Phage Orf family recombinases:conservation of activities and involvement of the central channel in DNA binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination

Learning together for and with the Martuwarra Fitzroy River

Co-production across scientific and Indigenous knowledge systems has become a cornerstone of research to enhance knowledge, practice, ethics, and foster sustainability transformations. However, the profound differences in world views and the complex and contested histories of nation-state colonisation on Indigenous territories, highlight both opportunities and risks for Indigenous people when engaging with knowledge co-production. This paper investigates the conditions under which knowledge co-production can lead to improved Indigenous adaptive environmental planning and management among remote land-attached Indigenous peoples through a case study with ten Traditional Owner groups in the Martuwarra (Fitzroy River) Catchment in Western Australia’s Kimberley region. The research team built a 3D map of the river and used it, together with an interactive table-top projector, to bring together both scientific and Indigenous spatial knowledge. Participatory influence mapping, aligned with Traditional Owner priorities to achieve cultural governance and management planning goals set out in the Fitzroy River Declaration, investigated power relations. An analytical framework, examining underlying mechanisms of social learning, knowledge promotion and enhancing influence, based on different theories of change, was applied to unpack the immediate outcomes from these activities. The analysis identified that knowledge co-production activities improved the accessibility of the knowledge, the experiences of the knowledge users, strengthened collective identity and partnerships, and strengthened Indigenous-led institutions. The focus on cultural governance and management planning goals in the Fitzroy River Declaration enabled the activities to directly affect key drivers of Indigenous adaptive environmental planning and management—the Indigenous-led institutions. The nation-state arrangements also gave some support to local learning and decision-making through a key Indigenous institution, Martuwarra Fitzroy River Council. Knowledge co-production with remote land-attached Indigenous peoples can improve adaptive environmental planning and management where it fosters learning together, is grounded in the Indigenous-led institutions and addresses their priorities

Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock: 2008

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012

OBJECTIVE: To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008. DESIGN: A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (1) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (1) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations. RESULTS: Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within 1 h of the recognition of septic shock (1B) and severe sepsis without septic shock (1C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (1B); infection source control with attention to the balance of risks and benefits of the chosen method within 12 h of diagnosis (1C); initial fluid resuscitation with crystalloid (1B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (1B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (1C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (1B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (1C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (1B); low tidal volume (1A) and limitation of inspiratory plateau pressure (1B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (1B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤100 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (1B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (1C); protocols for weaning and sedation (1A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (1B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (1C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) 180 mg/dL, targeting an upper blood glucose ≤180 mg/dL (1A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (1B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (1B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-10 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C). CONCLUSIONS: Strong agreement existed among a large cohort of international experts regarding many level 1 recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients

YbcN binding to SSB.

<p>(<b>A</b>) Interaction of YbcN with SSB in far western assays. SSB protein (0.5, 1.9, 3.8 µg) separated on 15% SDS-PAGE was blotted and probed with 20 µg MBP-YbcN (lanes a-c) or GST-YbcN (lanes d-f). Interactions were detected with antibodies specific for either the MBP or GST domains. (<b>B</b>) Interaction of GST-YbcN with SSB as detected by ELISA. SSB (0.5 µg) bound to Immulon-1 microtitre plates was exposed either to GST-YbcN, GST-Orf or GST proteins (0.0195-10 µg). Positive interactions were detected by addition of anti-GST antibody-HRP conjugate. Data are the mean and standard deviation of four independent experiments. (<b>C</b>) YbcN binding to SSBΔC10 and SSB113 proteins in far western assays. 0.5, 1.9, 3.8 µg SSB (lanes a-c), SSBΔC10 (lanes d-f) and SSB113 (lanes g-i) separated on 15% SDS-PAGE were blotted and probed with 20 µg MBP-YbcN. Interactions were detected with antibodies specific for MBP (row i). Row ii shows the efficiency of SSB, SSBΔC10 and SSB113 transfer onto blotted membranes as revealed by staining with amido black. Panel i (lanes a-c) is reproduced from (A) to facilitate comparisons with MBP-YbcN binding to wild-type SSB. (<b>D</b>) Interaction of GST-YbcN with SSB, SSBΔC10, SSB113 and SSBΔ115-144 as detected by ELISA. SSB wild-type and mutant proteins (0.5 µg) bound to microtitre plates were exposed to GST-YbcN (0.078-2.5 µg) and probed as in (B). Data are the mean and standard deviation of two independent experiments. (<b>E</b>) Yeast two-hybrid analysis of YbcN interactions with SSB and SSBΔC10. Experiments were performed using constructs fused to the GAL4 DNA-binding or activating domains. The β-galactosidase activities were the mean and standard deviation of three independent experiments. The vertical bars represent the β-galactosidase activity (Miller units) and values for YbcN-SSBΔC10 are given for pGBKT7-53 and pGADT7-T constructs in both orientations.</p

CD spectra and SLS analysis of MBP-Orf mutant proteins.

<p>(<b>A</b>) SLS analysis of selected MBP-Orf mutant proteins. Proteins at 0.2 mg/ml in 100 mM Tris-HCl pH7 at 25°C were analyzed in a Zetasizer µV. (<b>B</b>) CD analysis of MBP-Orf mutant proteins. CD spectra (180-260 nm) were obtained for MBP-Orf proteins in ultrapure water at 20°C and analysis performed using CDSSTR, Contin/LL and Selcon3 programs from the CDPro suite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102454#pone.0102454-Sreerama1" target="_blank">[54]</a>.</p

Comparison of MBP-YbcN and MBP-Orf binding to DNA.

<p>(<b>A</b>) Binding to ssDNA and dsDNA. Gel mobility shift assays contained 0, 62.5, 125, 250 and 500 nM MBP-Orf or MBP-YbcN proteins, 5 mM EDTA and either 0.3 nM of ³²P-labelled 50 nt (SS₅₀) ssDNA (lanes a-e and k-o) or 50 bp dsDNA (lanes f-j and p-t). (<b>B</b>) MBP-YbcN and MBP-Orf binding to ssDNA. Binding reactions contained 0.3 nM SS₅₀ with 0, 7.81, 15.625, 31.25, 62.5, 125, 250 and 500 nM protein and either 5 mM EDTA or 1 mM MgCl₂. Data are the mean and standard deviation of two independent experiments. (<b>C</b>) Binding to mismatch and bubble DNA. Gel mobility shift assays contained 250 nM MBP-Orf (O) or MBP-YbcN (Y) proteins, 5 mM EDTA and 0.15 nM of ³²P-labelled 60 nt (SS₆₀) ssDNA (lanes a-c), 60 bp (DS₆₀) dsDNA (lanes d-f), 1 bp (MM_G:G) mismatch (lanes g-i), 5 bp (BB₅) bubble (lanes j-l), 13 bp (BB₁₃) bubble (lanes m-o) and 20 bp (BB₂₀) bubble (lanes p-r).</p

Conservation of genomic organization and protein architecture among selected members of the Orf family.

<p>(<b>A</b>) Conserved gene order of <i>orf</i> relative to the replication and lysis genes of lambdoid phages and prophages. Genes are shown as boxes and colored according to their order in the <i>λ </i><i>ninR</i> region; in almost all cases they are transcribed from left to right. Orthologous genes are highlighted by name and color. Those that failed to match any of the <i>λ</i> genes in this region are shown as white boxes (the residue length of unannotated open-reading frames is indicated). Additional abbreviations refer to gene products that match the HNH nuclease domain family (HNH), DNA/RNA helicase superfamily II (SFII) or phage head proteins (head). The percentage identity to <i>λ</i> Orf is shown for each Orf homolog and listed in order of similarity. Many of the prophages are reservoirs for insertion sequences (shaded in grey); note that additional genes encoded by these elements are not shown. The putative 64-residue polypeptide between Q and S in <i>λ</i> was also omitted. (<b>B</b>) Conserved primary structure of <i>λ</i> Orf, DLP12 YbcN and <b><i>φ</i></b>ETA Orf20 proteins. <i>λ</i> Orf is aligned with homologous representatives from <i>Y. pestis</i> (<i>Ype</i>), <i>E. coli</i> O157:H7 prophage Stx2-converting phage VT2-Sa and <i>E. coli</i> H phage HK620. DLP12 YbcN is aligned with a prophage homolog from <i>P. luminescens</i> (<i>Plu</i>). <i>Staphylococcus aureus </i><b><i>φ</i></b>ETA Orf20 is aligned with a homolog from <i>Enterococcus faecalis</i> (<i>Efa</i>). Conserved residues within each subset are highlighted in blue (basic), red (acidic) and lilac (others); those occurring in both Orf/YbcN and Orf/Orf20 alignments are marked with an asterisk. Secondary elements from the crystal structure of <i>λ</i> Orf <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102454#pone.0102454-Maxwell1" target="_blank">[12]</a> are indicated above the alignment, as are the site-directed mutants Q45A, K48A, W50A, R103E, V106E and W137A. A pair of cysteines from the HNH motif are highlighted in orange.</p

Predicting Outcomes After Distal Radius Fracture: A 24-Center International Clinical Trial of Older Adults

© 2019 American Society for Surgery of the Hand Purpose: Current evidence on predictors of outcomes after distal radius fracture is often based on retrospective analyses or may be confounded by fracture type. Using data from the Wrist and Radius Injury Surgical Trial (WRIST), a 24-site randomized study of distal radius fracture treatment, in which all fractures are severe enough to warrant surgery, we set out to perform a secondary data analysis to explore predictors of better or worse hand outcomes. Methods: The primary outcome measure was the Michigan Hand Outcomes Questionnaire (MHQ) summary score 12 months after treatment. We used a regression tree analysis with recursive partitioning to identify subgroups of participants who experienced similar outcomes (ie, MHQ score) and to determine which baseline or treatment factors they had in common. Results: Factors most predictive of 12-month MHQ score were pain at enrollment, education, age, and number of comorbidities. Specifically, participants who had a high school education or less and also reported severe pain had the lowest MHQ scores. Conversely, participants with less pain and more education and who were age 87 years or younger with one or no comorbid condition had the highest MHQ scores. Treatment type or radiographic measurements assessed on post-reduction films did not affect 12-month outcomes. Conclusions: These results identified patient characteristics that can be used by surgeons to identify subgroups of patients who may experience similar hand outcomes. Type of study/level of evidence: Prognostic III