We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Two types of drug synergy, genetic and promiscuous, are explored in S. cerevisiae. The results suggest that promiscuous synergy predominates, and that propensity to synergize is an intrinsic drug property with the potential to accelerate the search for synergistic drug combinations.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

fThe emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col r ) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col r in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col r clinical isolates. Col r was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col r strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col r mechanisms may be dependent on the genetic background.T he increasing occurrence of multidrug-resistant (MDR) Klebsiella pneumoniae has expanded reliance on last-line therapies like colistin, a cationic antimicrobial peptide, for effective treatment (1). Of concern is the growing recovery of colistin-resistant (Col r ) strains from clinical settings (2-4). Colistin disrupts membrane integrity through displacement of cations like Mg 2ϩ and Ca 2ϩ in the outer membrane, leading to cell lysis (5). Resistance mechanisms described to date involve lipopolysaccharide (LPS) modification, particularly through derivatization of lipid A phosphate moieties with a sugar or ethanolamine. These modifications reduce the electrostatic affinity between the cationic colistin and anionic LPS. Mutations in the transcriptional regulatory systems controlling these LPS modifications are a common genetic mechanism leading to colistin resistance. For example, the PhoPQ and PmrAB two-component regulatory systems (TCRS) regulate expression of the gene (pmrC) that codes for the addition of phosphoethanolamine (pETN) and genes encoding biosynthesis and lipid A transfer of 4-amino-4-deoxy-L-arabinose (Ara4N) (pmrHFIJKLM) (Fig. 1A). Other regulatory components in this pathway include PmrD and MgrB, two connector proteins that convey feedback between the PmrAB and PhoPQ TCRS ...

Recent scientific advances have significantly contributed to our understanding of the complex connection between the microbiome and cancer. Our bodies are continuously exposed to microbial cells, both resident and transient, as well as their byproducts, including toxic metabolites. Circulation of toxic metabolites may contribute to cancer onset or progression at locations distant from where a particular microbe resides. Moreover, microbes may migrate to other locations in the human body and become associated with tumor development. Several case-control metagenomics studies suggest that dysbiosis in the commensal microbiota is also associated with inflammatory disorders and various cancer types throughout the body. Although the microbiome influences carcinogenesis through mechanisms independent of inflammation and immune system, the most recognizable link is between the microbiome and cancer via the immune system, as the resident microbiota plays an essential role in activating, training, and modulating the host immune response. Immunologic dysregulation is likely to provide mechanistic explanations as to how our microbiome influences cancer development and cancer therapies. In this review, we discuss recent developments in understanding the human gut microbiome's relationship with cancer and the feasibility of developing novel cancer diagnostics based on microbiome profiles. .

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

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Phenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best-known within gene families, where an important shared function may only be revealed by mutating all family members. Here we describe the ‘Green Monster’ technology enabling the precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein (GFP) reporter gene, is subjected to repeated rounds of mating, meiosis, and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci within single cells. The Green Monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To demonstrate the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 adenosine triphosphate-binding cassette transporters within clades associated with multi-drug resistance.