A dissected non-ribosomal peptide synthetase maintains activity.

Non-ribosomal peptide synthetases (NRPSs) generate chemically complex compounds and their modular architecture suggests that changing their domain organization can predictably alter their products. Ebony, a small three-domain NRPS, catalyzes the formation of ß-alanine containing amides from biogenic amines. To examine the necessity of interdomain interactions, we modeled and docked domains of Ebony to reveal potential interfaces between them. Testing the same domain combinations in vitro showed that 8 % of activity was preserved after Ebony was dissected into a di-domain and a detached C-terminal domain, suggesting that sufficient interaction was maintained after dissection. Our work creates a model to identify domain interfaces necessary for catalysis, an important step toward utilizing Ebony as a combinatorial engineering platform for novel amides.

Persistent anxiety among high school students: Survey results from the second year of the COVID pandemic.

INTRODUCTION: National mental health surveys have demonstrated increased stress and depressive symptoms among high-school students during the first year of the COVID-19 pandemic, but objective measures of anxiety after the first year of the pandemic are lacking. METHODS: A 25-question survey including demographics, the Generalized Anxiety Disorder-7 scale (GAD-7) a validated self-administered tool to evaluate anxiety severity, and questions on achievement goals and future aspirations was designed by investigators. Over a 2-month period, all students from grade 9-12 in a single high-school (n = 546) were invited to complete an online survey after electronic parental consent and student assent. Bi-variate and chi-square analyses examined demographic differences in anxiety scores and the impact on outcomes; qualitative analyses examined related themes from open-ended questions. RESULTS: In total, 155/546 (28%) completed the survey. Among students with binary gender classifications, 54/149 (36%) had GAD-7 scores in the moderate or severe anxiety range (scores≥10), with a greater proportion among females than males (47% vs 21%, P<0.001). Compared to students with GAD-7<10, those with ≥ 10 were more likely to strongly agree that the pandemic changed them significantly (51% vs 28%, p = 0.05), made them mature faster (44% vs 16%, p = 0.004), and affected their personal growth negatively (16% vs 6%, p = 0.004). Prominent themes that emerged from open-ended responses on regrets during the pandemic included missing out on school social or sports events, missing out being with friends, and attending family events or vacations. CONCLUSION: In this survey of high school students conducted 2 years after the onset of COVID-19 in the United States, 47% of females and 21% of males reported moderate or severe anxiety symptoms as assessed by the GAD-7. Whether heightened anxiety results in functional deficits is still uncertain, but resources for assessment and treatment should be prioritized.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Cobamide remodeling.

Cobamides are a family of structurally-diverse cofactors which includes vitamin B12 and over a dozen natural analogs. Within the nucleotide loop structure, cobamide analogs have variable lower ligands that fall into three categories: benzimidazoles, purines, and phenols. The range of cobamide analogs that can be utilized by an organism is dependent on the specificity of its cobamide-dependent enzymes, and most bacteria are able to utilize multiple analogs but not all. Some bacteria have pathways for cobamide remodeling, a process in which imported cobamides are converted into compatible analogs. Here we discuss cobamide analog diversity and three pathways for cobamide remodeling, mediated by amidohydrolase CbiZ, phosphodiesterase CbiR, and some homologs of cobamide synthase CobS. Remodeling proteins exhibit varying degrees of specificity for cobamide substrates, reflecting different strategies to ensure that imported cobamides can be utilized.

Escherichia coli Nissle 1917 secondary metabolism: aryl polyene biosynthesis and phosphopantetheinyl transferase crosstalk.

Escherichia coli Nissle 1917 (EcN) is a Gram-negative bacterium that is used to treat inflammatory bowel diseases. The probiotic character of EcN is not well-understood, but its ability to produce secondary metabolites plays an important role in its activity. The EcN genome encodes for an aryl polyene (APE) biosynthetic gene cluster (BGC), and APE products have a role in biofilm formation. We show here that this unusual polyketide assembly line synthase produces four APE molecules which are likely cis/trans isomers. Within the APE BGC, two acyl carrier proteins are involved in biosynthesis. Acyl carrier proteins require activation by post-translational modification with a phosphopantetheinyl transferase (PPTase). Through analysis of single, double, and triple mutants of three PPTases, the PPTase-BGC crosstalk relationship in EcN was characterized. Understanding PPTase-BGC crosstalk is important for the engineering of secondary metabolite production hosts and for targeting of PPTases with new antibiotics. KEY POINTS: • Escherichia coli Nissle 1917 biosynthesizes four aryl polyene isoforms. • Phosphopantetheinyl transferase crosstalk is important for biosynthesis.

California and Oregon NICU Wildfire Disaster Preparedness Tools.

The 2020 wildfire season was devastating to the Western United States and affected the region's NICUs. In this study, we ask the question, "what tools/strategies do medical professionals deem as important and most helpful as they are preparing for wildfire disaster response?" It is a follow up to our previous study: Learning from Wildfire Disaster Experience in California NICUs. We reevaluated how California NICUs dealt with the 2020 wildfires and expanded to Oregon and Southwest (SW) Washington NICUs. We conducted a survey with eleven Oregon and SW Washington NICUs about their wildfire evacuation preparedness. We also interviewed two neonatologists about their wildfire disaster experience evacuating their NICU or preparing to evacuate. Our findings suggest there is more work needed to fully prepare NICUs for wildfire disasters. We hope that by bringing light to the strategies used by affected clinicians, we can educate and support future NICU disaster preparedness responses.

Direct Cobamide Remodeling via Additional Function of Cobamide Biosynthesis Protein CobS from Vibrio cholerae.

Vitamin B12 belongs to a family of structurally diverse cofactors with over a dozen natural analogs, collectively referred to as cobamides. Most bacteria encode cobamide-dependent enzymes, many of which can only utilize a subset of cobamide analogs. Some bacteria employ a mechanism called cobamide remodeling, a process in which cobamides are converted into other analogs to ensure that compatible cobamides are available in the cell. Here, we characterize an additional pathway for cobamide remodeling that is distinct from the previously characterized ones. Cobamide synthase (CobS) is an enzyme required for cobamide biosynthesis that attaches the lower ligand moiety in which the base varies between analogs. In a heterologous model system, we previously showed that Vibrio cholerae CobS (VcCobS) unexpectedly conferred remodeling activity in addition to performing the known cobamide biosynthesis reaction. Here, we show that additional Vibrio species perform the same remodeling reaction, and we further characterize VcCobS-mediated remodeling using bacterial genetics and in vitro assays. We demonstrate that VcCobS acts upon the cobamide pseudocobalamin directly to remodel it, a mechanism which differs from the known remodeling pathways in which cobamides are first cleaved into biosynthetic intermediates. This suggests that some CobS homologs have the additional function of cobamide remodeling, and we propose the term "direct remodeling" for this process. This characterization of yet another pathway for remodeling suggests that cobamide profiles are highly dynamic in polymicrobial environments, with remodeling pathways conferring a competitive advantage. IMPORTANCE Cobamides are widespread cofactors that mediate metabolic interactions in complex microbial communities. Few studies directly examine cobamide profiles, but several have shown that mammalian gastrointestinal tracts are rich in cobamide analogs. Studies of intestinal bacteria, including beneficial commensals and pathogens, show variation in the ability to produce and utilize different cobamides. Some bacteria can convert imported cobamides into compatible analogs in a process called remodeling. Recent discoveries of additional cobamide remodeling pathways, including this work, suggest that remodeling is an important factor in cobamide dynamics. Characterization of such pathways is critical in understanding cobamide flux and nutrient cross-feeding in polymicrobial communities, and it facilitates the establishment of microbiome manipulation strategies via modulation of cobamide profiles.

Delivery room oxygen physiology and respiratory interventions for newborns with cyanotic congenital heart disease.

OBJECTIVE: To characterize pulse oxygen saturation (SpO2) trajectories and respiratory interventions after birth for newborns with cyanotic congenital heart disease (CCHD). STUDY DESIGN: Retrospective single-site study of newborns ≥32 weeks gestation with CCHD: single ventricle with critical aortic obstruction (SV-CAO), critical pulmonic obstruction (CPO), transposition of the great arteries (TGA). Minute-to-minute SpO2 values and respiratory interventions were summarized and compared. RESULTS: Two hundred infants were enrolled. SpO2 at each minute differed across groups (p < 0.01), with the lowest values in TGA. All interventions were most frequent in TGA (p < 0.01). Continuous positive airway pressure was provided in 22% SV-CAO, 23% CPO, and 66% TGA. Positive pressure ventilation occurred in 7% SV-CAO, 14% CPO, and 33% TGA. Intubation occurred in 4% SV-CAO, 10% CPO, and 53% TGA. CONCLUSION: We defined SpO2 trajectories and delivery room respiratory interventions for three CCHD phenotypes. These results inform delivery room management of these high-risk populations.

Learning from Wildfire Disaster Experience in California NICUs.

Wildfires have been affecting California greatly, and vulnerable patients in neonatal intensive care units (NICUs) are not exempt. Our aim was to learn how personnel working in NICUs of California hospitals handled issues of neonatal transfer during wildfire disasters in recent years, with an ultimate goal to share lessons learned with healthcare teams on disaster preparedness. We identified California fires through newspaper articles and the CalFire.gov list. We determined which hospitals were affected and contacted members of the healthcare team through connections via the California Perinatal Quality Care Collaborative (CPQCC) database. We audio recorded interviews over phone or remote conferencing software or by written survey. We coded and analyzed transcripts and survey responses. While describing disaster preparedness, equipment (such as bassinets and backpacks), ambulance access/transport and documentation/charting were noted as important and essential. Teamwork, willingness to do other tasks that are not part of typical job descriptions, and unconventional strategies contribute to the success of keeping NICU babies safe when California wildfire strikes. Healthcare teams developed ingenious and surprising ways to evacuate NICU babies.

Epithelial Migration and Non-adhesive Periderm Are Required for Digit Separation during Mammalian Development.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

Specificity of cobamide remodeling, uptake and utilization in Vibrio cholerae.

Cobamides are a group of compounds including vitamin B12 that can vary at the lower base position of the nucleotide loop. They are synthesized de novo by only a subset of prokaryotes, but some organisms encode partial biosynthesis pathways for converting one variant to another (remodeling) or completing biosynthesis from an intermediate (corrinoid salvaging). Here, we explore the cobamide specificity in Vibrio cholerae through examination of three natural variants representing major cobamide groups: commercially available cobalamin, and isolated pseudocobalamin and p-cresolylcobamide. We show that BtuB, the outer membrane corrinoid transporter, mediates the uptake of all three variants and the intermediate cobinamide. Our previous work suggested that V. cholerae could convert pseudocobalamin produced by cyanobacteria into cobalamin. In this work, cobamide specificity in V. cholerae is demonstrated by remodeling of pseudocobalamin and salvaging of cobinamide to produce cobalamin. Cobamide remodeling in V. cholerae is distinct from the canonical pathway requiring amidohydrolase CbiZ, and heterologous expression of V. cholerae CobS was sufficient for remodeling. Furthermore, function of V. cholerae cobamide-dependent methionine synthase MetH was robustly supported by cobalamin and p-cresolylcobamide, but not pseudocobalamin. Notably, the inability of V. cholerae to produce and utilize pseudocobalamin contrasts with enteric bacteria like Salmonella.

NOT Gate Genetic Circuits to Control Gene Expression in Cyanobacteria.

To downregulate gene expression in cyanobacteria, we constructed NOT gate genetic circuits using orthogonal promoters and their cognate repressors regulated translationally by synthetic riboswitches. Four NOT gates were tested and characterized in five cyanobacterial strains using fluorescent reporter-gene assays. In comparison to alternative systems used to downregulate gene expression in cyanobacteria, these NOT gates performed well, reducing YFP reporter expression by 4 to 50-fold. We further evaluated these NOT gates by controlling the expression of the ftsZ gene, which encodes a prokaryotic tubulin homologue that is required for cell division and is essential for Synechococcus elongatus PCC 7942. These NOT gates would facilitate cyanobacterial genetic engineering or the study of essential cellular processes.

An Amoebal Grazer of Cyanobacteria Requires Cobalamin Produced by Heterotrophic Bacteria.

Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs.IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.

Symmetry of cardiac function assessment.

Both right and left ventricles are developed from two adjacent segments of the primary heart tube. Though they are different with regard to shape and power, they mirror each other in terms of behavior. This is the first level of symmetry in cardiac function assessment. Both cardiac muscle contraction and relaxation are active. This constructs the second level of symmetry in cardiac function assessment. Combination of the two levels will help to find some hidden indexes or approaches to evaluate cardiac function. In this article, four major indexes from echocardiography were analyzed under this principal, another seventeen indexes or measurement approaches came out of the shadow, which is very helpful in the assessment of cardiac function, especially for the right cardiac function and diastolic cardiac function.

Regulation of gene expression in diverse cyanobacterial species by using theophylline-responsive riboswitches.

Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-ß-d-thiogalactopyranoside induction of a lacI(q)-Ptrc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Cleavage-resistant fusion proteins of the M(2) muscarinic receptor and Gα(i1). Homotropic and heterotropic effects in the binding of ligands.

BACKGROUND: G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor-G protein complex, but their usefulness depends upon the stability of the fusion. METHODS: Fusion proteins of the M(2) muscarinic receptor and the α-subunit of G(i1) were expressed in CHO and Sf9 cells, extracted in digitonin-cholate, and examined for their binding properties and their electrophoretic mobility on western blots. RESULTS: Receptor fused to native α(i1) underwent proteolysis near the point of fusion to release a fragment with the mobility of α(i1). The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[(3)H]methylscopolamine was 60% of that for [(3)H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. CONCLUSIONS: A stable fusion protein of the M(2) receptor and truncated α(i1) resembles the native receptor-G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. GENERAL SIGNIFICANCE: Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.