Efficient Algorithms for Identifying Loop Formation and Computing θ Value for Solving Minimum Cost Flow Network Problems

While the minimum cost flow (MCF) problems have been well documented in many publications, due to its broad applications, little or no effort have been devoted to explaining the algorithms for identifying loop formation and computing the value needed to solve MCF network problems. This paper proposes efficient algorithms, and MATLAB computer implementation, for solving MCF problems. Several academic and real-life network problems have been solved to validate the proposed algorithms; the numerical results obtained by the developed MCF code have been compared and matched with the built-in MATLAB function Linprog() (Simplex algorithm) for further validation

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

An <i>O</i>-Methyltransferase Is Required for Infection of Tick Cells by <i>Anaplasma phagocytophilum</i>

<div><p><i>Anaplasma phagocytophilum</i>, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by <i>Ixodes</i> spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of <i>A</i>. <i>phagocytophilum</i> resulted in an insertion within the coding region of an <i>o</i>-methyltransferase (<i>omt</i>) family 3 gene. In wild-type bacteria, expression of <i>omt</i> was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. <i>In vitro</i> methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the <i>apo</i> (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn²⁺ bound (PDB_ID:4PCA), and SAM- Mn²⁺ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in <i>A</i>. <i>phagocytophilum</i> that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.</p></div

Expression of the OMT during infection of ISE6 cells.

<p>qRT_PCR to track the expression of the <i>omt</i> gene in wild-type <i>A</i>. <i>phagocytophilum</i> during adhesion and invasion of ISE6 cells in comparison to <i>omt</i> gene transcripts detected in bacteria interacting with HL-60 cells. Wild-type bacteria were purified from HL-60 cells and inoculated onto cell layers of ISE6 cells or mixed with suspended HL-60 cells. RNA was purified at the indicated times post inoculation, and qRT-PCR was performed. The bars represent the average of the fold change normalized to <i>msp5</i> (blue) or to <i>rpoB</i> (red), and vertical lines represent the standard error of the mean. Up-regulation of <i>omt</i> transcription was seen as early as 30 min, reaching 34-fold change at 2 hr. At 4 hr there was no detectable difference in bacterial gene expression between the two host cell types (0.97 fold change).</p

Pathways that are altered in ΔOMT during binding to and internalization into ISE6 cells.

<p>Pathways that are altered in ΔOMT during binding to and internalization into ISE6 cells.</p

<i>A</i>. <i>phagocytophilum</i> HZ proteins that are differentially abundant in the ΔOMT, according to iTRAQ results.

<p>*Average of peptides used for the quantification of the proteins.</p><p><i>**</i>Ratios <1.0 are less abundant in ΔOMT and ratios >1.0 are more abundant in ΔOMT.</p><p><i>A</i>. <i>phagocytophilum</i> HZ proteins that are differentially abundant in the ΔOMT, according to iTRAQ results.</p

Effects of the mutation on the growth of <i>A</i>. <i>phagocytophilum</i> in tick cell culture.

<p>Growth curves representing the replication of ΔOMT (solid blue line) and wild-type (dashed red line) bacteria in ISE6 (top) or HL-60 cells (bottom). ΔOMT and wild-type bacteria were purified from HL-60 cells and inoculated into ISE6 or HL-60 cultures. The number of bacteria was estimated by determining the copy number of the <i>msp5</i> gene. Each data point represents the average number of bacteria from triplicate samples, and vertical bars indicate the standard deviation. Statistical differences were evaluated by repeated measures ANOVA. ΔOMT bacteria were not able to replicate in ISE6 cells (top) and had decreased already by day 3, which was significantly different from the replication of wild-type bacteria in ISE6 cells (P = 0.008). This is in contrast to ΔOMT growth within HL-60 cells (bottom), in which there was no significant difference between mutant and wild-type bacteria numbers (P = 0.504).</p

<i>In vitro</i> methylation of recombinant <i>A</i>. <i>phagocytophilum</i> proteins by rOMT and catalytic effect of metal ions.

<p>A recombinant version of the complete OMT was produced in <i>E</i>. <i>coli</i> Rosetta 2(DE3) pLysS and purified by column affinity chromatography. The eluted rOMT was visualized by Coomassie blue staining after electrophoresis in a 4–16% gel for 1 hr. The expected size of rOMT is indicated on the left; the left lane contains lysate from uninduced <i>E</i>. <i>coli</i> cells carrying the plasmid encoding OMT. The right lane contains rOMT His-tag purified from <i>E</i>. <i>coli</i> carrying the plasmid encoding OMT, following induction with IPTG. B) Enzymatic activity of the rOMT was determined in a methylation assay that measured fluorescence resulting from production of resorufin in each sample. Average fluorescence from three replicates of each sample was plotted against assay time indicated on the X-axis. The blue dotted line represents the reaction using Msp4 as the substrate, which produced a curve expected from an enzymatic reaction. The reactions with APH_0406 (black line), TypA (dotted red line), and p44-16b (dotted gray line) as substrates produced only a minimal increase in fluorescence that was not significant. Green dotted line: negative control. C) Enzymatic activity under different concentrations of additional manganese (as MnCl₂). (Red = 16 mM, Dark Blue = 8 mM, Green = 2 mM, Purple = 0.5 mM, Brown = 0 mM, Light Blue = Negative control) was tested to determine if Mn²⁺ (included at a concentration of 10 mM in the assay kit) was a limiting factor for the OMT. Higher concentrations of MnCl₂ resulted in a proportional increase in velocity of the reaction, indicating that Mn²⁺ was a co-factor and required at concentrations greater than 10 mM for optimal enzyme activity. Reactions with 16 mM additional MnCl₂ (17 mM total Mn²⁺) were completed in 90 min with fluorescence levels 25 times higher than with only 10 mM. The line graphs represent the averages from 3 replicates. Standard deviations for substrate testing ranged from 5%–10%, whereas standard deviations for Mn²⁺ assays were <5% at all concentrations used.</p