucrose, rhamnose, xylose, etc.).

The formation and preservation of protoplasts depend on Temperature, medium pH, lytic Enzyme Concentration, incubation time with the enzyme, as well as the age and growth phase of the culture being protoplastized.

The GENETIC APPARATUS OF protoplasts contains all the information necessary to restore (regenerate) the cell wall and to revert to the cellular form with its characteristic Morphology (reversion). The reversion process also depends on medium composition, temperature, pH, and the presence of Vitamins, Trace Elements, and protective proteins (such as gelatin or blood Serum proteins). For cell wall regeneration, protoplasts require contact with a supporting scaffold; therefore, regeneration is conducted on nutrient agar rather than in liquid media.

An effective inducer of protoplast fusion is polyethylene glycol (PEG). The surface of cells and protoplasts is negatively charged and surrounded by a layer of Water. PEG acts by reducing the surface charge and removing water, which creates conditions for close contact and membrane adhesion. Membrane rupture occurs at the contact points, and the Contents of the two neighboring protoplasts merge. PEG is a universal agent that induces the fusion of protoplasts from various microbial species while also facilitating their transformation and transfection. Most commonly, a polymer with an average molecular weight of 1,000–600 Da at a concentration of 30–50% (w/v) is used. In this case, osmotic stabilizers can be omitted, as PEG itself performs their function. Fusion is inhibited by high concentrations of Na⁺, K⁺, Cl^-, and NO₃^- ions and trace amounts of EDTA. Protoplast fusion takes place even at 4°C, and its efficiency increases when the temperature is raised to 30°C. Centrifugation enhances the fusion frequency.

Protoplast fusion experiments typically employ genetically marked strains, often carrying auxotrophic and antibiotic-resistance mutations. The fusion products, known as fusants, are selected on selective media.

Protoplastization and protoplast regeneration can lead to plasmid loss, chromosomal rearrangements, and mutations, some of which may prove beneficial.