Showing papers by "James J. Collins published in 2016"

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip

Abstract: A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.

Comparison of Cas9 activators in multiple species

Abstract: Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.

“Deadman” and “Passcode” microbial kill switches for bacterial containment

Abstract: Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Abstract: Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

Comparison of Cas9 activators in multiple species

Abstract: Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms To whom correspondence should be addressed alex.chavez@wyss.harvard.edu and gchurch@genetics.med.harvard.edu. Contributions: A.C. and M.T. conceived of the study. A.C., M.T., B.W.P., and R.C. designed and performed experiments. S.J.H., R.J.C., and J.B. performed experiments.B.E.C, B.E.H and N.P. designed and performed all experiments in Drosophila Melanogaster. D.T-O and E.J.K.K. performed RNA-seq experiments and analyzed data. J.J.C and G.M.C. supervised the study. A.C. and M.T. wrote the manuscript with support from all authors. Conflict of interest: G.M.C. has equity in Editas and Caribou Biosciences. Accession codes: Primary accessions: Gene Expression Omnibus GSE80611 HHS Public Access Author manuscript Nat Methods. Author manuscript; available in PMC 2016 November 23. Published in final edited form as: Nat Methods. 2016 July ; 13(7): 563–567. doi:10.1038/nmeth.3871. A uhor M anscript

Chemogenomics and orthology-based design of antibiotic combination therapies

Abstract: Combination antibiotic therapies are being increasingly used in the clinic to enhance potency and counter drug resistance. However, the large search space of candidate drugs and dosage regimes makes the identification of effective combinations highly challenging. Here, we present a computational approach called INDIGO, which uses chemogenomics data to predict antibiotic combinations that interact synergistically or antagonistically in inhibiting bacterial growth. INDIGO quantifies the influence of individual chemical-genetic interactions on synergy and antagonism and significantly outperforms existing approaches based on experimental evaluation of novel predictions in Escherichia coli Our analysis revealed a core set of genes and pathways (e.g. central metabolism) that are predictive of antibiotic interactions. By identifying the interactions that are associated with orthologous genes, we successfully estimated drug-interaction outcomes in the bacterial pathogens Mycobacterium tuberculosis and Staphylococcus aureus, using the E. coli INDIGO model. INDIGO thus enables the discovery of effective combination therapies in less-studied pathogens by leveraging chemogenomics data in model organisms.

A role for the bacterial GATC methylome in antibiotic stress survival

Abstract: Antibiotic resistance is an increasingly serious public health threat. Understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets. We explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single-molecule real-time sequencing to characterize genomic methylation kinetics. We find that Escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at GATC sites. Although the adenine methylome remains stable during drug stress, without GATC methylation, methyl-dependent mismatch repair (MMR) is deleterious and, fueled by the drug-induced error-prone polymerase Pol IV, overwhelms cells with toxic DNA breaks. In multiple E. coli strains, including pathogenic and drug-resistant clinical isolates, DNA adenine methyltransferase deficiency potentiates antibiotics from the β-lactam and quinolone classes. This work indicates that the GATC methylome provides structural support for bacterial survival during antibiotic stress and suggests targeting bacterial DNA methylation as a viable approach to enhancing antibiotic activity.

Creating Single-Copy Genetic Circuits

Abstract: Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use.

Stem cell progeny contribute to the schistosome host-parasite interface

Abstract: Schistosomes infect more than 200 million of the world's poorest people. These parasites live in the vasculature, producing eggs that spur a variety of chronic, potentially life-threatening, pathologies exacerbated by the long lifespan of schistosomes, that can thrive in the host for decades. How schistosomes maintain their longevity in this immunologically hostile environment is unknown. Here, we demonstrate that somatic stem cells in Schistosoma mansoni are biased towards generating a population of cells expressing factors associated exclusively with the schistosome host-parasite interface, a structure called the tegument. We show cells expressing these tegumental factors are short-lived and rapidly turned over. We suggest that stem cell-driven renewal of this tegumental lineage represents an important strategy for parasite survival in the context of the host vasculature.

Tissue Degeneration following Loss of Schistosoma mansoni cbp1 Is Associated with Increased Stem Cell Proliferation and Parasite Death In Vivo

Abstract: Schistosomiasis is second only to malaria in terms of the global impact among diseases caused by parasites. A striking feature of schistosomes are their ability to thrive in their hosts for decades. We have previously demonstrated that stem cells, called neoblasts, promote homeostatic tissue maintenance in adult schistosomes and suggested these cells likely contribute to parasite longevity. Whether these schistosome neoblasts have functions independent of homeostatic tissue maintenance, for example in processes such as tissue regeneration following injury, remains unexplored. Here we characterize the schistosome CBP/p300 homolog, Sm-cbp1. We found that depleting cbp1 transcript levels with RNA interference (RNAi) resulted in increased neoblast proliferation and cell death, eventually leading to organ degeneration. Based on these observations we speculated this increased rate of neoblast proliferation may be a response to mitigate tissue damage due to increased cell death. Therefore, we tested if mechanical injury was sufficient to stimulate neoblast proliferation. We found that mechanical injury induced both cell death and neoblast proliferation at wound sites, suggesting that schistosome neoblasts are capable of mounting proliferative responses to injury. Furthermore, we observed that the health of cbp1(RNAi) parasites progressively declined during the course of our in vitro experiments. To determine the fate of cbp1(RNAi) parasites in the context of a mammalian host, we coupled RNAi with an established technique to transplant schistosomes into the mesenteric veins of uninfected mice. We found transplanted cbp1(RNAi) parasites were cleared from vasculature of recipient mice and were incapable of inducing measurable pathology in their recipient hosts. Together our data suggest that injury is sufficient to induce neoblast proliferation and that cbp1 is essential for parasite survival in vivo. These studies present a new methodology to study schistosome gene function in vivo and highlight a potential role for schistosome neoblasts in promoting tissue repair following injury.

NF-YB Regulates Spermatogonial Stem Cell Self-Renewal and Proliferation in the Planarian Schmidtea mediterranea

Abstract: Gametes are the source and carrier of genetic information, essential for the propagation of all sexually reproducing organisms. Male gametes are derived from a progenitor stem cell population called spermatogonial stem cells (SSCs). SSCs give rise to male gametes through the coordination of two essential processes: self-renewal to produce more SSCs, and differentiation to produce mature sperm. Disruption of this equilibrium can lead to excessive proliferation of SSCs, causing tumorigenesis, or can result in aberrant differentiation, leading to infertility. Little is known about how SSCs achieve the fine balance between self-renewal and differentiation, which is necessary for their remarkable output and developmental potential. To understand the mechanisms of SSC maintenance, we examine the planarian homolog of Nuclear Factor Y-B (NF-YB), which is required for the maintenance of early planarian male germ cells. Here, we demonstrate that NF-YB plays a role in the self-renewal and proliferation of planarian SSCs, but not in their specification or differentiation. Furthermore, we characterize members of the NF-Y complex in Schistosoma mansoni, a parasitic flatworm related to the free-living planarian. We find that the function of NF-YB in regulating male germ cell proliferation is conserved in schistosomes. This finding is especially significant because fecundity is the cause of pathogenesis of S. mansoni. Our findings can help elucidate the complex relationship between self-renewal and differentiation of SSCs, and may also have implications for understanding and controlling schistosomiasis.

Transformation by Simian Virus 40 of Spleen Cells from a Hyperimmune Rabbit: Evidence for Synthesis of Immunoglobulin by the Transformed Cells (cloning/coprecipitation/autoradiography/differentiated)

Abstract: Spleen cells derived from a rabbit hyper- immunized with a Type III pneumococcal vaccine were exposed to simian virus 40 in vitro. After 114 days, trans- formed cells growing on the surface of one culture dish were observed and a cell line was established. The trans- formed cells had a morphology characteristic of cells transformed by simian virus 40, contained the simian virus 40-specific T antigen, and yielded infectious simian virus 40 upon cultivation with indicator cells in the pres- ence of Sendai fusion factor. Transformed cells incorpo- rated labeled amino acid into protein with the antigenic properties of rabbit immunoglobulin G. We have previously attempted to obtain a virus-transformed cell line capable of indefinite growth in vitro and continuous production of specific antibody (1). Lymph-node cells from immunized rats were exposed to simian virus 40 (SV40); four transformed cell lines were obtained, but none was found to synthesize immunoglobulin or immunoglobulin fragments. Attempts to establish a relationship between the transformed cells and lymphoid cells were inconclusive, and the identity of the target cell for transformation remained uncertain. In order to increase the likelihood of successful transformation of antibody-producing cells, resort was made, in the present experiments, to cells from a rabbit hyperimmunized with a pneumococcal vaccine. Rabbits injected intravenously with pneumococcal vaccines produce large amounts of antibody of restricted heterogeneity (2). Furthermore, marked plasma- cell infiltration of the spleen has been demonstrated (3). This report describes the infection with SV40 of spleen cells from one such rabbit, and the subsequent establishment of a trans- formed cell line capable of producing rabbit immunoglobulin of the IgG class. In the accompanying manuscript, the presence of a protein of markedly restricted heterogeneity that spe- cifically binds the immunizing antigen is demonstrated (4).

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

Abstract: The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.

Precise Cas9 targeting enables genomic mutation prevention

Abstract: Here we present a generalized method of guide RNA tuning that enables Cas9 to discriminate between two target sites that differ by a single nucleotide polymorphism. We employ our methodology to generate a novel in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

Transformation by Simian Virus 40 of Spleen Cells from a Hyperimmune Rabbit: Demonstration of Production of Specific Antibody to the Immunizing Antigen (immunoadsorbent/isoelectric focusing/pneumococcal capsular polysaccharide)

Abstract: Extracts of spleen cells obtained from a rabbit hyperimmunized with Type III pneumococcal vaccine and transformed with simian virus 40, were sub- jected to agarose gel electrophoresis in the presence of an (125)S3 polysaccharide-protein conjugate. Binding of radioactivity in the gamma globulin region was observed. Extracts and media obtained from labeled cell cultures contained a protein that bound to an S3, but not to an S8 or inactivated S3 immunoadsorbent. After elution, the bound protein showed a single band on isoelectric focusing which corresponded in isoelectric point to one of the serum anti- S3 antibodies of the donor rabbit. These observations strongly suggest that a normal committed lymphoid cell may be brought into continuous culture by virus trans- formation and yet retain its ability to synthesize specific antibody. In the accompanying paper, we showed that spleen cells of a rabbit hyperimmunized with Type III pneumococcal vaccine were transformed with simian virus 40 (SV40) and that the transformed cells synthesized IgG (1). In this paper, a frac- tion isolated from culture fluid and cell extract by a specific immunoadsorbent was shown to consist of a single major band by isoelectric focusing.

The Effect of Compositional Context on Synthetic Gene Networks

Abstract: It is well known that synthetic gene expression is highly sensitive to how genetic elements (promoter structure, spacing regions between promoter and cod- ing sequences, ribosome binding sites, etc) are spatially configured An important topic that has received far less attention is how the compositional context, or spatial arrangement, of entire genes within a synthetic gene network affects their individual expression levels In this paper we show, both quantitatively and qualitatively, that compositional context significantly alters transcription levels in synthetic gene networks We demonstrate that key characteristics of gene induction, such as ultra-sensitivity and dynamic range, strongly depend on compositional context We postulate that supercoiling can be used to explain this interference and validate this hypothesis through modeling and a series of in vitro supercoiling relaxation experiments This compo- sitional interference enables a novel form of feedback in synthetic gene networks We illustrate the use of this feedback by redesigning the toggle switch to incorporate compositional context We show the context-optimized toggle switch has improved threshold detection and memory properties

Mortality risk among workers with exposure to dioxins.

Abstract: Background In several studies, dioxin exposure has been associated with increased risk from several causes of death. Aims To compare the mortality experience of workers exposed to dioxins during trichlorophenol (TCP) and pentachlorophenol (PCP) production to that of the general population and to examine mortality risk by estimated exposure levels. Methods A retrospective cohort study which followed up workers' vital status from 1940 to 2011, with serum surveys to support estimation of historical dioxin exposure levels. Results Among the 2192 study subjects, there were nine deaths in TCP workers from acute non-lymphatic leukaemia [standardized mortality ratio (SMR) = 2.88, 95% confidence interval (CI) 1.32- 5.47], four mesothelioma deaths (SMR = 5.12, 95% CI 1.39-13.10) and four soft tissue sarcoma (STS) deaths (SMR = 3.08, 95% CI 0.84-7.87). In PCP workers, there were eight deaths from non-Hodgkin's lymphoma (SMR = 1.92, 95% CI 0.83-3.79), 150 from ischaemic heart disease (SMR = 1.20, 95% CI 1.01-7.89) and five from stomach ulcers (SMR = 3.38, 95% CI 1.10-7.89). There were no trends of increased mortality with increased dioxin exposure except for STS and 2,3,7,8-tetrachlorodibenzo-p-dioxin levels. This finding for STS should be interpreted with caution due to the small number of deaths and the uncertainty in diagnosis and nosology. Conclusions While some causes of death were greater than expected, this study provides little evidence of increased risk when dioxin exposures are considered.

Deadman and passcode microbial kill switches

Abstract: Provided herein are systems, methods and compositions for rendering cells or the expression of an effector protein sensitive to a predetermined condition. In one aspect, cells can be rendered dependent upon the presence of an environmental agent, e.g. , an exogenous agent, without which the cell will default to expression of a death protein and be killed. In another aspect, cells can be rendered sensitive to the presence of a set of predetermined conditions such that cells will only grow when two or more necessary exogenous agents are supplied, and without either of which, the cells are killed. In this aspect, hybrid transcription factors provide a vast array of possible predetermined conditions.