Organ-on-a-chip models for elucidating the cellular biology of infectious diseases

Infectious diseases are caused by the invasion of pathogens into a host. To explore the mechanisms of pathogen infections and cellular responses, human models that can accurately recapitulate human pathophysiology are needed. Organ-on-a-chip is a type of advanced in vitro model system that cultures cells in microfluidic devices to replicate physiologically relevant microenvironments such as 3D structures, shear stress, and mechanical stimulation. Recently, organ-on-a-chips have been widely adopted to examine the pathophysiology of infectious diseases in detail. Here, we will summarize recent advances in infectious disease research of visceral organs such as the lung, intestine, liver, and kidneys, using organ-on-a-chips.     

Genomics, systems biology and drug development for infectious diseases

Although a variety of drugs are available for many infectious diseases that predominantly affect the developing world reasons remain for continuing to search for new chemotherapeutics. First, the development of microbial resistance has made some of the most effective and inexpensive drug regimes unreliable and dangerous to use on severely ill patients. Second, many existing antimicrobial drugs show toxicity or are too expensive for countries where the per capita income is in the order of hundreds of dollars per year. In recognition of this, new publicly and privately financed drug discovery efforts have been established to identify and develop new therapies for diseases such as tuberculosis, malaria and AIDS. This in turn, has intensified the need for tools to facilitate drug identification for those microbes whose molecular biology is poorly understood, or which are difficult to grow in the laboratory. While much has been written about how functional genomics can be used to find novel protein targets for chemotherapeutics this review will concentrate on how genome-wide, systems biology approaches may be used following whole organism, cell-based screening to understand the mechanism of drug action or to identify biological targets of small molecules. Here we focus on protozoan parasites, however, many of the approaches can be applied to pathogenic bacteria or parasitic helminths, insects or disease-causing fungi.     

Heat-labile proteases in molecular biology applications

Thermolabile proteases were identified in three Gram-negative psychrotrophic bacteria. The protease from the psychrotrophic strain A9 was purified and its application to common molecular biology techniques was demonstrated. Heat-stable molecular biology enzymes (Taq polymerase and PvuII) were digested by a heat-labile protease, which was then inactivated by a mild heat treatment. The clear benefit of using heat-labile proteases arises in situations where further reactions may be accomplished without an intermediate purification step, thereby saving time and avoiding the possibility of product loss.     

Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.     

The molecular biology of inflammatory bowel diseases

IBDs (inflammatory bowel diseases) are a group of diseases affecting the gastrointestinal tract. The diseases are multifactorial and cover genetic aspects: susceptibility genes, innate and adaptive responses to inflammation, and structure and efficacy of the mucosal protective barrier. Animal models of IBD have been developed to gain further knowledge of the disease mechanisms. These topics form an overlapping background to enable an improved understanding of the molecular features of these diseases. A series of articles is presented based on the topics covered at the Biochemical Society Focused Meeting The Molecular Biology of Inflammatory Bowel Diseases.     

High molecular weight DNA assembly in vivo for synthetic biology applications

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.     

Serotonin receptors - from molecular biology to clinical applications

Serotonin (5-hydroxytryptamine) is an ubiquitary monoamine acting as one of the neurotransmitters at synapses of nerve cells. Serotonin acts through several receptor types and subtypes. The profusion of 5-HT receptors should eventually allow a better understanding of the different and complex processes in which serotonin is involved. Its role is expected in the etiology of several diseases, including depression, schizophrenia, anxiety and panic disorders, migraine, hypertension, pulmonary hypertension, eating disorders, vomiting and irritable bowel syndromes. In the past 20 years, seven distinct families of 5-HT receptors have been identified and various subpopulations have been described for several of them. Increasing number of 5-HT receptors has made it difficult to unravel the role of 5-HT receptor subpopulations due to the lack of suitable selective agents. The present review describes the different populations and nomenclature of recently discovered 5-HT receptors and their pharmacological relevance.     

Molecular biology and the diagnosis, epidemiology and pathogenesis of infectious diseases

In this short review, the impact of molecular biology on microbiology in general is described. Specifically, molecular biology is increasingly enlarging the available choice of methods for the diagnosis of microbial disease. In situ hybridization seems to be a particularly promising procedure. In epidemiology, an interesting facet is the high mutation rate of RNA viruses. In pathogenesis, molecular biology will help to elucidate pathways of infection and the targeting of pathogenic macromolecules within the cell and within an organism.