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Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Rhinovirus (genus enterovirus) infections are responsible for many of the severe exacerbations of asthma and chronic obstructive pulmonary disease. Other members of the genus can cause life-threatening acute neurological infections. There is currently no antiviral drug approved for the treatment of such infections. We have identified a series of potent, broad-spectrum antiviral compounds that inhibit the replication of the human rhinovirus, Coxsackie virus, poliovirus, and enterovirus-71. The mechanism of action of the compounds has been established as inhibition of a lipid kinase, PI4KIIIβ. Inhibition of hepatitis C replication in a replicon assay correlated with enterovirus inhibition. KEYWORDS: antiviral, enterovirus, HCV, inhibitor, PI4KIIIβ O ver the last 15 years, evidence has gathered that viral infections of the respiratory tract are a major cause of exacerbations in both chronic obstructive pulmonary disease (COPD) 1 and asthma. 2 Of these infections, half to two-thirds are caused by human rhinovirus (HRV), the most well-known etiologic agent of the common cold.3 While these infections are merely inconvenient in otherwise healthy individuals, exacerbation of the symptoms of COPD and asthma are serious, leading to very large numbers of hospitalizations and significant mortality. In fact, the prevalence of COPD is increasing rapidly, and it is predicted to become the third leading cause of death globally by 2030. 4 Hence, the identification of compounds that can interfere with rhinovirus replication may lead to drugs that have important clinical value in the management of a growing medical need.In the past, several drug candidates have been progressed into clinical trials for the treatment of HRV infections. These include Rupintrivir, 5 a viral 3C protease inhibitor, and a number of compounds that prevent virus entry into cells by binding to the viral capsid: Pirodavir, 6 Pleconaril, 7 and BTA798. 8 Only the latter of these is currently in clinical development, the others having been terminated due to lack of clinical efficacy or unacceptable side effects.As part of an antiviral program directed toward hepatitis C (HCV), we identified a series of imidazo-pyrazines with modest (micromolar) activity in a genotype 1b replicon screen ( Figure 1). These compounds were derived from a BioFocus SoftFocus library (SFK28) initially designed as potential kinase inhibitors.Additional screening of the imidazo-pyrazines against a panel of other viruses revealed similar activity against the Enteroviruses, a genus of the Picornaviridae family, including HRV, polio virus (PV), and Coxsackie virus (CV). In most cases, the compounds were 3−5-fold more active against enteroviruses, which, in common with HCV, are positive-sense singlestranded RNA (+ssRNA) viruses. The compounds proved inactive against other RNA, DNA, and retroviruses and were not cytotoxic at antiviral concentrations, indicating a specific and selective antiviral effect.Enteroviruses present a range of threats to human health, from the common cold ...

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Although antimicrobial resistance is an increasingly significant public health concern, there have only been two new classes of antibiotics approved for human use since the 1960s. Understanding the mechanisms of action of antibiotics is critical for novel antibiotic discovery, though novel approaches are needed that do not exclusively rely on experiments. Molecular dynamics simulation is a computational tool that uses simple models of the atoms in a system to discover nanoscale insights into the dynamic relationship between mechanism and biological function. Such insights can lay the framework for elucidating mechanism of action and optimizing antibiotic templates. Antimicrobial peptides represent a promising solution to escalating antimicrobial resistance given their lesser tendency to induce resistance than small molecule antibiotics. Simulations of these agents have already revealed how they interact with bacterial membranes and the underlying physiochemical features directing their structure and function. In this Minireview, we discuss how traditional molecular dynamics simulation works, and its role and potential for the development of new antibiotic candidates with an emphasis on antimicrobial peptides.