Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Effectiveness of knowledge brokering and recommendation dissemination for influencing healthcare resource allocation decisions: A cluster randomised controlled implementation trial

BackgroundImplementing evidence into clinical practice is a key focus of healthcare improvements to reduce unwarranted variation. Dissemination of evidence-based recommendations and knowledge brokering have emerged as potential strategies to achieve evidence implementation by influencing resource allocation decisions. The aim of this study was to determine the effectiveness of these two research implementation strategies to facilitate evidence-informed healthcare management decisions for the provision of inpatient weekend allied health services.Methods and findingsThis multicentre, single-blinded (data collection and analysis), three-group parallel cluster randomised controlled trial with concealed allocation was conducted in Australian and New Zealand hospitals between February 2018 and January 2020. Clustering and randomisation took place at the organisation level where weekend allied health staffing decisions were made (e.g., network of hospitals or single hospital). Hospital wards were nested within these decision-making structures. Three conditions were compared over a 12-month period: (1) usual practice waitlist control; (2) dissemination of written evidence-based practice recommendations; and (3) access to a webinar-based knowledge broker in addition to the recommendations. The primary outcome was the alignment of weekend allied health provision with practice recommendations at the cluster and ward levels, addressing the adoption, penetration, and fidelity to the recommendations. The secondary outcome was mean hospital length of stay at the ward level. Outcomes were collected at baseline and 12 months later. A total of 45 clusters (n = 833 wards) were randomised to either control (n = 15), recommendation (n = 16), or knowledge broker (n = 14) conditions. Four (9%) did not provide follow-up data, and no adverse events were recorded. No significant effect was found with either implementation strategy for the primary outcome at the cluster level (recommendation versus control β 18.11 [95% CI −8,721.81 to 8,758.02] p = 0.997; knowledge broker versus control β 1.24 [95% CI −6,992.60 to 6,995.07] p = 1.000; recommendation versus knowledge broker β −9.12 [95% CI −3,878.39 to 3,860.16] p = 0.996) or ward level (recommendation versus control β 0.01 [95% CI 0.74 to 0.75] p = 0.983; knowledge broker versus control β −0.12 [95% CI −0.54 to 0.30] p = 0.581; recommendation versus knowledge broker β −0.19 [−1.04 to 0.65] p = 0.651). There was no significant effect between strategies for the secondary outcome at ward level (recommendation versus control β 2.19 [95% CI −1.36 to 5.74] p = 0.219; knowledge broker versus control β −0.55 [95% CI −1.16 to 0.06] p = 0.075; recommendation versus knowledge broker β −3.75 [95% CI −8.33 to 0.82] p = 0.102). None of the control or knowledge broker clusters transitioned to partial or full alignment with the recommendations. Three (20%) of the clusters who only received the written recommendations transitioned from nonalignment to partial alignment. Limitations include underpowering at the cluster level sample due to the grouping of multiple geographically distinct hospitals to avoid contamination.ConclusionsOwing to a lack of power at the cluster level, this trial was unable to identify a difference between the knowledge broker strategy and dissemination of recommendations compared with usual practice for the promotion of evidence-informed resource allocation to inpatient weekend allied health services. Future research is needed to determine the interactions between different implementation strategies and healthcare contexts when translating evidence into healthcare practice.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12618000029291.

In a cluster randomized controlled implementation trial, Dr. Mitchell N Sarkies and colleagues examine the effectiveness of knowledge brokering and recommendation dissemination in influencing healthcare resource allocation decisions in Australia and New Zealand.     

Hillslope Nutrient Dynamics Following Upland Riparian Vegetation Disturbance

We investigated the effects of removing near-stream Rhododendron and of the natural blowdown of canopy trees on nutrient export to streams in the southern Appalachians. Transects were instrumented on adjacent hillslopes in a first-order watershed at the Coweeta Hydrologic Laboratory (35°03′N, 83°25′W). Dissolved organic carbon (DOC), K⁺, Na⁺, Ca²⁺, Mg²⁺, NO₃ ⁻-N, NH₄ ⁺-N, PO₄ ³⁻-P, and SO₄ ²⁻ were measured for 2 years prior to disturbance. In August 1995, riparian Rhododendron on one hillslope was cut, removing 30% of total woody biomass. In October 1995, Hurricane Opal uprooted nine canopy trees on the other hillslope, downing 81% of the total woody biomass. Over the 3 years following the disturbance, soilwater concentrations of NO₃ ⁻-N tripled on the cut hillslope. There were also small changes in soilwater DOC, SO₄ ²⁻, Ca²⁺, and Mg²⁺. However, no significant changes occurred in groundwater nutrient concentrations following Rhododendron removal. In contrast, soilwater NO₃ ⁻-N on the storm-affected hillslope showed persistent 500-fold increases, groundwater NO₃ ⁻-N increased four fold, and streamwater NO₃ ⁻-N doubled. Significant changes also occurred in soilwater pH, DOC, SO₄ ²⁻, Ca²⁺, and Mg²⁺. There were no significant changes in microbial immobilization of soil nutrients or water outflow on the storm-affected hillslope. Our results suggest that Rhododendron thickets play a relatively minor role in controlling nutrient export to headwater streams. They further suggest that nutrient uptake by canopy trees is a key control on NO₃ ⁻-N export in upland riparian zones, and that disruption of the root–soil connection in canopy trees via uprooting promotes significant nutrient loss to streams. Received 30 January 2001; accepted 25 July 2002.     

Characterization and evolution of two bacteriome‐inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini)

Like other plant sap‐sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with ‘Candidatus Sulcia muelleri’ was detected in PCR assays using primers specific for the Bacteroidetes. Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola, fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that ‘Ca. Sulcia muelleri’ and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. ‘Candidatus Purcelliella pentastirinorum’ was proposed as the novel gammaproteobacterial symbiont.     

Sexual Orientation Disparities in Weight Status in Adolescence: Findings From a Prospective Study

A growing number of studies among adult women have documented disparities in overweight adversely affecting lesbian and bisexual women, but few studies have examined sexual orientation–related patterns in weight status among men or adolescents. We examined sexual orientation group trends in BMI (kg/m²), BMI Z‐scores, and overweight using 56,990 observations from 13,785 adolescent females and males in the Growing Up Today Study (GUTS), a large prospective cohort of US youth. Participants provided self‐reported information from six waves of questionnaire data collection from 1998 to 2005. Gender‐stratified linear regression models were used to estimate BMI and BMI Z‐scores and modified Poisson regression models to estimate risk ratios for overweight, controlling for age and race/ethnicity, with heterosexuals as the referent group. Among females, we observed fairly consistently elevated BMI in all sexual orientation minority groups relative to heterosexual peers. In contrast, among males we documented a sexual‐orientation‐by‐age interaction indicating steeper increases in BMI with age from early‐to‐late adolescence in heterosexuals relative to sexual orientation minorities. Additional prospective research is needed to understand the determinants of observed sexual orientation disparities and to inform appropriate preventive and treatment interventions. The long‐term health consequences of overweight are well‐documented and over time are likely to exact a high toll on populations with elevated rates.     

Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide,BMAP-18

Background

Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.

Methodology/Principal Findings

BMAP-18 showed strong growth inhibitory activity against several species and life cycle stages of African trypanosomes, fish trypanosomes and Leishmania parasites in vitro. When compared to native BMAP-27, the truncated BMAP-18 peptide showed reduced cytotoxicity on a wide variety of mammalian and insect cells and on Sodalis glossindius, a bacterial symbiont of the tsetse vector. The fluorescent stain rhodamine 123 was used in immunofluorescence microscopy and flow cytometry experiments to show that BMAP-18 at low concentrations rapidly disrupted mitochondrial potential without obvious alteration of parasite plasma membranes, thus inducing death by apoptosis. Scanning electron microscopy revealed that higher concentrations of BMAP-18 induced membrane lesions in the parasites as early as 15 minutes after exposure, thus killing them by necrosis. In addition to direct killing of parasites, BMAP-18 was shown to inhibit LPS-induced secretion of tumour necrosis factor alpha (TNF-α), a cytokine that is associated with inflammation and cachexia (wasting) in sleeping sickness patients. As a prelude to in vivo applications, high affinity antibodies to BMAP-18 were produced in rabbits and used in immuno-mass spectrometry assays to detect the intact peptide in human blood and plasma.

Conclusions/Significance

BMAP-18, a truncated form of the potent antimicrobial BMAP-27, showed low toxicity to mammalian cells, insect cells and the tsetse bacterial symbiont Sodalis glossinidius while retaining an ability to kill a variety of species and life cycle stages of pathogenic kinetoplastid parasites in vitro. BMAP-18 also inhibited secretion of TNF-α, an inflammatory cytokine that plays a role in the cachexia associated with African sleeping sickness. These findings support the idea that BMAP-18 should be explored as a candidate for therapy of economically important trypanosome-infected hosts, such as cattle, fish and humans, and for paratransgenic expression in Sodalis glossinidius, a bacterial symbiont in the tsetse vector, as a strategy for interference with trypanosome transmission.     

Expanding horizons for herbaceous ecosystem restoration: the Grassy Groundcover Restoration Project

Summary The Grassy Groundcover Restoration Project (GGRP) has sown thirteen 1 ha plots of species‐rich grassland or herbaceous understorey in previously weedy agricultural paddocks in a range of rural locations across southern and western Victoria, Australia. The sown plots are intended as both experimental trials and ‘core’ areas for the restoration of herbaceous communities native to these regions. Approximately 200 species were grown in Seed Production Areas (SPAs) and successfully sown in the field. Species were most successfully established on areas that were scalped prior to seeding, and least successful on plots that were pre‐treated with 1, 2 or 3 years of traditional herbicide weed control. Weed presence was lowest in scalped plots and highest in non‐scalped plots. Long‐term monitoring will be required to understand the development trajectories and degree of persistence of the sown communities, but in the shorter term (3–6 years of post‐seeding) an average of 80% of sown species have established and remain as adult populations. Surveys indicate that in scalped plots (n = 130) vegetation composition, structure and quality has been maintained. Conversely, composition, structure and quality have declined markedly in non‐scalped plots (n = 130). Formal surveys and field observations have also revealed that all sites provide a range of habitats which have been colonized by fauna from a variety of trophic levels. The implications of building on these trials to realize complex grassy ecosystem restoration at larger scales are discussed including the securing of sufficient quantities of high‐quality seed, the use of mechanized broad‐scale direct‐seeding techniques and the effectiveness of using complex mixtures of species early in the restoration cycle.