Biogenesis of a bacterial organelle: the carboxysome assembly pathway.

The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, we describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Our results demonstrate that, unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. We propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.

Genome editing retraces the evolution of toxin resistance in the monarch butterfly.

Identifying the genetic mechanisms of adaptation requires the elucidation of links between the evolution of DNA sequence, phenotype, and fitness1. Convergent evolution can be used as a guide to identify candidate mutations that underlie adaptive traits2-4, and new genome editing technology is facilitating functional validation of these mutations in whole organisms1,5. We combined these approaches to study a classic case of convergence in insects from six orders, including the monarch butterfly (Danaus plexippus), that have independently evolved to colonize plants that produce cardiac glycoside toxins6-11. Many of these insects evolved parallel amino acid substitutions in the α-subunit (ATPα) of the sodium pump (Na+/K+-ATPase)7-11, the physiological target of cardiac glycosides12. Here we describe mutational paths involving three repeatedly changing amino acid sites (111, 119 and 122) in ATPα that are associated with cardiac glycoside specialization13,14. We then performed CRISPR-Cas9 base editing on the native Atpα gene in Drosophila melanogaster flies and retraced the mutational path taken across the monarch lineage11,15. We show in vivo, in vitro and in silico that the path conferred resistance and target-site insensitivity to cardiac glycosides16, culminating in triple mutant 'monarch flies' that were as insensitive to cardiac glycosides as monarch butterflies. 'Monarch flies' retained small amounts of cardiac glycosides through metamorphosis, a trait that has been optimized in monarch butterflies to deter predators17-19. The order in which the substitutions evolved was explained by amelioration of antagonistic pleiotropy through epistasis13,14,20-22. Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities2,6-11,15,17-19.

The Mount Sinai international enhancement of social work leadership program: The past and the future.

Developed in 1988, the Mount Sinai International Enhancement of Social Work Leadership Program brings 4-6 social workers from several countries each year to the Mount Sinai Hospital in New York City, where they meet with leaders from the hospital, community based organizations and graduate schools of social work, to enhance their leadership ability, strengthen management and research skills, and build upon global social work relationships. This article reviews the results of a survey conducted in 2016 to assess whether the visiting scholars met established learning objectives of the Program. Survey outcomes, presented in quantitative and qualitative terms, show positive results, and the scholars reported that the Program was extremely beneficial. The Program is viewed through the lens of two select adult learning theories: Social Learning Theory, which incorporates collaboration and learning from others, and Transformative Learning Theory, which is comprised of self-reflection and individualized learning. The inclusion of these theories in the implementation of the Program will be discussed. An analysis of the survey's outcomes, through pre- and post-Program participation and learning, facilitates assessment of potential programmatic adjustments to help evaluate long-term viability of the Program and potential duplication by other academic medical centers.

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins.

Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.

Effects of a Psychosocial Transitional Care Model on Hospitalizations and Cost of Care for High Utilizers.

Evidence of care coordination programs to reduce readmissions is limited. We examined whether a social work transitional care model reduced hospital utilization and costs with a retrospective cohort study conducted from 9/3/2010-8/31/2012. Patients enrolled in the Preventable Admissions Care Team (PACT) program were matched to controls. PACT patients received follow-up from a social worker to address psychosocial strain. PACT reduced thirty-day readmission rate by 34% (p = <0.001), Sixty-day hospitalization rate by 22% (p = 0.004); ninety-day hospitalization rate by 19% (p = 0.006), and but not 180-day hospitalization rate. Inpatient costs thirty days post-index were $2.7 million for PACT patients and $3.6 million for controls.

Taste evolution in an herbivorous drosophilid.

Plant secondary metabolites pose a challenge for generalist herbivorous insects because they are not only potentially toxic, they also may trigger aversion. On the contrary, some highly specialized herbivorous insects evolved to use these same compounds as 'token stimuli' for unambiguous determination of their host plants. Two questions that emerge from these observations are how recently derived herbivores evolve to overcome this aversion to plant secondary metabolites and the extent to which they evolve increased attraction to these same compounds. In this study, we addressed these questions by focusing on the evolution of bitter taste preferences in the herbivorous drosophilid Scaptomyza flava, which is phylogenetically nested deep in the paraphyletic Drosophila. We measured behavioral and neural responses of S. flava and a set of non-herbivorous species representing a phylogenetic gradient (S. pallida, S. hsui, and D. melanogaster) towards host- and non-host derived bitter plant compounds. We observed that S. flava evolved a shift in bitter detection, rather than a narrow shift towards glucosinolates, the precursors of mustard-specific defense compounds. In a dye-based consumption assay, S. flava exhibited shifts in aversion toward the non-mustard bitter, plant-produced alkaloids caffeine and lobeline, and reduced aversion towards glucosinolates, whereas the non-herbivorous species each showed strong aversion to all bitter compounds tested. We then examined whether these changes in bitter preferences of S. flava could be explained by changes in sensitivity in the peripheral nervous system and compared electrophysiological responses from the labellar sensilla of S. flava, S. pallida, and D. melanogaster. Using scanning electron microscopy, we also created a map of labellar sensilla in S. flava and S. pallida. We assigned each sensillum to a functional sensilla class based on their morphology and initial response profiles to bitter and sweet compounds. Despite a high degree of conservation in the morphology and spatial placement of sensilla between S. flava and S. pallida, electrophysiological studies revealed that S. flava had reduced sensitivity to glucosinolates to varying degrees. We found this reduction only in I type sensilla. Finally, we speculate on the potential role that evolutionary genetic changes in gustatory receptors between S. pallida and S. flava may play in driving these patterns. Specifically, we hypothesize that the evolution of bitter receptors expressed in I type sensilla may have driven the reduced sensitivity observed in S. flava, and ultimately, its reduced bitter aversion. The S. flava system showcases the importance of reduced aversion to bitter defense compounds in relatively young herbivorous lineages, and how this may be achieved at the molecular and physiological level.

Engineering bacterial microcompartment shells: chimeric shell proteins and chimeric carboxysome shells.

Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorganic carbon fixation (carboxysomes) or organic carbon utilization (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. We undertook both approaches to altering shell properties using the carboxysome as a model system. There are two types of carboxysomes, α and ß. In both, the predominant shell protein(s) contain a single copy of the BMC domain (pfam00936), but they are significantly different in primary structure. Indeed, phylogenetic analysis shows that the two types of carboxysome shell proteins are more similar to their counterparts in metabolosomes than to each other. We solved high resolution crystal structures of the major shell proteins, CsoS1 and CcmK2, and the presumed minor shell protein CcmK4, representing both types of cyanobacterial carboxysomes and then tested the interchangeability. The in vivo study presented here confirms that both engineering pores to mimic those of other shell proteins and the construction of chimeric shells is feasible.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and ß, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the ß-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for ß-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function.

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39±2nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.

Towards understanding region-specificity of triplet repeat diseases: coupled immunohistology and mass spectrometry imaging.

Many trinucleotide repeat disorders exhibit region-specific toxicity within tissues, the basis of which cannot be explained by traditional methods. For example, in Huntington's Disease (HD), the toxic disease-causing protein is ubiquitously expressed. However, only the medium spiny neurons in the striatum are initially targeted for death. Many changes are likely to initiate in these cells at an intracellular and microstructural level long before there is a measureable phenotype, but why some regions of the brain are more susceptible to death is unknown. This chapter describes a method to detect functional changes among brain regions and cell types, and link them directly with region-specific physiology. Due to the neurodegeneration that accompanies many triplet repeat disorders, we focus on the brain, although the methods described in this chapter can be translated to other tissue types. We integrate immunohistology and traditional mass spectrometry with a novel mass spectrometry imaging technique, called nanostructure initiated mass spectrometry (NIMS). When used together, these tools offer unique insights into region-specific physiology of the brain, and a basis for understanding the region-specific toxicity associated with triplet repeat disorders.

Outcomes of endodontic therapy in general practice: a study by the Practitioners Engaged in Applied Research and Learning Network.

BACKGROUND: The authors undertook a study involving members of a dental practice-based research network to determine the outcome and factors associated with success and failure of endodontic therapy. METHODS: Members in participating practices (practitioner-investigators [P-Is]) invited the enrollment of all patients seeking treatment in the practice who had undergone primary endodontic therapy and restoration in a permanent tooth three to five years previously. If a patient had more than one tooth so treated, the P-I selected as the index tooth the tooth treated earliest during the three- to five-year period. The authors excluded from the study any teeth that served as abutments for removable partial dentures or overdentures, third molars and teeth undergoing active orthodontic endodontic therapy. The primary outcome was retention of the index tooth. Secondary outcomes, in addition to extraction, that defined failure included clinical or radiographic evidence (or both) of periapical pathosis, endodontic retreatment or pain on percussion. RESULTS: P-Is in 64 network practices enrolled 1,312 patients with a mean (standard deviation) time to follow-up of 3.9 (0.6) years. During that period, 3.3 percent of the index teeth were extracted, 2.2 percent underwent retreatment, 3.6 percent had pain on percussion and 10.6 percent had periapical radiolucencies for a combined failure rate of 19.1 percent. The presence of preoperative periapical radiolucency with a diagnosis of either irreversible pulpitis or necrotic pulp was associated with failure after multivariate analysis, as were multiple canals, male sex and Hispanic/Latino ethnicity. CONCLUSIONS: These results suggest that failure rates for endodontic therapy are higher than previously reported in general practices, according to results of studies based on dental insurance claims data. CLINICAL IMPLICATIONS: The results of this study can help guide the practitioner in deciding the most appropriate course of therapy for teeth with irreversible pulpitis, necrotic pulp or periapical periodontitis.