Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

Histone H2B monoubiquitination (H2Bub1) is a dynamic posttranslational modification which are linked to DNA damage and plays a key role in a wide variety of regulatory transcriptional programs. Cancer cells exhibit a variety of epigenetic changes, particularly any aberrant H2Bub1 has frequently been associated with the development of tumors. Nevertheless, our understanding of the mechanisms governing the histone H2B deubiquitination and their associated functions during stem cell differentiation remain only partially understood. In this study, we wished to investigate the role of deubiquitinating enzymes (DUBs) on H2Bub1 regulation during stem cell differentiation. In a search for potential DUBs for H2B monoubiquitination, we identified Usp7, a ubiquitin-specific protease that acts as a negative regulator of H2B ubiquitination during the neuronal differentiation of mouse embryonic carcinoma cells. Loss of function of the Usp7 gene by a CRISPR/Cas9 system during retinoic acid-mediated cell differentiation contributes to the increase in H2Bub1. Furthermore, knockout of the Usp7 gene particularly elevated the expression of neuronal differentiation related genes including astryocyte-specific markers and oligodendrocyte-specific markers. In particular, glial lineage cell-specific transcription factors including oligodendrocyte transcription factor 2, glial fibrillary acidic protein, and SRY-box transcription factor 10 was significantly upregulated during neuronal differentiation. Thus, our findings suggest a novel role of Usp7 in gliogenesis in mouse embryonic carcinoma cells.

Drug repurposing for metabolic disorders: Scientific, technological and economic issues.

Obesity, diabetes, and other metabolic disorders place a huge burden on both the physical health and financial well-being of the community. While the need for effective treatment of metabolic disorders remains urgent and the reality is that traditional drug development involves high costs and a very long time with many pre-clinical and clinical trials, the need for drug repurposing has emerged as a potential alternative. Scientific evidence has shown the anti-diabetic and anti-obesity effects of old drugs, which were initially utilized for the treatment of inflammation, depression, infections, and even cancers. The drug library used modern technological methods to conduct drug screening. Computational molecular docking, genome-wide association studies, or omics data mining are advantageous and unavoidable methods for drug repurposing. Drug repurposing offers a promising avenue for economic efficiency in healthcare, especially for less common metabolic diseases, despite the need for rigorous research and validation. In this chapter, we aim to explore the scientific, technological, and economic issues surrounding drug repurposing for metabolic disorders. We hope to shed light on the potential of this approach and the challenges that need to be addressed to make it a viable option in the treatment of metabolic disorders, especially in the future fight against metabolic disorders.

Current progress in high-throughput screening for drug repurposing.

High-throughput screening (HTS) is a simple, rapid and cost-effective solution to determine active candidates from large library of compounds. HTS is gaining attention from Pharmaceuticals and Biotechnology companies for accelerating their drug discovery programs. Conventional drug discovery program is time consuming and expensive. In contrast drug repurposing approach is cost-effective and increases speed of drug discovery as toxicity profile is already known. The present chapter highlight HTS technology including microplate, microfluidics, lab-on-chip, organ-on-chip for drug repurposing. The current chapter also highlights the application of HTS for bacterial infections and cancer.

USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells.

Cisplatin is a chemotherapy drug that causes a plethora of DNA lesions and inhibits DNA transcription and replication, resulting in the induction of apoptosis in cancer cells. However, over time, patients develop resistance to cisplatin due to repeated treatment and thus the treatment efficacy is limited. Therefore, identifying an alternative therapeutic strategy combining cisplatin treatment along with targeting factors that drive cisplatin resistance is needed. CRISPR/Cas9 system-based genome-wide screening for the deubiquitinating enzyme (DUB) subfamily identified USP28 as a potential DUB that governs cisplatin resistance. USP28 regulates the protein level of microtubule-associated serine/threonine kinase 1 (MAST1), a common kinase whose expression is elevated in several cisplatin-resistant cancer cells. The expression level and protein turnover of MAST1 is a major factor driving cisplatin resistance in many cancer types. Here we report that the USP28 interacts and extends the half-life of MAST1 protein by its deubiquitinating activity. The expression pattern of USP28 and MAST1 showed a positive correlation across a panel of tested cancer cell lines and human clinical tissues. Additionally, CRISPR/Cas9-mediated gene knockout of USP28 in A549 and NCI-H1299 cells blocked MAST1-driven cisplatin resistance, resulting in suppressed cell proliferation, colony formation ability, migration and invasion in vitro. Finally, loss of USP28 destabilized MAST1 protein and attenuated tumor growth by sensitizing cells to cisplatin treatment in mouse xenograft model. We envision that targeting the USP28-MAST1 axis along with cisplatin treatment might be an alternative therapeutic strategy to overcome cisplatin resistance in cancer patients.

ßTrCP1 promotes SLC35F2 protein ubiquitination and inhibits cancer progression in HeLa cells.

The solute carrier family 35 F2 (SLC35F2) belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, we identified ßTrCP1 interacting with and reducing the SLC35F2 protein level. ßTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between ßTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of ßTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, we demonstrate that ßTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of ßTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, we envision that ßTrCP1 may be a potential target for cancer therapeutics.

Fate of pesticides in agricultural runoff treatment systems: Occurrence, impacts and technological progress.

The levels of pesticides in air, water, and soil are gradually increasing due to its inappropriate management. In particular, agricultural runoff inflicts the damages on the ecosystem and human health at massive scale. Present study summarizes 70 studies in which investigations on removal or treatment of pesticides/insecticides/herbicides are reported. A bibliometric analysis was also done to understand the recent research trends through the analysis of 2218 publications. The specific objectives of this study are as follows: i) to inventorize the characteristics details of agriculture runoff and analyzing the occurrence and impacts of pesticides, ii) analyzing the role and interaction of pesticides in different environmental segments, iii) investigating the fate of pesticides in agriculture runoff treatment systems, iv) summarizing the experiences and findings of most commonly technology deployed for pesticides remediation in agriculture runoff including target pesticide(s), specifications, configuration of technological intervention. Among the reported technologies for pesticide treatment in agriculture runoff, constructed wetland was at the top followed by algal or photobioreactor. Among various advanced oxidation processes, photo Fenton method is mainly used for pesticides remediation such as triazine, methyl parathion, fenuron and diuron. Algal bioreactors are extensively used for a wide range of pesticides treatment including 2,4-Dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, alachlor, diuron, chlorpyrifos, endosulfan, and imidacloprid; especially at lower hydraulic retention time of 2-6 h. This study highlights that hybrid approaches can offers potential opportunities for effective removal of pesticides in a more viable manner.

Phage for drug delivery vehicles.

Viruses being the natural carriers of gene have been widely used as drug delivery systems. However, the commonly used eukaryotic viruses such as adenoviruses, retroviruses, and lentiviruses, besides efficiently targeting the cells, can also stimulate immunological response or disrupt tumour suppressor genes leading to cancer. Consequently, there has been an increase interest in the scientific fraternity towards exploring other alternatives, which are safer and equally efficient for drug delivery. Bacteriophages, in this context have been at the forefront as an efficient, reliable, and safer choice. Novel phage dependent technologies led the foundation of peptide libraries and provides way to recognising abilities and targeting of specific ligands. Hybridisation of phage with inorganic complexes could be an appropriate strategy for the construction of carrying bioinorganic carriers. In this chapter, we have tried to cover major advances in the phage species that can be used as drug delivery vehicles.

USP19 Negatively Regulates p53 and Promotes Cervical Cancer Progression.

p53 is a tumor suppressor gene activated in response to cellular stressors that inhibits cell cycle progression and induces pro-apoptotic signaling. The protein level of p53 is well balanced by the action of several E3 ligases and deubiquitinating enzymes (DUBs). Several DUBs have been reported to negatively regulate and promote p53 degradation in tumors. In this study, we identified USP19 as a negative regulator of p53 protein level. We demonstrate a direct interaction between USP19 and p53 by pull down assay. The overexpression of USP19 promoted ubiquitination of p53 and reduced its protein half-life. We also demonstrate that CRISPR/Cas9-mediated knockout of USP19 in cervical cancer cells elevates p53 protein levels, resulting in reduced colony formation, cell migration, and cell invasion. Overall, our results indicate that USP19 negatively regulates p53 protein levels in cervical cancer progression.

Halomonas gemina sp. nov. and Halomonas llamarensis sp. nov., two siderophore-producing organisms isolated from high-altitude salars of the Atacama Desert.

Introduction: This study aimed to identify and characterize novel siderophore-producing organisms capable of secreting high quantities of the iron-binding compounds. In the course of this, two not yet reported halophilic strains designated ATCHAT and ATCH28T were isolated from hypersaline, alkaline surface waters of Salar de Llamará and Laguna Lejía, respectively. The alkaline environment limits iron bioavailability, suggesting that native organisms produce abundant siderophores to sequester iron. Methods: Both strains were characterized by polyphasic approach. Comparative analysis of the 16S rRNA gene sequences revealed their affiliation with the genus Halomonas. ATCHAT showed close similarity to Halomonas salicampi and Halomonas vilamensis, while ATCH28T was related closest to Halomonas ventosae and Halomonas salina. The ability of both strains to secrete siderophores was initially assessed using the chromeazurol S (CAS) liquid assay and subsequently further investigated through genomic analysis and NMR. Furthermore, the effect of various media components on the siderophore secretion by strain ATCH28T was explored. Results: The CAS assay confirmed the ability of both strains to produce iron-binding compounds. Genomic analysis of strain ATCHAT revealed the presence of a not yet reported NRPS-dependant gene cluster responsible for the secretion of siderophore. However, as only small amounts of siderophore were secreted, further investigations did not lie within the scope of this study. Via NMR and genomic analysis, strain ATCH28T has been determined to produce desferrioxamine E (DFOE). Although this siderophore is common in various terrestrial microorganisms, it has not yet been reported to occur within Halomonas, making strain ATCH28T the first member of the genus to produce a non-amphiphilic siderophore. By means of media optimization, the produced quantity of DFOE could be increased to more than 1000 µM. Discussion: Phenotypic and genotypic characteristics clearly differentiated both strains from other members of the genus Halomonas. Average nucleotide identity (ANI) values and DNA-DNA relatedness indicated that the strains represented two novel species. Therefore, both species should be added as new representatives of the genus Halomonas, for which the designations Halomonas llamarensis sp. nov. (type strain ATCHAT = DSM 114476 = LMG 32709) and Halomonas gemina sp. nov. (type strain ATCH28T = DSM 114418 = LMG 32708) are proposed.

Applications of bioinformatics in epigenetics.

Epigenetic modifications such as DNA methylation, post-translational chromatin modifications and non-coding RNA-mediated mechanisms are responsible for epigenetic inheritance. Change in gene expression due to these epigenetic modifications are responsible for new traits in different organisms leading to various diseases including cancer, diabetic kidney disease (DKD), diabetic nephropathy (DN) and renal fibrosis. Bioinformatics is an effective approach for epigenomic profiling. These epigenomic data can be analyzed by a large number of bioinformatics tools and software. Many databases are available online, which comprises huge amount of information regarding these modifications. Recent methodologies include many sequencing and analytical techniques to extrapolate different types of epigenetic data. This data can be used to design drugs against diseases linked to epigenetic modifications. This chapter briefly highlights different epigenetics databases (MethDB, REBASE, Pubmeth, MethPrimerDB, Histone Database, ChromDB, MeInfoText database, EpimiR, Methylome DB, and dbHiMo), and tools (compEpiTools, CpGProD, MethBlAST, EpiExplorer, and BiQ analyzer), which are being utilized to retrieve the data and mechanistically analysis of epigenetics modifications.

The expression and mutation of BRCA1/2 genes in ovarian cancer: a global systematic study.

INTRODUCTION: This systematic review was designed to summarize the findings on expression and mutation of BRCA1/2 genes in ovarian cancer (OC) patients, focusing on mutation detection technology and taking clinical decisions for better treatment. AREAS COVERED: We conducted a systematic review by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses document selection guidelines for the document selection process and the PICOT standard for developing the keywords to search for. A total of 5729 publications were included, and 50 articles were put into the final screening. The results showed that Next-Generation Sequencing was a breakthrough technology in detecting Breast Cancer 1/2 (BRCA1/2) gene mutations because of its efficacy and affordability. Other technologies are also being applied now for mutation detection. The most prominent associations of BRCA1/2 gene mutations were age, heredity, and family history. Furthermore, mutations of BRCA1/2 could improve survival rate and overall survival. There is no sufficient study available to conclude a systematic analysis for the expression of BRCA1/2 gene in OC. EXPERT OPINION: Research will continue to develop more diagnostic techniques based on the expression and mutation of BCRA1/2 genes for OC in the near future.

Exploring the Potential of Stem Cell-Based Therapy for Aesthetic and Plastic Surgery.

Over the last decade, stem cell-associated therapies are widely used because of their potential in self-renewable and multipotent differentiation ability. Stem cells have become more attractive for aesthetic uses and plastic surgery, including scar reduction, breast augmentation, facial contouring, hand rejuvenation, and anti-aging. The current preclinical and clinical studies of stem cells on aesthetic uses also showed promising outcomes. Adipose-derived stem cells are commonly used for fat grafting that demonstrated scar improvement, anti-aging, skin rejuvenation properties, etc. While stem cell-based products have yet to receive approval from the FDA for aesthetic medicine and plastic surgery. Moving forward, the review on the efficacy and potential of stem cell-based therapy for aesthetic and plastic surgery is limited. In the present review, we discuss the current status and recent advances of using stem cells for aesthetic and plastic surgery. The potential of cell-free therapy and tissue engineering in this field is also highlighted. The clinical applications, advantages, and limitations are also discussed. This review also provides further works that need to be investigated to widely apply stem cells in the clinic, especially in aesthetic and plastic contexts.

Dysbiosis of human microbiome and infectious diseases.

Since birth, the human body gets colonized by various communities of symbiotic or commensal microorganisms and they persist till the death of an individual. The human microbiome is comprised of the genomes of microorganisms such as viruses, archaea, eukaryotes, protozoa, and, most remarkably, bacteria. The development of "omics" technologies gave way to the Human Microbiome Project (HMP) which aimed at exploring the collection of microbial genes and genomes inhabiting the human body. Eubiosis, i.e., a healthy and balanced composition of such microbes contributes to the metabolic function, protection against pathogens and provides nutrients and energy to the host. Whereas, an imbalance in the diversity of microorganisms, termed dysbiosis, greatly influences the state of health and disease. This chapter summarizes the impact of gut bacteria on the well-being of humans and highlights the protective role played by the human microbiota during bacterial and viral infections. The condition of dysbiosis and how it plays a role in the establishment of various infections and metabolic disorders such as Clostridioides difficile infection (CFI), inflammatory bowel disease (IBD), cancer, periodontitis, and obesity are described in detail. Further, treatments such as fecal transplantation, probiotics, prebiotics, phage therapy, and CRISPR/Cas system, which target gut microbiota during digestive diseases are also discussed.

Gut microbiota in gastrointestinal diseases.

Gut microbiota is a highly dense population of different kinds of bacteria residing in the gut which co-evolves with the host. It engages in a number of metabolic and immunological activities. Gut microbiota is associated with maintenance of health, and unbalanced microbiota contributes in the development of several diseases. Alteration of beneficial gut microbiota population triggers gastrointestinal diseases including irritable bowel syndrome, inflammatory bowel disease, celiac disease, colorectal cancer, and many others. Gut microbiota can be affected by multiple factors such as diet, stress, genetic variations. In this chapter, we highlight how gut microbiota plays a key role in pathogenesis of gastrointestinal disease.

Current approaches in CRISPR-Cas9 mediated gene editing for biomedical and therapeutic applications.

A single gene mutation can cause a number of human diseases that affect the quality of life. Until the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems, it was challenging to correct a gene mutation to avoid a disease by reverting phenotypes. The advent of CRISPR technology has changed the field of gene editing, given its simplicity and intrinsic programmability, surpassing the limitations of both zinc-finger nuclease and transcription activator-like effector nuclease and becoming the method of choice for therapeutic gene editing by overcoming the bottlenecks of conventional gene-editing techniques. Currently, there is no commercially available medicinal cure to correct a gene mutation that corrects and reverses the abnormality of a gene's function. Devising reprogramming strategies for faithful recapitulation of normal phenotypes is a crucial aspect for directing the reprogrammed cells toward clinical trials. The CRISPR-Cas9 system has been promising as a tool for correcting gene mutations in maladies including blood disorders and muscular degeneration as well as neurological, cardiovascular, renal, genetic, stem cell, and optical diseases. In this review, we highlight recent developments and utilization of the CRISPR-Cas9 system in correcting or generating gene mutations to create model organisms to develop deeper insights into diseases, rescue normal gene functionality, and curb the progression of a disease. Delivery of CRISPR-components being a pivotal aspect in proving its effectiveness, various proven delivery systems have also been briefly discussed.

Microfluidics based point-of-care for disease diagnostics.

Microfluidics platform is widely used for several basic biological to advanced biotechnological applications. It reduces the expenditure of reagent consumption by readily reducing the volume of the reaction system. It is being used for early diagnosis of diseases, detection of pathogens, cancer markers, high-throughput screening and many such applications. Currently, microfluidics and lab-on-chip is integrated together with sample preparation, extraction, analysis and detection of biomarkers for disease diagnosis. This technology offers low-cost, rapid, sensitive and paper-based lateral flow mode of detection which is user-friendly and scalable. In this chapter, we highlight recent developments in microfluidics platform for disease diagnosis.

Advances in microfluidics devices and its applications in personalized medicines.

Microfluidics is an exponentially growing area and is being used for numerous applications from basic science to advanced biotechnology and medicines. Microfluidics provides a platform to the research community for studying and building new strategies for the diagnosis and therapeutics applications. In the last decade, microfluidic have enriched the field of diagnostics by providing new solutions which was not possible with conventional detection and treatment methods. Microfluidics has the ability to precisely control and perform high-throughput functions. It has been proven as an efficient and rapid method for biological sample preparation, analysis and controlled drug delivery system. Microfluidics plays significant role in personalized medicine. These personalized medicines are used for medical decisions, practices and other interventions as well as for individual patients based on their predicted response or risk of disease. This chapter highlights microfluidics in developing personalized medical applications for its applications in diseases such as cancer, cardiovascular disease, diabetes, pulmonary disease and several others.

CYLD destabilizes NoxO1 protein by promoting ubiquitination and regulates prostate cancer progression.

The NADPH oxidase (Nox) family of enzymes is solely dedicated in the generation of reactive oxygen species (ROS). ROS generated by Nox are involved in multiple signaling cascades and a myriad of pathophysiological conditions including cancer. As such, ROS seem to have both detrimental and beneficial roles in a number of cellular functions, including cell signaling, growth, apoptosis and proliferation. Regulatory mechanisms are required to control the activity of Nox enzymes in order to maintain ROS balance within the cell. Here, we performed genome-wide screening for deubiquitinating enzymes (DUBs) regulating Nox organizer 1 (NoxO1) protein expression using a CRISPR/Cas9-mediated DUB-knockout library. We identified cylindromatosis (CYLD) as a binding partner regulating NoxO1 protein expression. We demonstrated that the overexpression of CYLD promotes ubiquitination of NoxO1 protein and reduces the NoxO1 protein half-life. The destabilization of NoxO1 protein by CYLD suppressed excessive ROS generation. Additionally, CRISPR/Cas9-mediated knockout of CYLD in PC-3 cells promoted cell proliferation, migration, colony formation and invasion in vitro. In xenografted mice, injection of CYLD-depleted cells consistently led to tumor development with increased weight and volume. Taken together, these results indicate that CYLD acts as a destabilizer of NoxO1 protein and could be a potential tumor suppressor target for cancer therapeutics.

CRISPR-based diagnostics for detection of pathogens.

The improved sensitivity and superior specificity associated with the use of molecular assays has improved the fate of disease diagnosis by bestowing the clinicians with outcomes that are both rapid and precise. In recent years, CRISPR has made considerable progress in in vitro diagnostic platform which has paved its way for developing rapid and sensitive CRISPR-based diagnostic tools. Improved perception and better understanding of diverse CRISPR-Cas systems has broadened the reach of CRISPR applications for not just early detection of pathogens but also for early onset of diseases such as cancer. The inherent allele specificity of CRISPR is the predominant reason for its application in designing a diagnostic-tool that is field-deployable, portable, sensitive, specific and rapid. In this chapter, we highlight various CRISPR-based diagnostic platforms, its applications, challenges and future prospects of the CRISPR-Cas system.

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities.

The triterpene squalene is a natural compound that has demonstrated an extraordinary diversity of uses in pharmaceutical, nutraceutical, and personal care industries. Emboldened by this range of uses, novel applications that can gain profit from the benefits of squalene as an additive or supplement are expanding, resulting in its increasing demand. Ever since its discovery, the primary source has been the deep-sea shark liver, although recent declines in their populations and justified animal conservation and protection regulations have encouraged researchers to identify a novel route for squalene biosynthesis. This renewed scientific interest has profited from immense developments in synthetic biology, which now allows fine-tuning of a wider range of plants, fungi, and microorganisms for improved squalene production. There are numerous naturally squalene producing species and strains; although they generally do not make commercially viable yields as primary shark liver sources can deliver. The recent advances made toward improving squalene output from natural and engineered species have inspired this review. Accordingly, it will cover in-depth knowledge offered by the studies of the natural sources, and various engineering-based strategies that have been used to drive the improvements in the pathways toward large-scale production. The wide uses of squalene are also discussed, including the notable developments in anti-cancer applications and in augmenting influenza vaccines for greater efficacy.