Showing papers by "James J. Collins published in 2015"

Highly efficient Cas9-mediated transcriptional programming

Abstract: The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Antibiotic efficacy is linked to bacterial cellular respiration

Abstract: Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes—the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy.

Cas9 gRNA engineering for genome editing, activation and repression

Abstract: We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Highly efficient Cas9-mediated transcriptional programming

Abstract: The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of changes to the Cas9 protein in addition to the fusion of a transcriptional activation domain (AD)1–5. However, the modest levels of gene activation achieved by current Cas9 activators have limited their potential applications. Here we describe the development of an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of induced pluripotent stem cells (iPSCs).

Synthetic biology devices for in vitro and in vivo diagnostics

Abstract: There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

Unraveling the physiological complexities of antibiotic lethality.

Abstract: We face an impending crisis in our ability to treat infectious disease brought about by the emergence of antibiotic-resistant pathogens and a decline in the development of new antibiotics. Urgent action is needed. This review focuses on a less well-understood aspect of antibiotic action: the complex metabolic events that occur subsequent to the interaction of antibiotics with their molecular targets and play roles in antibiotic lethality. Independent lines of evidence from studies of the action of bactericidal antibiotics on diverse bacteria collectively suggest that the initial interactions of drugs with their targets cannot fully account for the antibiotic lethality and that these interactions elicit the production of reactive oxidants including reactive oxygen species that contribute to bacterial cell death. Recent challenges to this concept are considered in the context of the broader literature of this emerging area of research. Possible ways that this new knowledge might be exploited to improve antibiotic therapy are also considered.

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage

Abstract: Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (?-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 min that then diverged into more distinct profiles at later time points. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products.

Chromatin regulation at the frontier of synthetic biology

Abstract: As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.

Engineered Phagemids for Nonlytic, Targeted Antibacterial Therapies.

Abstract: The increasing incidence of antibiotic-resistant bacterial infections is creating a global public health threat. Because conventional antibiotic drug discovery has failed to keep pace with the rise of resistance, a growing need exists to develop novel antibacterial methodologies. Replication-competent bacteriophages have been utilized in a limited fashion to treat bacterial infections. However, this approach can result in the release of harmful endotoxins, leading to untoward side effects. Here, we engineer bacterial phagemids to express antimicrobial peptides (AMPs) and protein toxins that disrupt intracellular processes, leading to rapid, nonlytic bacterial death. We show that this approach is highly modular, enabling one to readily alter the number and type of AMPs and toxins encoded by the phagemids. Furthermore, we demonstrate the effectiveness of engineered phagemids in an in vivo murine peritonitis infection model. This work shows that targeted, engineered phagemid therapy can serve as a viable, no...

Humanized microbiota mice as a model of recurrent Clostridium difficile disease

Abstract: Clostridium difficile disease is the leading antibiotic-associated cause of diarrhea and nosocomial acquired infection in the western world. The per annum burden in the USA alone amounts to 250,000 cases with 14,000 ascribed deaths and medical costs in excess of a billion dollars. Novel models for the study of C. difficile infection are therefore pertinent. Germ free C57BL/6 mice gavaged with a healthy human fecal microbiota maintained a stable “humanized” microbiota over multiple generations when housed under specific pathogen-free (SPF) conditions. As with mice containing a conventional microbiota, treatment with a five-antibiotic cocktail followed by a single dose of clindamycin renders the animals susceptible to C. difficile infection (CDI). Interestingly, after recovery from the initial CDI infection, a single intraperitoneal injection of clindamycin is sufficient to induce CDI relapse. Relapse of CDI can be induced up to 35 days postinfection after recovery from the initial infection, and multiple episodes of relapse can be induced. This model enables the study of recurrent C. difficile disease in a host containing a human-derived microbiota. Probiotic treatments using human-derived microbes, either prophylactic or curative, can be tested within the model. The identification and testing of human-derived microbial communities within a humanized microbiota mouse model may enable a higher rate of successful transfer of bacteria-based treatments from the lab to human patients due to the microbes involved initiating from, and being adapted to, the human GI tract.

DNA sense-and-respond protein modules for mammalian cells

Abstract: We generated synthetic protein components that can detect specific DNA sequences and subsequently trigger a desired intracellular response. These modular sensors exploit the programmability of zinc-finger DNA recognition to drive the intein-mediated splicing of an artificial trans-activator that signals to a genetic circuit containing a given reporter or response gene. We used the sensors to mediate sequence recognition-induced apoptosis as well as to detect and report a viral infection. This work establishes a synthetic biology framework for endowing mammalian cells with sentinel capabilities, which provides a programmable means to cull infected cells. It may also be used to identify positively transduced or transfected cells, isolate recipients of intentional genomic edits and increase the repertoire of inducible parts in synthetic biology.

Cas9 gRNA engineering for genome editing, activation and repression

Abstract: We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Association of a Novel ACTA1 Mutation With a Dominant Progressive Scapuloperoneal Myopathy in an Extended Family

Abstract: Importance New genomic strategies can now be applied to identify a diagnosis in patients and families with previously undiagnosed rare genetic conditions. The large family evaluated in the present study was described in 1966 and now expands the phenotype of a known neuromuscular gene. Objective To determine the genetic cause of a slowly progressive, autosomal dominant, scapuloperoneal neuromuscular disorder by using linkage and exome sequencing. Design, Setting, and Participants Fourteen affected individuals in a 6-generation family with a progressive scapuloperoneal disorder were evaluated. Participants were examined at pediatric, neuromuscular, and research clinics from March 1, 2005, to May 31, 2014. Exome and linkage were performed in genetics laboratories of research institutions. Main Outcomes and Measures Examination and evaluation by magnetic resonance imaging, ultrasonography, electrodiagnostic studies, and muscle biopsies (n = 3). Genetic analysis included linkage analysis (n = 17) with exome sequencing (n = 7). Results Clinical findings included progressive muscle weakness in an initially scapuloperoneal and distal distribution, including wrist extensor weakness, finger and foot drop, scapular winging, mild facial weakness, Achilles tendon contractures, and diminished or absent deep tendon reflexes. Both age at onset and progression of the disease showed clinical variability within the family. Muscle biopsy specimens demonstrated type I fiber atrophy and trabeculated fibers without nemaline rods. Analysis of exome sequences within the linkage region (4.8 megabases) revealed missense mutation c.591C>A p.Glu197Asp in a highly conserved residue in exon 4 of ACTA1 . The mutation cosegregated with disease in all tested individuals and was not present in unaffected individuals. Conclusions and Relevance This family defines a new scapuloperoneal phenotype associated with an ACTA1 mutation. A highly conserved protein, ACTA1 is implicated in multiple muscle diseases, including nemaline myopathy, actin aggregate myopathy, fiber-type disproportion, and rod-core myopathy. To our knowledge, mutations in Glu197 have not been reported previously. This residue is highly conserved and located in an exposed position in the protein; the mutation affects the intermolecular and intramolecular electrostatic interactions as shown by structural modeling. The mutation in this residue does not appear to lead to rod formation or actin accumulation in vitro or in vivo, suggesting a different molecular mechanism from that of other ACTA1 diseases.

Migration, language diversity and education policy: A contextualized analysis of inequality, risk and state effects

Abstract: No Child Left Behind is federal education legislation consisting of implementation programs intended to reconcile the goals of insuring equality while promoting competition in public education in t...

Lymphatic and hematopoietic cancers among benzene-exposed workers

Abstract: Objective:High benzene exposure is related to acute nonlymphocytic leukemia. Recently, myelodysplastic syndrome has been observed at low benzene exposure levels.Methods:We updated a mortality study of workers with benzene exposure examining acute nonlymphocytic leukemia and myelodysplastic syndrome.

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea.

Abstract: Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.

Matrix-assisted spectrophotometry

Abstract: Disclosed herein are multiwell plates suitable for spectrophotometry for low-volume liquid samples. For the multiwell plates, the bottom of at least one well has a layer of porous matrix disposed thereon, or is comprised of a layer of porous matrix. The layer of porous matrix permits a low-volume liquid sample to distribute evenly across the porous matrix. Also disclosed herein are methods of performing a photometric or spectrophotometric measurement on a liquid sample having a small volume, by using a multiwell plate comprising a layer of porous matrix.

Literacy Practices, Linguistic Anthropology and Social Inequality

Abstract: This paper discusses my own long-term research efforts to understand the interaction of schooled literacy, language diversity, and social inequality. It draws on semiotic and Marxian traditions to investigate language diversity and social inequality in contemporary European and North American settings. Focusing especially on racialization practices and class dynamics, the arguments uses early studies of minority language and schooling in order to frame a recent study of federal education policy and migrant experiences of schooling and language hierarchy. The study draws from a year of sociolinguistic and ethnographic research among multilingual Oaxacan migrant families and communities in upstate New York, with particular focus on their children’s experience with multilingual repertoires and monolingual language polices in schooling. (Author 2012) Drawing on data from classroom lessons, district curriculum decisions, and state and federal education policies, I analyze a set of ‘state effects’ (Trouillot 2001) as they operate across different institutional sites, arguing that such effects are ways the neoliberal states attempt to regulate globalized class and racial dynamics, by shaping educational subjects who are stripped of linguistic diversity, ethnic solidarity, and class awareness. This neoliberal project, which directly contributes to the reproduction of social inequality (Menken 2008), is opposed by a variety of domestic-familial linguistic practices as well as by political movements that challenge monolingual education policy.

Systematic Identification of Factors for Provirus Silencing in Embryonic Stem Cells

Abstract: Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs) Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28 Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome

Chromatin regulation at the frontier of synthetic biology

Abstract: National Institute of General Medical Sciences (U.S.) (Ruth L. Kirschstein Postdoctoral Fellowship )

Correction: Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development

Abstract: The authors would like to clarify the figure presentation of Figure 7. A corrected legend for Figure 7 is provided here. Figure 7 Some prohormone genes are expressed differentially in the CNS of sexual and asexual planarians.

Práticas de letramento, antropologia linguística e desigualdade social: casos etnográficos e compromissos teóricos

Abstract: This paper discusses my efforts during several decades of research to understand the interaction of schooled literacy, language diversity, and social inequality. It draws on semiotic and Marxian traditions to investigate language diversity and social inequality in contemporary European and North American settings. Focusing especially on racialization practices and class dynamics, the arguments present early studies of minority language and schooling, which build toward and frame a recent study of federal education policy and immigrant experiences of schooling and language hierarchy. That study draws from sociolinguistic and ethnographic research among multilingual migrant families and communities in upstate New York, with particular focus on children’s experience with multilingual repertoires and monolingual language polices in schooling (COLLINS, 2012). Examining federal education policy and debates and comparing classroom interaction processes involving different ethnolinguistic groups, I identify two “state effects” (TROUILLOT, 2001) as they operate across different institutional sites. I argue that such effects are ways in which contemporary states attempt to regulate globalized class and racial dynamics. By shaping educational subjects whose social and linguistic characteristics, and especially their class characteristics, are both obscured and employed in school-related categorizations and school-based communicative processes, such effects contribute to the hegemonic reproduction of social, linguistic and educational inequalities (HYMES, 1996; MENKEN, 2008).