Emergency physician resource utilization varies by years of experience.

Objectives: One of the most pivotal decisions an emergency physician (EP) makes is whether to admit or discharge a patient. The emergency department (ED) work-up leading to this decision involves several resource-intensive tests. Previous studies have demonstrated significant differences in EP resource utilization, measured by lab tests, advanced imaging (magnetic resonance imaging [MRI], computed tomography [CT], ultrasound), consultations, and propensity to admit a patient. However, how an EP's years of experience may impact their resource utilization and propensity to admit patients has not been well characterized. This study seeks to better understand how EPs' years of experience, post-residency, relates to their use of advanced imaging and patient disposition. Methods: Ten years of ED visits were analyzed for this study from a single, academic tertiary care center in the urban Northeast United States. The primary outcomes were utilization of advanced imaging during the visit (CT, MRI, or formal ultrasound) and whether the patient was admitted. EP years of experience was categorized into 0-2 years, 3-5 years, 6-8 years, 9-11 years, and 12 or more years. Patient age, sex, Emergency Severity Index (ESI), and the attending EP's years of experience were collected. The relationship between EP years of experience and each outcome was assessed with a linear mixed model with a random effect for provider and patient age, sex, and ESI as covariates. Results: A total of 460,937 visits seen by 65 EPs were included in the study. Over one-third (37.6%) of visits had an advanced imaging study ordered and nearly half (49.5%) resulted in admission. Compared to visits with EPs with 0-2 years of experience, visits with EPs with 3-5 or 6-8 years of experience had significantly lower odds of advanced imaging occurring. Visits seen by EPs with more than 2 years of experience had lower odds of admission than visits by EPs with 0-2 years of experience. Conclusion: More junior EPs tend to order more advanced imaging studies and have a higher propensity to admit patients. This may be due to less comfort in decision-making without advanced imaging or a lower risk tolerance. Conversely, the additional clinical experience of the most senior EPs, with greater than 9 years of experience, likely impacts their resource utilization patterns such that their use of advanced imaging does not significantly differ from the most junior EPs.

Hybrid assemblies of microbiome Blastocystis protists reveal evolutionary diversification reflecting host ecology.

The most prevalent microbial eukaryote in the human gut is Blastocystis, an obligate commensal protist also common in many other vertebrates. Blastocystis is descended from free-living stramenopile ancestors; how it has adapted to thrive within humans and a wide range of hosts is unclear. Here, we cultivated six Blastocystis strains spanning the diversity of the genus and generated highly contiguous, annotated genomes with long-read DNA-seq, Hi-C, and RNA-seq. Comparative genomics between these strains and two closely related stramenopiles with different lifestyles, the lizard gut symbiont Proteromonas lacertae and the free-living marine flagellate Cafeteria burkhardae, reveal the evolutionary history of the Blastocystis genus. We find substantial gene content variability between Blastocystis strains. Blastocystis isolated from an herbivorous tortoise has many plant carbohydrate metabolizing enzymes, some horizontally acquired from bacteria, likely reflecting fermentation within the host gut. In contrast, human-isolated Blastocystis have gained many heat shock proteins, and we find numerous subtype-specific expansions of host-interfacing genes, including cell adhesion and cell surface glycan genes. In addition, we observe that human-isolated Blastocystis have substantial changes in gene structure, including shortened introns and intergenic regions, as well as genes lacking canonical termination codons. Finally, our data indicate that the common ancestor of Blastocystis lost nearly all ancestral genes for heterokont flagella morphology, including cilia proteins, microtubule motor proteins, and ion channel proteins. Together, these findings underscore the huge functional variability within the Blastocystis genus and provide candidate genes for the adaptations these lineages have undergone to thrive in the gut microbiomes of diverse vertebrates.

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition.

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through liquid-liquid phase separation. Here, we find that the phase separation of Caenorhabditis elegans SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation. We identify the SAD-1 kinase as a regulator of SYD-2 phase separation and determine presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. Using phosphoproteomics, we find SAD-1 phosphorylates SYD-2 on 3 sites that are critical to activate phase separation. Mechanistically, SAD-1 phosphorylation relieves a binding interaction between 2 folded domains in SYD-2 that inhibits phase separation by an intrinsically disordered region (IDR). We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, activating its phase separation and active zone assembly.

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition.

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through a liquid-liquid phase separation. Here, we find that the phase separation of SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation. Using phosphoproteomics, we identify the SAD-1 kinase to phosphorylate SYD-2 and a number of other substrates. Presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. We determine SAD-1 phosphorylation of SYD-2 at three sites is critical to activate its phase separation. Mechanistically, phosphorylation relieves a binding interaction between two folded SYD-2 domains that inhibits phase separation by an intrinsically disordered region. We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, enabling its phase separation and active zone assembly.

Assessment of enzymatically synthesized DNA for gene assembly.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

Evaluating the persistence and stability of a DNA-barcoded microbial system in a mock home environment.

Recent advancements in engineered microbial systems capable of deployment in complex environments have enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures, enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system composed of germination-deficient Bacillus subtilis and Saccharomyces cerevisiae spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated proteins (Cas) detection (Sherlock). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together, we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications. Graphical Abstract.

Variability in cell-free expression reactions can impact qualitative genetic circuit characterization.

Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning is required and a high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, laboratories, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here, we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument and material batch. Although the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability. Graphical Abstract.

Cell-Free Protein Systems from Yersinia pestis are Functional and Growth-Temperature Dependent.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92- are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Finding functions of phase separation in the presynapse.

Synapses are the basic units of neuronal communication. Understanding how synapses assemble and function is therefore essential to understanding nervous systems. Decades of study have identified many molecular components and functional mechanisms of synapses. Recently, an additional level of synaptic protein organization has been identified: phase separation. In the presynapse, components of the central active zone and a synaptic vesicle-clustering factor have been shown to form liquid-liquid phase-separated condensates or hydrogels. New in vivo functional studies have directly tested how phase separation impacts both synapse formation and function. Here, we review this emerging evidence for in vivo functional roles of phase separation at the presynapse and discuss future functional studies necessary to understand its complexity.

Structural and Biosynthetic Diversity of Nonulosonic Acids (NulOs) That Decorate Surface Structures in Bacteria.

Nonulosonic acids (NulOs) are a diverse family of 9-carbon α-keto acid sugars that are involved in a wide range of functions across all branches of life. The family of NulOs includes the sialic acids as well as the prokaryote-specific NulOs. Select bacteria biosynthesize the sialic acid N-acetylneuraminic acid (Neu5Ac), and the ability to produce this sugar and its subsequent incorporation into cell-surface structures is implicated in a variety of bacteria-host interactions. Furthermore, scavenging of sialic acid from the environment for energy has been characterized across a diverse group of bacteria, mainly human commensals and pathogens. In addition to sialic acid, bacteria have the ability to biosynthesize prokaryote-specific NulOs, of which there are several known isomers characterized. These prokaryotic NulOs are similar in structure to Neu5Ac but little is known regarding their role in bacterial physiology. Here, we discuss the diversity in structure, the biosynthesis pathways, and the functions of bacteria-specific NulOs. These carbohydrates are phylogenetically widespread among bacteria, with numerous structurally unique modifications recognized. Despite the diversity in structure, the NulOs are involved in similar functions such as motility, biofilm formation, host colonization, and immune evasion.

The ChpR transcriptional regulator of Sinorhizobium meliloti senses 3,5,6-trichloropyridinol, a degradation product of the organophosphate pesticide chlorpyrifos.

The global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.

Opposite Surfaces of the Cdc15 F-BAR Domain Create a Membrane Platform That Coordinates Cytoskeletal and Signaling Components for Cytokinesis.

Many eukaryotes assemble an actin- and myosin-based cytokinetic ring (CR) on the plasma membrane (PM) for cell division, but how it is anchored there remains unclear. In Schizosaccharomyces pombe, the F-BAR protein Cdc15 links the PM via its F-BAR domain to proteins in the CR's interior via its SH3 domain. However, Cdc15's F-BAR domain also directly binds formin Cdc12, suggesting that Cdc15 may polymerize a protein network directly adjacent to the membrane. Here, we determine that the F-BAR domain binds Cdc12 using residues on the face opposite its membrane-binding surface. These residues also bind paxillin-like Pxl1, promoting its recruitment with calcineurin to the CR. Mutation of these F-BAR domain residues results in a shallower CR, with components localizing ∼35% closer to the PM than in wild type, and aberrant CR constriction. Thus, F-BAR domains serve as oligomeric membrane-bound platforms that can modulate the architecture of an entire actin structure.

Assembly of synaptic active zones requires phase separation of scaffold molecules.

The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system1. Every presynapse builds a core 'active zone' structure, where ion channels cluster and synaptic vesicles release their neurotransmitters2. Although the composition of active zones is well characterized2,3, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-α) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division.

In many organisms, positive and negative signals cooperate to position the division site for cytokinesis. In the rod-shaped fission yeast Schizosaccharomyces pombe, symmetric division is achieved through anillin/Mid1-dependent positive cues released from the central nucleus and negative signals from the DYRK-family polarity kinase Pom1 at cell tips. Here we establish that Pom1's kinase activity prevents septation at cell tips even if Mid1 is absent or mislocalized. We also find that Pom1 phosphorylation of F-BAR protein Cdc15, a major scaffold of the division apparatus, disrupts Cdc15's ability to bind membranes and paxillin, Pxl1, thereby inhibiting Cdc15's function in cytokinesis. A Cdc15 mutant carrying phosphomimetic versions of Pom1 sites or deletion of Cdc15 binding partners suppresses division at cell tips in cells lacking both Mid1 and Pom1 signals. Thus, inhibition of Cdc15-scaffolded septum formation at cell poles is a key Pom1 mechanism that ensures medial division.

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis.

F-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.

CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species.

BACKGROUND: Bacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context. RESULTS: Herein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer. CONCLUSIONS: Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide.

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.