Vascular Graft Infections Treated With Bioabsorbable Antibiotic Beads.

INTRODUCTION: Inguinal vascular surgical site infections (VSSI) and infected prosthetic grafts remain a critical problem in vascular surgery. Prior clinical reports suggest antibiotic-impregnated beads may be used to attempt salvage of the graft and improve outcomes, especially if explant would result in major amputation or mortality. Described is our institutional experience managing inguinal VSSI using bioabsorbable, antibiotic-impregnated beads compared to inguinal VSSI managed with debridement alone. METHODS: Patients with VSSIs after lower-extremity procedures were identified through the institutional database and departmental registries from 2014 to 2023. Cases were excluded if they did not involve an inguinal wound infection or an operation for VSSI management. Outcomes, including amputation-free survival, reinfection, and re-operation for infection were recorded, along with microbial isolates. Basic descriptive statistics, Kaplan-Meier, and Multiple variable Cox proportional hazards analyses were performed. RESULTS: There were 43 patients identified (23 with intravenous antibiotics and debridement alone, and 20 treated with intravenous antibiotics, debridement, and antibiotic beads). The two groups differed significantly in their Szilagyi classification, with thirteen patients (65%) with class III infections in the antibiotic bead group compared with one (7%) in the debridement alone group. There was no significant difference in amputation-free survival for those cases that received debridement and antibiotic beads versus debridement alone (P = 0.20) or amputation-free survival between Szilagyi classifications (P = 0.47) despite a higher representation of Szilagyi III cases in the antibiotic bead group (P = 0.0001). Patients with graft infections treated with beads experienced similar survival outcomes to patients with subcutaneous infections treated with debridement alone (P = 0.21). CONCLUSIONS: The efficacy of bioabsorbable antibiotic beads in VSSIs remains controversial. While this cohort study demonstrated an increased risk of re-infection, this is confounded by the antibiotic bead group having a higher frequency of graft infections. Antibiotic beads appear to be safe for use in patients with vascular graft infections and may help achieve outcomes comparable to patients without graft involvement. Further studies with larger patient populations and similar infection severity between groups are needed.

Total hip arthroplasty for fractured neck of femur does not restore preoperative hip-specific function, health-related quality of life, or level of fitness.

PURPOSE: The primary aim was to assess whether a total hip arthroplasty (THA) was able to restore health-related quality of life (HRQoL) following an intracapsular hip fracture. The secondary aims were to assess changes in hip-specific function, fitness/frailty, mortality risk, complications and revision risk, and factors independently associated with these. METHODS: This retrospective cohort study included all patients aged ≥ 50 years admitted with a hip fracture from the emergency department at a single centre during a 42-month period. Patient demographics, perioperative variables, complications, revision, and mortality were collected. Patient-reported outcome measures (PROMs) were assessed at final follow-up. RESULTS: Among 250 identified patients, 189 (75.6%) were women with a mean age of 70.3 (range 50-94 years). Mean follow-up was 2.3 (SD 1.1) years. The implant and patient survival rates at 2 years were both 95.5% (95% confidence intervals (CI) +/- 2.7). Older age (hazard ratio [HR] 1.22, 95% CI 1.12-1.33, p < 0.001) and male sex (HR 3.33, 95% CI 1.15-10.0, p = 0.026) were independently associated with mortality. There were 19 (7.6%) postoperative complications that included 6 (2.4%) periprosthetic fractures, 5 (2.0%) deep infections, and 8 (3.2%) dislocations, of which 13 underwent revision. Increasing time to theatre (HR 1.02, 95% CI 1.01-1.03, p = 0.017) was independently associated with a postoperative complication. Postoperative PROMs were available for 166 (66.4%) patients. There were significant (p < 0.001) deteriorations in EuroQol-5D (Mean difference [MD] 0.192, 95% CI 0.133-0.252), Oxford hip score (MD 2.5, 95% CI 1.5-3.6), and fitness (Rockwood score MD 0.7, 95% CI 0.5-0.8) relative to preoperative levels of function. CONCLUSION: THA may be the treatment of choice in a physically active patient with the aim of restoring their HRQoL, hip function, and fitness, but this was not observed. Furthermore, there was a high complication rate which was associated with increasing time to theatre. LEVEL OF EVIDENCE: III, retrospective cohort study.

Gender Differences in Aortic Anatomic Severity Grade and Long-Term Survival Following Elective Abdominal Aortic Aneurysm Repair at a Single Tertiary Center.

BACKGROUND: Anatomic severity grade (ASG) score is utilized to assess preoperative abdominal aortic aneurysms (AAA) and provide a quantitative data on its anatomic complexity. The aim of this study is to determine the anatomical differences and long-term survival between male and female patients undergoing elective AAA repair. METHODS: All patients undergoing intact AAA repair from 2007 to 2014 were included. ASG scores were calculated based on preoperative anatomical characteristics including aortic neck, aneurysm, and iliac artery. Standard univariate analysis was used to evaluate patient and anatomical characteristics. Kaplan-Meier survival curves were used to evaluate long-term survival at 1 and 5 years. RESULTS: A total of 379 patients were identified, of which, majority of them were males (80%). Females were on average 3 years older (mean [SD]: 74.32 [8.63] vs. 71.92 [8.64] years) and were more likely to undergo open repair (29.7% vs. 17.5%) (both P < 0.05). Both groups had similar comorbidities. The mean long-term follow-up (S.D.) was 6.21 (3.81) years. No significant difference was seen between males versus females in long-term survival at both 1 year (86.3% vs. 92.8, P = 0.06) and 5 year (68.5% vs. 72.7%, P = 0.38). In regard to the anatomical characteristics, females had shorter aortic neck length (mean in mm [S.D.]: 17.67 [1.41] vs. 27.20 [15.76]), increased tortuosity index [mean (S.D.): 1.11 (0.07) vs. 1.09 (0.07)]) and higher calcification [mean % (S.D.): 17.12 (21.17) vs. 10.59 (16.82)] (All P < 0.05). In contrast, males had larger aortic neck (mean in mm (S.D.): 23.81 (4.17) vs. 22.41 (4.16)] and iliac artery [mean in mm (S.D.): 7.70 (1.91) vs. 6.28 (1.67)] diameter (both P < 0.05). The mean total ASG score was significantly higher among females versus males [mean (S.D.): 17.23 (4.01) vs. 15.67 (3.96), P = 0.003]. After stratifying by ASG score ≥15, females had significantly lower survival at 1 year compared to males (82.6% vs. 92.1%, P = 0.04). However, this difference disappeared at 5 years. CONCLUSIONS: The data demonstrate that females present at an older age with more complex AAA anatomy than males. Based on anatomical complexities, females were more likely to undergo open repair, with a corresponding increase in 1-year mortality, but not at 5 year. The data suggest that care processes for optimization of aortic surgery in females are needed to improve 1-year survival.

ProX from marine Synechococcus spp. show a sole preference for glycine-betaine with differential affinity between ecotypes.

Osmotic stress, caused by high or fluctuating salt concentrations, is a crucial abiotic factor affecting microbial growth in aquatic habitats. Many organisms utilize common responses to osmotic stress, generally requiring active extrusion of toxic inorganic ions and accumulation of compatible solutes to protect cellular machinery. We heterologously expressed and purified predicted osmoprotectant, proline/glycine betaine-binding proteins (ProX) from two phylogenetically distinct Synechococcus spp. MITS9220 and WH8102. Homologues of this protein are conserved only among Prochlorococcus LLIV and Synechococcus clade I, III and CRD1 strains. Our biophysical characterization show Synechococcus ProX exists as a dimer, with specificity solely for glycine betaine but not to other osmoprotectants tested. We discovered that MITS9220_ProX has a 10-fold higher affinity to glycine betaine than WH8102_ProX, which is further elevated (24-fold) in high salt conditions. The stronger affinity and effect of ionic strength on MITS9220_ProX glycine betaine binding but not on WH8102_ProX alludes to a novel regulatory mechanism, providing critical functional insights into the phylogenetic divergence of picocyanobacterial ProX proteins that may be necessary for their ecological success.

Functional characterisation of substrate-binding proteins to address nutrient uptake in marine picocyanobacteria.

Marine cyanobacteria are key primary producers, contributing significantly to the microbial food web and biogeochemical cycles by releasing and importing many essential nutrients cycled through the environment. A subgroup of these, the picocyanobacteria (Synechococcus and Prochlorococcus), have colonised almost all marine ecosystems, covering a range of distinct light and temperature conditions, and nutrient profiles. The intra-clade diversities displayed by this monophyletic branch of cyanobacteria is indicative of their success across a broad range of environments. Part of this diversity is due to nutrient acquisition mechanisms, such as the use of high-affinity ATP-binding cassette (ABC) transporters to competitively acquire nutrients, particularly in oligotrophic (nutrient scarce) marine environments. The specificity of nutrient uptake in ABC transporters is primarily determined by the peripheral substrate-binding protein (SBP), a receptor protein that mediates ligand recognition and initiates translocation into the cell. The recent availability of large numbers of sequenced picocyanobacterial genomes indicates both Synechococcus and Prochlorococcus apportion >50% of their transport capacity to ABC transport systems. However, the low degree of sequence homology among the SBP family limits the reliability of functional assignments using sequence annotation and prediction tools. This review highlights the use of known SBP structural representatives for the uptake of key nutrient classes by cyanobacteria to compare with predicted SBP functionalities within sequenced marine picocyanobacteria genomes. This review shows the broad range of conserved biochemical functions of picocyanobacteria and the range of novel and hypothetical ABC transport systems that require further functional characterisation.

Functional traits reveal the presence and nature of multiple processes in the assembly of marine fish communities.

Functional traits can be used to identify the importance of various community assembly mechanisms such as ecological drift, environmental filtering, and limiting similarity. These processes act in concert, not isolation, and different processes may act upon separate traits, potentially concealing the ecological signal of one or more of the mechanisms. Nine functional attributes of marine fish were used to identify changes in the importance of various mechanisms in the assembly of marine fish communities over a latitudinal gradient along the Western Australian coast. Complementary null modelling approaches were used to test the relative importance of assembly processes (ecological drift, environmental filtering, and limiting similarity) in structuring fish communities. Ecological drift was found to be a major driver of the structure of fish communities, and dispersal limitation was strongest in the tropical region, with homogenising dispersal strongest in the temperate region. Dispersion of functional traits identified environmental filtering acting on most traits incorporated in this study, in addition to limiting similarity acting on traits associated with acquisition of trophic resources. The coexistence of Western Australian marine fishes thus results from concurrent ecological drift, environmental filtering, and limiting similarity structuring the communities. The observed ecological drift may be the result of priority effects and/or context-dependent biotic interactions. Both niche complementarity and predator avoidance may be the drivers of the observed limiting similarity in the communities.

Endovascular Repair of the Ascending Aorta for an Anastomotic Saphenous Vein Graft Aneurysm.

The ascending aorta is the final segment of the aorta to be explored with endovascular stent grafts. With a patient population of increasingly advanced age and disease, there are situations where traditional open repair for ascending aneurysms or dissections may be prohibitive. However, the ascending aorta has multiple hostile characteristics that make endovascular treatment challenging. There is also a lack of approved specialized devices in the United States for this aortic territory. We demonstrate the feasibility of adapting an abdominal aortic graft to the ascending aorta for the treatment of a saphenous vein graft aneurysm with a discussion of the technical considerations for the operation.

Reduced Tolerance and Asymmetrical Crosstolerance to Effects of the Indole Quinuclidinone Analog PNR-4-20, a G Protein-Biased Cannabinoid 1 Receptor Agonist in Mice: Comparisons with Δ9-Tetrahydrocannabinol and JWH-018.

Most cannabinoid 1 receptor (CB1R) agonists will signal through both G protein-dependent and -independent pathways in an unbiased manner. Recruitment of ß-arrestin 2 desensitizes and internalizes receptors, producing tolerance that limits therapeutic utility of cannabinoids for chronic conditions. We developed the indole quinuclidinone (IQD) analog (Z)-2-((1-(4-fluorobenzyl)-1H-indol-3-yl)methylene)quinuclidin-3-one (PNR-4-20) as a novel G protein-biased agonist at CB1Rs, and the present studies determine if repeated administration of PNR-4-20 produces lesser tolerance to in vivo effects compared with unbiased CB1R agonists Δ9-tetrahydrocannabinol (Δ9-THC) and 1-pentyl-3-(1-naphthoyl)indole (JWH-018). Adult male National Institutes of Health Swiss mice were administered comparable doses of PNR-4-20 (100 mg/kg), Δ9-THC (30 mg/kg), or JWH-018 (3 mg/kg) once per day for five consecutive days to determine tolerance development to hypothermic, antinociceptive, and cataleptic effects. Persistence of tolerance was then determined after a drug abstinence period. We found that unbiased CB1R agonists Δ9-THC and JWH-018 produced similar tolerance to these effects, but lesser tolerance was observed with PNR-4-20 for hypothermic and cataleptic effects. Tolerance to the effects of PNR-4-20 completely recovered after drug abstinence, while residual tolerance was always observed with unbiased CB1R agonists. Repeated treatment with PNR-4-20 and Δ9-THC produced asymmetric crosstolerance to hypothermic effects. Importantly, binding studies suggest PNR-4-20 produced significantly less downregulation of CB1Rs relative to Δ9-THC in hypothalamus and thalamus of chronically treated mice. These studies suggest that the G protein-biased CB1R agonist PNR-4-20 produces significantly less tolerance than unbiased cannabinoid agonists, and that the IQD analogs should be investigated further as a novel molecular scaffold for development of new therapeutics.

The tamoxifen derivative ridaifen-B is a high affinity selective CB2 receptor inverse agonist exhibiting anti-inflammatory and anti-osteoclastogenic effects.

Selective estrogen receptor modulators (SERMs) target estrogen receptors (ERs) to treat breast cancer and osteoporosis. Several SERMs exhibit anti-cancer activity not related to ERs. To discover novel anti-cancer drugs acting via ER-independent mechanisms, derivatives of the SERM tamoxifen, known as the "ridaifen" compounds, have been developed that exhibit reduced or no ER affinity, while maintaining cytotoxicity. Tamoxifen and other SERMs bind to cannabinoid receptors with moderate affinity. Therefore, ER-independent effects of SERMs might be mediated via cannabinoid receptors. This study determined whether RID-B, a first generation ridaifen compound, exhibits affinity and/or activity at CB1 and/or CB2 cannabinoid receptors. RID-B binds with high affinity (Ki = 43.7 nM) and 17-fold selectivity to CB2 over CB1 receptors. RID-B acts as an inverse agonist at CB2 receptors, modulating G-protein and adenylyl cyclase activity with potency values predicted by CB2 affinity. Characteristic of an antagonist, RID-B co-incubation produces a parallel-rightward shift in the concentration-effect curve of CB2 agonist WIN-55,212-2 to inhibit adenylyl cyclase activity. CB2 inverse agonists are reported to exhibit anti-inflammatory and anti-ostoeclastogenic effects. In LPS-activated macrophages, RID-B exhibits anti-inflammatory effects by reducing levels of nitric oxide (NO), IL-6 and IL-1α, but not TNFα. Only reduction of NO concentration by RID-B is mediated by cannabinoid receptors. RID-B also exhibits pronounced anti-osteoclastogenic effects, reducing the number of osteoclasts differentiating from primary bone marrow macrophages in a cannabinoid receptor-dependent manner. In summary, the tamoxifen derivative RID-B, developed with reduced affinity for ERs, is a high affinity selective CB2 inverse agonist with anti-inflammatory and anti-osteoclastogenic properties.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development.

The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in ß-arrestin knockout mice suggest that interaction of certain GPCRs, including µ-, δ-, κ-opioid and hCB1Rs, with ß-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to ß-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no ß-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to ß-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced ß-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to similar treatment with CP-55,940. PNR-4-20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non-biased full agoinst JWH-018 or Δ9-THC. Finally, studies of a structurally similar analog PNR- 4-02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to ß-arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically.

Antinociceptive effects of the 6-O-sulfate ester of morphine in normal and diabetic rats: Comparative role of mu- and delta-opioid receptors.

This study determined the antinociceptive effects of morphine and morphine-6-O-sulfate (M6S) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ORs) and delta-opioid receptors (delta-ORs) in the antinociceptive action of these opioids. In vitro characterization of mu-OR and delta-OR-mediated signaling by M6S and morphine in stably transfected Chinese hamster ovary (CHO-K1) cells showed that M6S exhibited a 6-fold higher affinity for delta-ORs and modulated G-protein and adenylyl cyclase activity via delta-ORs more potently than morphine. Interestingly, while morphine acted as a full agonist at delta-ORs in both functional assays examined, M6S exhibited either partial or full agonist activity for modulation of G-protein or adenylyl cyclase activity, respectively. Molecular docking studies indicated that M6S but not morphine binds equally well at the ligand binding site of both mu- and delta-ORs. In vivo analgesic effects of M6S and morphine in both normal and streptozotocin-induced diabetic Sprague-Dawley rats utilizing the hot water tail flick latency test showed that M6S produced more potent antinociception than morphine in both normal rats and diabetic rats. This difference in potency was abrogated following antagonism of delta- but not mu- or kappa (kappa-ORs) opioid receptors. During 9days of chronic treatment, tolerance developed to morphine-treated but not to M6S-treated rats. Rats that developed tolerance to morphine still remained responsive to M6S. Collectively, this study demonstrates that M6S is a potent and efficacious mu/delta opioid analgesic with a delayed tolerance profile when compared to morphine in both normal and diabetic rats. PERSPECTIVE: This study demonstrates that M6S acts at both mu- and delta-ORs, and adds to the growing evidence that the use of mixed mu/delta opioid agonists in pain treatment may have clinical benefit.

Education of Physicians and Implementation of a Formal Referral System Can Improve Cardiac Rehabilitation Referral and Participation Rates after Percutaneous Coronary Intervention.

BACKGROUND: Cardiac rehabilitation (CR) is an effective preventive measure that remains underutilised in the United States. The study aimed to determine the CR referral rate (RR) after percutaneous coronary intervention (PCI) at an academic tertiary care centre, identify barriers to referral, and evaluate awareness of CR benefits and indications (CRBI) among cardiologists. Subsequently, it aimed to evaluate if an intervention consisting of physicians' education about CRBI and implementation of a formal CR referral system could improve RR and consequently participation rate (PR). METHODS: Data were retrospectively collected for all consecutive patients who underwent PCI over 12 months. Referral rate was determined and variables were compared for differences between referred and non-referred patients. A questionnaire was distributed among the physicians in the Division of Cardiology to assess awareness of CRBI and referral practice patterns. After implementation of the intervention, data were collected retrospectively for consecutive patients who underwent PCI in the following six months. Referral rate and changes in PRs were determined. RESULTS: Prior to the intervention, RR was 17.6%. Different barriers were identified, but the questionnaire revealed lack of physicians' awareness of CRBI and inconsistent referral patterns. After the intervention, RR increased to 88.96% (Odds Ratio 37.73, 95% CI 21.34-66.70, p<0.0001) and PR increased by 32.8% to reach 26%. Personal endorsement of CRBI by cardiologists known to patients increased CR program graduation rate by 35%. CONCLUSIONS: Cardiologists' awareness of CRBI increases CR RR and their personal endorsement improves PR and compliance. Education of providers and implementation of a formal referral system can improve RR and PR.

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen.

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 µM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors.

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R=R(2)=H, R(1)=F) and 13 (R=COOCH3, R(1)=R(2)=H) exhibited high affinity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.

Marine picocyanobacterial PhnD1 shows specificity for various phosphorus sources but likely represents a constitutive inorganic phosphate transporter.

Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.

Risk factors for SARS-CoV-2 infection in primary and secondary school students and staff in England in the 2020/2021 school year: a longitudinal study.

OBJECTIVES: Investigate risk factors for SARS-CoV-2 infections in school students and staff. METHODS: In the 2020/2021 school year, we administered polymerase chain reaction, antibody tests, and questionnaires to a sample of primary and secondary school students and staff, with data linkage to COVID-19 surveillance. We fitted logistic regression models to identify the factors associated with infection. RESULTS: We included 6799 students and 5090 staff in the autumn and 11,952 students and 4569 staff in the spring/summer terms. Infections in students in autumn 2020 were related to the percentage of students eligible for free school meals. We found no statistical association between infection risk in primary and secondary schools and reported contact patterns between students and staff in either period in our study. Using public transports was associated with increased risk in autumn in students (adjusted odds ratio = 1.72; 95% confidence interval 1.31-2.25) and staff. One or more infections in the same household during either period was the strongest risk factor for infection in students and more so among staff. CONCLUSION: Deprivation, community, and household factors were more strongly associated with infection than contacts patterns at school; this suggests that the additional school-based mitigation measures in England in 2020/2021 likely helped reduce transmission risk in schools.

Novel functional insights into a modified sugar-binding protein from Synechococcus MITS9220.

Paradigms of metabolic strategies employed by photoautotrophic marine picocyanobacteria have been challenged in recent years. Based on genomic annotations, picocyanobacteria are predicted to assimilate organic nutrients via ATP-binding cassette importers, a process mediated by substrate-binding proteins. We report the functional characterisation of a modified sugar-binding protein, MsBP, from a marine Synechococcus strain, MITS9220. Ligand screening of MsBP shows a specific affinity for zinc (KD ~ 1.3 µM) and a preference for phosphate-modified sugars, such as fructose-1,6-biphosphate, in the presence of zinc (KD ~ 5.8 µM). Our crystal structures of apo MsBP (no zinc or substrate-bound) and Zn-MsBP (with zinc-bound) show that the presence of zinc induces structural differences, leading to a partially-closed substrate-binding cavity. The Zn-MsBP structure also sequesters several sulphate ions from the crystallisation condition, including two in the binding cleft, appropriately placed to mimic the orientation of adducts of a biphosphate hexose. Combined with a previously unseen positively charged binding cleft in our two structures and our binding affinity data, these observations highlight novel molecular variations on the sugar-binding SBP scaffold. Our findings lend further evidence to a proposed sugar acquisition mechanism in picocyanobacteria alluding to a mixotrophic strategy within these ubiquitous photosynthetic bacteria.

Epidemiology of SARS-CoV-2 infection among staff and students in a cohort of English primary and secondary schools during 2020-2021.

Background: There remains uncertainty about the epidemiology of SARS-CoV-2 among school students and staff and the extent to which non-pharmaceutical-interventions reduce the risk of school settings. Methods: We conducted an open cohort study in a sample of 59 primary and 97 secondary schools in 15 English local authority areas that were implementing government guidance to schools open during the pandemic. We estimated SARS-CoV-2 infection prevalence among those attending school, antibody prevalence, and antibody negative to positive conversion rates in staff and students over the school year (November 2020-July 2021). Findings: 22,585 staff and students participated. SARS-CoV-2 infection prevalence among those attending school was highest during the first two rounds of testing in the autumn term, ranging from 0.7% (95% CI 0.2, 1.2) among primary staff in November 2020 to 1.6% (95% CI 0.9, 2.3) among secondary staff in December 2020. Antibody conversion rates were highest in the autumn term. Infection patterns were similar between staff and students, and between primary and secondary schools. The prevalence of nucleoprotein antibodies increased over the year and was lower among students than staff. SARS-CoV-2 infection prevalence in the North-West region was lower among secondary students attending school on normal school days than the regional estimate for secondary school-age children. Interpretation: SARS-CoV-2 infection prevalence in staff and students attending school varied with local community infection rates. Non-pharmaceutical interventions intended to prevent infected individuals attending school may have partially reduced the prevalence of infection among those on the school site. Funding: UK Department of Health and Social Care.

Battling the known unknowns: a synoptic review of aquatic plastics research from Australia, the United Kingdom and China.

Plastic pollution is a global environmental and human health issue, with plastics now ubiquitous in the environment and biota. Despite extensive international research, key knowledge gaps ("known unknowns") remain around ecosystem-scale and human health impacts of plastics in the environment, particularly in limnetic, coastal and marine systems. Here we review aquatic plastics research in three contrasting geographic and cultural settings, selected to present a gradient of heavily urbanised (and high population density) to less urbanised (and low population density) areas: China, the United Kingdom (UK), and Australia. Research from each country has varying environmental focus (for example, biota-focussed studies in Australia target various bird, fish, turtle and seal species, while UK and China-based studies focus on commercially important organisms such as bivalves, fish and decapods), and uses varying methods and reporting units (e.g. mean, median or range). This has resulted in aquatic plastics datasets that are hard to compare directly, supporting the need to converge on standardised sampling methods, and bioindicator species. While all the study nations show plastics contamination, often at high levels, datasets are variable and do not clearly demonstrate pollution gradients.

Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for anti-cancer drug development.

AIMS: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development. MAIN METHODS: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively. KEY FINDINGS: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [3H]WIN-55,212-2, but not CB1/CB2R radioligand [3H]CP-55,940. Homologous receptor binding demonstrated that [3H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [3H]WIN-55,212-2, whereas two ligands enhanced [3H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [3H]WIN-55,212-2 displacement exhibited high nanomolar affinity (Ki) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity. SIGNIFICANCE: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS.