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De novo peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable de novo antiparallel and parallel coiled-coil heterotetramers, i.e. , A 2 B 2 systems, to afford control in downstream applications. The switches are incorporated by adding protein kinase A phosphorylation sites, R-R-X-S, with the phosphoacceptor serine residues placed to maximize disruption of the coiled-coil interfaces. The unphosphorylated peptides assemble as designed and unfold reversibly when heated. Addition of kinase to the assembled states unfolds them with half-lives of ≤5 min. Phosphorylation is reversed by Lambda Protein Phosphatase resulting in tetramer reassembly. We envisage that the new de novo designed coiled-coil components, the switches, and a mechanistic model for them will be useful in synthetic biology, biomaterials, and biotechnology applications.

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Recently, studies have begun to identify oil-degrading bacteria and host-taxon specific bacterial assemblages associated with the coral holobiont, including deep-sea cold-water corals, which are thought to provide metabolic functions and additional carbon sources to their coral hosts. Here, we describe the identification of Marinobacter on the soft tissue of Lophelia pertusa coral polyps by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). L. pertusa samples from three reef sites in the northeast Atlantic (Logachev, Mingulay and Pisces) were collected at depth by vacuum seal to eliminate contamination issues. After decalcification, histological processing and sagittal sectioning of the soft coral polyp tissues, the 16S rRNA-targeted oligonucleotide HRP-labelled probe Mrb-0625-a, and Cyanine 3 (Cy3)-labelled tyramides, were used to identify members of the hydrocarbon-degrading genus Marinobacter. Mrb-0625-a-hybridized bacterial cell signals were detected in different anatomical sites of all polyps collected from each of the three reef sites, suggesting a close, possibly intimate, association between them, but the purpose of which remains unknown. We posit that Marinobacter, and possibly other hydrocarbon-degrading bacteria associated with Lophelia, may confer the coral with the ability to cope with toxic levels of hydrocarbons in regions of natural oil seepage and where there is an active oil and gas industry presence., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The surface of marine eukaryotic phytoplankton can harbour communities of hydrocarbon-degrading bacteria; however, this algal-bacterial association has, hitherto, been only examined with non-axenic laboratory cultures of micro-algae. In this study, we isolated an operationally-defined community of phytoplankton, of cell size 50-70 μm, from a natural community in sea surface waters of a subarctic region in the northeast Atlantic. Using MiSeq 16S rRNA sequencing, we identified several recognized ( Alcanivorax , Marinobacter , Oleispira , Porticoccus , Thalassospira ) and putative hydrocarbon degraders ( Colwelliaceae , Vibrionaceae ) tightly associated with the phytoplankton population. We combined fluorescence in situ hybridisation with flow-cytometry (FISH-Flow) to examine the association of Marinobacter with this natural eukaryotic phytoplankton population. About 1.5% of the phytoplankton population contained tightly associated Marinobacter . The remaining Marinobacter population were loosely associated with either eukaryotic phytoplankton cells or non-chlorophyll particulate material. This work is the first to show the presence of obligate, generalist and putative hydrocarbonoclastic bacteria associated with natural populations of eukaryotic phytoplankton directly from sea surface water samples. It also highlights the suitability of FISH-Flow for future studies to examine the spatial and temporal structure and dynamics of these and other algal-bacterial associations in natural seawater samples.

Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces ('phycosphere') has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Preclinical radiation biology has become increasingly sophisticated due to the implementation of advanced small animal image guided radiation platforms into laboratory investigation. These small animal radiotherapy devices enable state-of-the-art image guided therapy (IGRT) research to be performed by combining high-resolution cone beam computed tomography (CBCT) imaging with an isocentric irradiation system. Such platforms are capable of replicating modern clinical systems similar to those that integrate a linear accelerator with on-board CBCT image guidance., Methods: In this study, we present a dosimetric evaluation of the small animal radiotherapy research platform (SARRP, Xstrahl Inc.) focusing on small field dosimetry. Physical dosimetry was assessed using ion chamber for calibration and radiochromic film, investigating the impact of beam focus size on the dose rate output as well as beam characteristics (beam shape and penumbra). Two film analysis tools) have been used to assess the dose output using the 0.5 mm diameter aperture., Results: Good agreement (between 1.7-3%) was found between the measured physical doses and the data provided by Xstrahl for all apertures used. Furthermore, all small field dosimetry data are in good agreement for both film reading methods and with our Monte Carlo simulations for both focal spot sizes. Furthermore, the small focal spot has been shown to produce a more homogenous beam with more stable penumbra over time., Conclusions: FilmQA Pro is a suitable tool for small field dosimetry, with a sufficiently small sampling area (0.1 mm) to ensure an accurate measurement. The electron beam focus should be chosen with care as this can potentially impact on beam stability and reproducibility.

Advanced radiotherapy techniques such as intensity modulated radiation therapy achieve highly conformal dose distributions within target tumor volumes through the sequential delivery of multiple spatially and temporally modulated radiation fields and have been shown to influence radiobiological response. The goals of this study were to determine the effect of hypoxia on the cell survival responses of different cell models (H460, DU145, A549, MDA231 and FADU) to modulated fields and to characterize the time dependency of signaling under oxic conditions, following reoxygenation and after prolonged hypoxia. Hypoxia was induced by incubating cells at 95% nitrogen and 5% carbon dioxide for 4 h prior to irradiation. The out-of-field response in MDA231 cells was oxygen dependent and therefore selected for co-culture studies to determine the signaling kinetics at different time intervals after irradiation under oxic and hypoxic conditions. Under both oxic and hypoxic conditions, significant increases in cell survival were observed in-field with significant decreases in survival observed out-of-field (P < 0.05), which were dependent on intercellular communication. The in-field response of MDA231 cells showed no significant time dependency up to 24 h postirradiation, while out-of-field survival decreased significantly during the first 6 h postirradiation (P < 0.05). While in-field responses were oxygen dependent, out-of-field effects were observed to be independent of oxygen, with similar or greater cell killing under hypoxic conditions. This study provides further understanding of intercellular signaling under hypoxic conditions and highlights the need for further refinement of established radiobiological models for future applications in advanced radiotherapies.

Polycyclovorans algicola strain TG408 is a recently discovered bacterium associated with marine eukaryotic phytoplankton and exhibits the ability to utilize polycyclic aromatic hydrocarbons (PAHs) almost exclusively as sole sources of carbon and energy. Here, we present the genome sequence of this strain, which is 3,653,213 bp, with 3,477 genes and an average G+C content of 63.8%., (Copyright © 2015 Gutierrez et al.)

Objective: To estimate the increased Medicaid expenditures associated with child maltreatment., Methods: Data on child maltreatment were collected from the National Survey of Child and Adolescent Well-Being, a nationally representative sample of cases investigated or assessed by local Child Protective Services agencies between October 1999 and December 2000. Medicaid claims data for 2000 to 2003 were obtained from the Medicaid Analytic Extract (MAX). Children from the National Survey of Child and Adolescent Well-Being who had Medicaid were matched to the MAX data by Social Security number or birthdate, gender, and zip code. Propensity score matching was used to select a comparison group from the MAX data. Two-part regression models were used to estimate the impact of child maltreatment on expenditures. Data with individual identifiers were obtained under confidentiality agreements with the collecting agencies., Results: Children who were identified as maltreated or as being at risk of maltreatment incurred, on average, Medicaid expenditures that were >$2600 higher per year compared with children not so identified. This finding accounted for ~9% of all Medicaid expenditures for children., Conclusions: Child maltreatment imposes a substantial financial burden on the Medicaid system. These expenses could be partially offset by increased investment in child maltreatment prevention.

Relatively little research has focused on the relationship between functional remission and symptomatic remission in mood and anxiety disorders. This study investigates the relationship and synchrony between symptomatic and functional remission in outpatients with major depressive disorder (MDD) and generalized anxiety disorder (GAD). Using data from three MDD (N=1419) and four GAD (N=1847) randomized, placebo-controlled duloxetine studies, we calculated the percentages of patients meeting symptomatic, functional, and combined functional-symptomatic remission criteria for each disorder. We also calculated mean depression [17-item Hamilton depression rating scale (HAMD₁₇), Montgomery-Asberg depression rating scale] scores and mean anxiety (Hamilton anxiety rating scale) scores for patients meeting Sheehan disability scale (SDS) functional remission and the mean SDS scores for patients with symptomatic remission. Among the patients with MDD, 38% achieved symptomatic remission (HAMD₁₇ ≤ 7), 32% achieved functional remission (SDS ≤ 6), and 23% achieved combined functional-symptomatic remission. Mean HAMD₁₇ and Montgomery-Asberg depression rating scale scores for patients with functional remission were approximately 6. Mean SDS total scores for patients with symptomatic remission were 7.1 (patients with HAMD₁₇ ≤ 7) and 8.6 (patients with Montgomery-Asberg depression rating scale ≤ 10). Among the patients with GAD, 30% achieved symptomatic remission (Hamilton anxiety rating scale ≤ 7), 45% achieved functional remission (SDS ≤ 6), and 25% achieved combined symptomatic-functional remission. The mean Hamilton anxiety rating scale score in GAD was approximately 8 for patients with functional remission and the mean SDS total score was approximately 4 in patients with symptomatic remission. The study shows that functional remission does not always move in tandem with symptom remission and provides useful anchor points or rules of thumb for evaluating symptomatic and functional remission in MDD and GAD.

Background: Influenza vaccination is recommended for adults with asthma., Purpose: This study estimates the effect of influenza vaccination on utilization of medical services and expenditures for acute and chronic respiratory conditions (ACRC) among adults with asthma., Methods: The sample was adults aged ≥18 years self-reporting asthma in the 2003 through 2006 Medical Expenditure Panel Survey (MEPS), covering four complete influenza seasons. The dependent variables were indicators for any ACRC claims within service category and ACRC expenditures. The main independent variable was an indicator of influenza vaccination. To control for selection bias in the observational data, a nonlinear instrumental variables approach was used. The instruments were indicators for influenza in the first year of MEPS and vaccination in the year prior to MEPS. Data were analyzed in 2009., Results: Adults with asthma vaccinated for influenza were 4.4 percentage points less likely to have an inpatient stay due to ACRC (95% CI = -10.8, -1.0). Influenza vaccination was associated with a $492 decrease (95% CI = -$1591, -$56) in annual ACRC nonprescription expenditures, a $224 increase (95% CI = $70, $360) in annual ACRC prescription expenditures, and a nonsignificant $216 decrease (95% CI = -$854, $248) in overall annual ACRC expenditures., Conclusions: Although there was no evidence that vaccination reduced overall ACRC expenditures, the study suggests that efforts to increase the percentage of adults with asthma who are vaccinated may bring substantial benefits in terms of reducing the prevalence and costs of hospitalization although raising prescription medication costs, possibly through improvement in compliance., (Copyright © 2010 American Journal of Preventive Medicine. All rights reserved.)