**1.3 Advantages and disadvantages of BACs**

Genes expressed from BACs mirror endogenous gene expression far more accurately than other cloning systems. The large size of BACs help to minimize site of integration effects, a phenomenon which has been defined as endogenous sequences (such as gene coding regions and distal regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes (Adamson, Jackson et al. 2011) in gene cloning technology. The larger sized BAC constructs contain enhancers and locus control regions, which leads to more accurate gene expression *in vivo* (Townes, Lingrel et al. 1985; Jones, Monks et al. 1995). 1995). The human genome BACs consist of the full gene structure, including untranslated regions, exons and introns, alternative promoters and splice sites and microRNA coding sequences. RNAs such as RNA splicing or microRNAs play very important role in gene regulation (Jackson and Standard 2007). Therefore the human genome BACs will ensure full mRNA processing and splicing when genes are transcribed, and produce the full complement of protein isoforms once mRNAs are translated. BACs can be transfected and expressed in mammalian cell lines although transfection efficiency and copy numbers are low (Magin-Lachmann, Kotzamanis et al. 2004; Sparwasser and Eberl 2007).

BACs also have a number of disadvantages. A construct containing a large genomic fragment is likely to contain non-related genes that may lead to indirect, non-specific gene expression and unanticipated changes in the cell phenotype; Secondly, compared to plasmids or other gene expression vectors, the generation and screening of recombinant BAC constructs can be time-consuming and labor-intensive. Also, the oversized BAC DNA constructs are more easily sheared and degraded during manipulation before transfection; and some random recombination events may occur, for example, LoxP sites may lead to random Cre-mediated recombination (Semprini, Troup et al. 2007). Finally, repeating homologous sequences in some BACs constructs may undergo intramolecular rearrangements, which reduce the recombination efficiency and increase the rate of falsepositive clones in some selection/counter-selection approaches (Narayanan 2008).

Overall, BACs have numerous advantages when compared to conventional plasmids. They protect the gene from site of integration effects and produce accurate regulation of transcription and translation. However, the large size results in technical difficulties when handling them as well as the potential non-specific gene expression. Therefore the application of BACs as a gene expression model system should be careful considered based on the pros and cons previously described.


Preface

This book presents and discusses recent developments in the field of drug delivery. Technological advancements in the field of pharmaceutical sciences have revolutionized the patient care industry. The manufacturing industry has seen a dramatic shift from developing simple traditional dosage forms, which included simple compressed tablets and syrups, to novel drug delivery systems and targeted therapeutics. Thus, it becomes essential to bridge the gap between the current research scenario and technical knowledge provided at the pharmaceutical institutions to maximize the skills of individuals involved at any level in this domain. Developing a robust, safe and effective dosage form has always been a challenge. It requires a multidisciplinary approach. Thorough information of basic principles in pharmaceutical sciences is necessary to impart robustness, enhance efficacy and develop smarter medicaments. This book includes chapters that cover topics such as physicochemical and biopharmaceutical characterization of drugs, design and development of smart dosage forms, and technological advancements such as artificial intelligence and its role in drug delivery.

The book is divided into two sections. The first section, "Drug Delivery System," focuses on different carrier systems. Chapters in this section are related to drug delivery through liposomes, protein and peptide delivery, smart drug delivery systems in the treatment of rheumatoid arthritis, phospholipid-based nano-drug delivery systems of phytoconstituents, aliphatic polyester nanoparticles for drug delivery systems, strategies to develop cyclodextrin-based nanosponges for smart drug delivery, smart drug delivery strategies for enhancing the bioavailability of nutraceuticals, and targeted nano-drug delivery systems for colon cancer. The second section, "Novel Tools and Techniques in Drug Delivery System," focuses on the utilization of recent techniques and software for developing effective dosage forms. One chapter in this section provides an overview of artificial intelligence in health care and another chapter provides valuable information on the application of statistical tools in the optimization and development of smart drug delivery systems. Chapters in both sections are written by experts in the field and the authors have put forth tremendous effort in compiling useful information that can be used by both researchers and academicians around the world.

The editor is thankful to his co-editors Dr. Juber Akhtar and Dr. Md. Faheem Haider for their support throughout the journey of the book. He is also grateful to all the authors for their excellent contributions.

> **Dr. Usama Ahmad** Faculty of Pharmacy, Integral University Lucknow, India

**Md. Faheem Haider and Juber Akhtar** Integral University, Lucknow, India


*Mycoplasma bovis* **Infections: Occurrence, Pathogenesis, Diagnosis and Control, Including Prevention and Therapy**

Editors

**Katarzyna Dudek Ewelina Szacawa**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Katarzyna Dudek National Veterinary Research Institute Poland

Ewelina Szacawa National Veterinary Research Institute Poland

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Pathogens* (ISSN 2076-0817) (available at: https://www.mdpi.com/journal/pathogens/special issues/mycoplasma bovis infections).

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4.6.1. (Ultra) High-Performance Liquid Chromatography ((U)HPLC)

During different stages of pharmaceutical development, including synthesis and isolation of API, dosage form development, and pharmacological testing, gas or liquid chromatographic methods of analysis are used for quantitative and/or qualitative purposes [133].

High-Performance Liquid Chromatography (HPLC) is based on the principles and theories of gas chromatography and is a rapid and efficient analytical procedure [134,135]. In the broadest sense, chromatographic separations can be classified as normal (NPC) and reversed-phase (RPC), where the stationary phase is more polar than the mobile phase for NPC, whereas the converse is true for RPC [136].

RP-HPLC is the method of choice for in vitro analysis of API due to the stability and reproducibility of stationary phases used for analyses [137]. A large number of aqueous components in the mobile phase, in addition to the ease of reproducing analytical methods in different laboratory settings, make RP-HPLC an ideal analytical method for product development and quality control purposes in the pharmaceutical industry [137].

The use of stationary phases that have a silica-based backbone chemically bonded to a variety of different organic functional groups, form the basis for separations in RP-HPLC [138]. Separation is achieved when the sample to be analysed partitions between an organic modifier used in the mobile phase and the stationary phase or column [139,140]. In general, non-polar compounds are attracted to hydrophobic stationary phases and polar compounds preferentially partition into polar mobile phase components [139]. The specificity and selectivity of an analytical method is the consequence of interaction(s) between analyte(s) and binding sites of the stationary phase and partitioning in the mobile phase [141]. Silica-based columns with C3, C4, C8, or C18 alkyl chains bonded to the backbone are the commonly used non-polar stationary phases in RP-HPLC. Silica-based phases are compatible, do not swell in water, and some organic solvents are, therefore, suitable for use with mobile phases comprised of these solvents [139,141,142].

The mobile phase in RP-HPLC is usually water or a buffered solution with an organic modifier such as methanol or acetonitrile added to the composition in order to reduce the polarity of the mobile phase, thereby facilitating the achievement of appropriate retention characteristics for the compounds of interest [138]. Other factors that affect the retention time of analytes include temperature, pH of the buffer and/or mobile phase, stationary phase properties, polarity of the mobile phase, flow rate, and mobile phase composition [139,140].


*4.1.9 New approaches for cancer therapy with mushroom bioactive compounds (vaccinotherapy)*

Vaccine preparations with preventive activities against liver and cervical cancer associated with hepatitis B and human papillomatosis infections have been developed in Belgium and USA [66]. Currently, there is no commercialized vaccine for the protection of existing tumors, metastases, or relapses [3, 19]. The addition of immunomodulating substances of natural and synthetic origin to vaccines has shown promising potential to sufficiently enhance their anticancer properties. Therefore,