Clavulanic Acid Decreases Cocaine Cue Reactivity in Addiction-Related Brain Areas, a Randomized fMRI Pilot Study.

Background: Preclinical studies show that clavulanic acid (CLAV) inhibits cocaine self-administration. This study investigates the effect of CLAV on regions of brain activation in response to cocaine cues during functional magnetic resonance imaging (fMRI) in participants with cocaine use disorder (CUD). Methods: A double-masked, placebo-controlled clinical trial with thirteen individuals with severe CUD who were randomized to treatment with CLAV (N = 10, 9 completers) 500 mg/day or matched placebo (PBO) (N = 3) for 3 days. fMRI was used to assess brain reactivity to 18 alternating six-second video clips of cocaine or neutral scenes. In this paradigm, participants were exposed to three different stimulus conditions: NEUTRAL, WATCH (passive watching), and DOWN (actively inhibiting craving while watching). Results: Participants who received CLAV demonstrated a significant reduction in brain activity in the anterior cingulate gyrus (p = 0.009) and the caudate (p = 0.018) in response to DOWN cocaine cues. There was a trend toward lessened cue reactivity in other regions implicated in CUD. Conclusion: CLAV reduced the response of the brain regions associated with motivation and emotional response during the DOWN condition compared to PBO, suggesting CLAV may strengthen voluntary efforts to avoid cocaine use. This pilot data supports the use of CLAV for CUD. (Trial registered in ClinicalTrials.gov NCT04411914).

Title: Obesogenic microbial signatures and the development of obesity in childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) is the most common childhood cancer with survival exceeding 90% for standard-risk groups. A debilitating side-effect of treatment is the development of overweight/obesity (OW/OB), which develops in approximately 40% of children by the end of treatment. The microbiome has been associated with the development of OW/OB. We examined fluctuations in the microbiome with the development of OW/OB during the first six months of treatment at diagnosis, and two subsequent timepoints (N = 62). Shotgun metagenomic sequencing was performed on Illumina Nextseq system, and taxa and functional pathways were extracted from sequences using kraken2 and humann2, respectively. An association of increased presence of several species (e.g., Klebsiella pneumoniae, Escherichia coli) was observed in children with OW/OB, while lean-promoting species (Veillonella, Haemophilus, and Akkermansia) were increased in children who maintained a normal weight. Pathway analysis revealed purine nucleotide biosynthesis, sugar nucleotide biosynthesis, and enzyme cofactor biosynthesis were positively correlated with Bacteroides spp. among children with OW/OB. We identified several taxa and functional pathways that may confer increased risk for the development of OW/OB. The associations observed in this pilot are preliminary and warrant further research in the microbiome and the development of OW/OB in childhood ALL.

Management and outcome of burn injuries during pregnancy: A systematic review and presentation of a comprehensive guideline.

Pregnant women are a vulnerable subgroup of burn patients, due to altered physiological state and possible adverse effect on the fetus. The aim of this study was to develop a guideline for a tailored treatment of pregnant patients with burns to optimally treat both mother and fetus. This study consists of two parts: the first part contains a systematic review that presents a comprehensive overview of the literature on the management and outcome of pregnant women who sustain severe burn injuries and based on the results of this review, a guideline on the general, obstetric and burn management was developed and presented in the second part. A total of 35 studies including 1395 patients were included. Although the clinical outcomes seemed to be similar to non-pregnant patients, one study showed that mortality might be higher in the pregnant population. Predictive factors for maternal and fetal mortality were a total burned surface area of over 40% and inhalation injury. Early surgery may lead to a higher chance of survival of mother and fetus. A comprehensive guideline on the general management of pregnant patients with burns, obstetric management and specific burn management is provided. We encourage international burn organizations and guideline committees to use and evaluate the presented guideline.

Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone.

OBJECTIVE: The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. DESIGN: Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%). RESULTS: We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles. CONCLUSION: We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.

Viva la VOSCE?

BACKGROUND: The COVID-19 pandemic lockdown precluded face-to-face final Objective Structured Clinical Examinations (OSCE) in the UK. RESULTS: In response, we rapidly developed and then successfully implemented a novel Virtual Objective Structured Clinical Examination (VOSCE). CONCLUSIONS: In this article we both describe and reflect on our experience as well as discuss the implications for future undergraduate assessment as the situation evolves.

Epidemiological risk factors for clinical malaria infection in the highlands of Western Kenya.

BACKGROUND: Understanding the complex heterogeneity of risk factors that can contribute to an increased risk of malaria at the individual and household level will enable more effective use of control measures. The objective of this study was to understand individual and household factors that influence clinical malaria infection among individuals in the highlands of Western Kenya. METHODS: This was a matched case-control study undertaken in the Western Kenya highlands. Clinical malaria cases were recruited from health facilities and matched to asymptomatic individuals from the community who served as controls. Each participant was screened for malaria using microscopy. Follow-up surveys were conducted with individual households to collect socio-economic data. The houses were also checked using pyrethrum spray catches to collect mosquitoes. RESULTS: A total of 302 malaria cases were matched to 604 controls during the surveillance period. Mosquito densities were similar in the houses of both groups. A greater percentage of people in the control group (64.6%) used insecticide-treated bed nets (ITNs) compared to the families of malaria cases (48.3%). Use of ITNs was associated with lower level of clinical malaria episodes (odds ratio 0.51; 95% CI 0.39-0.68; P < 0.0001). Low income was the most important factor associated with higher malaria infections (adj. OR 4.70). Use of malaria prophylaxis was the most important factor associated with less malaria infections (adj OR 0.36). Mother's (not fathers) employment status (adj OR 0.48) and education level (adj OR 0.54) was important malaria risk factor. Houses with open eaves was an important malaria risk factor (adj OR 1.72). CONCLUSION: The identification of risk factors for clinical malaria infection provides information on the local malaria epidemiology and has the potential to lead to a more effective and targeted use of malaria control measures. These risk factors could be used to assess why some individuals acquire clinical malaria whilst others do not and to inform how intervention could be scaled at the local level.

Extraskeletal Calcifications in Hutchinson-Gilford Progeria Syndrome.

PURPOSE: Children with Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disease, exhibit extraskeletal calcifications detected by radiographic analysis and on physical examination. The aim of this study was to describe the natural history and pathophysiology of these abnormal calcifications in HGPS, and to determine whether medications and/or supplements tested in clinical trials alter their development. METHODS: Children from two successive clinical trials administering 1) lonafarnib (n = 26) and 2) lonafarnib + pravastatin + zoledronic acid (n = 37) were studied at baseline (pre-therapy), one year on therapy, and at end-of-therapy (3.3-4.3 years after the baseline visit). Calcium supplementation (oral calcium carbonate) was administered during the first year of the second trial and was subsequently discontinued. Information on calcifications was obtained from physical examinations, radiographs, and serum and urinary biochemical measures. The mineral content of two skin-derived calcifications was determined by x-ray diffraction. RESULTS: Extraskeletal calcifications were detected radiographically in 12/39 (31%) patients at baseline. The odds of exhibiting calcifications increased with age (p = 0.045). The odds were unaffected by receipt of lonafarnib, pravastatin, and zoledronate therapies. However, administration of calcium carbonate supplementation, in conjunction with all three therapeutic agents, significantly increased the odds of developing calcifications (p = 0.009), with the odds plateauing after the supplement's discontinuation. Composition analysis of calcinosis cutis showed hydroxyapatite similar to bone. Although serum calcium, phosphorus, and parathyroid hormone (PTH) were within normal limits at baseline and on-therapy, PTH increased significantly after lonafarnib initiation (p < 0.001). Both the urinary calcium/creatinine ratio and tubular reabsorption of phosphate (TRP) were elevated at baseline in 22/39 (56%) and 31/37 (84%) evaluable patients, respectively, with no significant changes while on-therapy. The mean calcium × phosphorus product (Ca × Pi) was within normal limits, but plasma magnesium decreased over both clinical trials. Fibroblast growth factor 23 (FGF23) was lower compared to age-matched controls (p = 0.03). CONCLUSIONS: Extraskeletal calcifications increased with age in children with HGPS and were composed of hydroxyapatite. The urinary calcium/creatinine ratio and TRP were elevated for age while FGF23 was decreased. Magnesium decreased and PTH increased after lonafarnib therapy which may alter the ability to mobilize calcium. These findings demonstrate that children with HGPS with normal renal function and an unremarkable Ca × Pi develop extraskeletal calcifications by an unidentified mechanism that may involve decreased plasma magnesium and FGF23. Calcium carbonate accelerated their development and is, therefore, not recommended for routine supplementation in these children.

Proposed standard model and consistent terminology for monitoring and outcome evaluation in different dietetic care settings: Results from the EU-sponsored IMPECD project.

BACKGROUND & AIMS: Dietetic interventions contribute to certain health objectives and other outcomes, but are mostly part of a multimodal and multidisciplinary approach what makes evaluating the actual effects of dietitians' involvement rather complex. Although monitoring and outcome evaluation (M&OE) can provide routine data to prove the effectiveness of dietetic interventions, this has not been established yet in different dietetic settings. METHODS: A comprehensive framework for M&OE in dietetics was developed by dietetic experts from five European higher education institutes for dietetics in the course of the EU sponsored project "Improvement of Education and Competences in Dietetics (IMPECD)". RESULTS: Firstly, clear definitions on M&OE are proposed to facilitate the use of consistent terminology, with a specific emphasis on the term "impact" covering macro-level outcomes such as cost-effectiveness. Secondly, the Dietetic Care Process (DCP) was merged into a logic model to demonstrate the position of M&OE in relation to intervention planning and implementation, in both group and individual settings. Thirdly, selecting the appropriate indicators is indispensable to monitor and evaluate outcomes, and requires a high level of dietitians' critical reasoning. A categorized overview of indicators is provided to support this process. Lastly, the consortium developed a checklist to give dietitians a handle on what elements could be included in their M&OE plan and trigger them to perform M&OE in practice. CONCLUSIONS: Innovative M&OE models may help dietitians to demonstrate their effectiveness in improving clinical outcomes and justify their role in health care.

Structural crossover in a model fluid exhibiting two length scales: Repercussions for quasicrystal formation.

We investigate the liquid state structure of the two-dimensional model introduced by Barkan et al. [Phys. Rev. Lett. 113, 098304 (2014)10.1103/PhysRevLett.113.098304], which exhibits quasicrystalline and other unusual solid phases, focusing on the radial distribution function g(r) and its asymptotic decay r→∞. For this particular model system, we find that as the density is increased there is a structural crossover from damped oscillatory asymptotic decay with one wavelength to damped oscillatory asymptotic decay with another distinct wavelength. The ratio of these wavelengths is ≈1.932. Following the locus in the phase diagram of this structural crossover leads directly to the region where quasicrystals are found. We argue that identifying and following such a crossover line in the phase diagram towards higher densities where the solid phase(s) occur is a good strategy for finding quasicrystals in a wide variety of systems. We also show how the pole analysis of the asymptotic decay of equilibrium fluid correlations is intimately connected with the nonequilibrium growth or decay of small-amplitude density fluctuations in a bulk fluid.

Does the addition of active body warming to in-line intravenous fluid warming prevent maternal hypothermia during elective caesarean section? A randomised controlled trial.

INTRODUCTION: Inadvertent perioperative hypothermia occurs frequently during elective caesarean section but perioperative active body warming is not widely used. There is a paucity of evidence of its use in the obstetric population, and no applicable guidelines. We set out to identify a superior active warming method for preventing inadvertent perioperative hypothermia. METHODS: Following ethical approval, 132 women presenting for uncomplicated elective caesarean section under spinal anaesthesia were recruited. All participants received in-line intravenous fluid warming and were randomised to one of three parallel groups: no active body warming; forced air warming; and conduction mattress warming. The primary outcome was the difference in mean core temperature, measured on admission to the recovery room, between study groups. Core temperature and thermal comfort were measured perioperatively at 15-min intervals. Estimated blood loss, haemoglobin change, length of hospital stay and neonatal core temperature were also recorded. RESULTS: One-hundred-and-thirty-one women completed the study. There was no significant difference in mean core temperature on admission to the recovery room (36.6°C vs. 36.6°C vs. 36.6°C, η2=0.005, P=0.74). Maternal hypothermia was prevented in all groups with only 0.3% hypothermic at any of the temperature measurements (3/1016). There was no difference in mean neonatal core temperature (36.3°C vs. 36.3°C vs. 36.3°C, η2=0.003, P=0.82); however, 59.4% (76/128) of all neonates were hypothermic. CONCLUSION: In-line intravenous fluid warming is sufficient to prevent maternal hypothermia and maintain core temperature. The addition of active body warming conferred no added benefit.

Dose-adapted post-transplant cyclophosphamide for HLA-haploidentical transplantation in Fanconi anemia.

We developed a haploidentical transplantation protocol with post-transplant cyclophosphamide (CY) for in vivo T-cell depletion (TCD) using a novel adapted-dosing schedule (25 mg/kg on days +3 and +4) for Fanconi anemia (FA). With median follow-up of 3 years (range, 37 days to 6.2 years), all six patients engrafted. Two patients with multiple pre-transplant comorbidities died, one from sepsis and one from sepsis with associated chronic GVHD. Four patients without preexisting comorbidities and early transplant referrals are alive with 100% donor chimerism and excellent performance status. We conclude that adjusted-dosing post-transplant CY is effective in in vivo TCD to promote full donor engraftment in patients with FA.

Antenatal Ultrasound Detection of Cleft in Western Australia from 2003 to 2012: A Follow-Up Study.

AIM: To investigate trends in the rate of antenatal detection of cleft lip and palate (CLP) patients referred to the CLP Unit at Princess Margaret Hospital for Children in Perth, Western Australia during the period 2003-2012 and compare data with a previously published report covering the years 1996-2003. METHODS: This is a single-center, retrospective survey of antenatal transabdominal ultrasound screenings of mothers of infants born between July 1, 2003 and June 30, 2012 that were referred to the CLP Unit at Princess Margaret Hospital. RESULTS: Detection rates of oral clefts increased significantly when compared with outcomes reported in the same population between 1996 and 2003 (P < .05). An overall detection rate of 71.7% (165/230) was achieved for clefts involving lip and palate. Detection of isolated cleft palate (1/99) and microform (0/8) remained elusive. Most detections (76.5%) were achieved at 15 to 20 weeks of gestational age, corresponding with routine anatomical screening. A further 16.8% were detected post-20 weeks of gestation. Scans were performed by specialist obstetricians, and sonography clinics reported a detection rate of 84.6% (55/65), whereas nonspecialist clinics reported a detection rate of only 67.1% (110/164). CONCLUSION: The antenatal detection rates of oral clefts involving the lip have improved to the extent that the majority of mothers are now being referred to a cleft unit in Western Australia prior to the births of their children. As a result of this improvement, antenatal counseling is now a common facet of cleft management.

Isospin Mixing Reveals

The thermonuclear ^{30}P(p,γ)^{31}S reaction rate is critical for modeling the final elemental and isotopic abundances of ONe nova nucleosynthesis, which affect the calibration of proposed nova thermometers and the identification of presolar nova grains, respectively. Unfortunately, the rate of this reaction is essentially unconstrained experimentally, because the strengths of key ^{31}S proton capture resonance states are not known, largely due to uncertainties in their spins and parities. Using the ß decay of ^{31}Cl, we have observed the ß-delayed γ decay of a ^{31}S state at E_{x}=6390.2(7) keV, with a ^{30}P(p,γ)^{31}S resonance energy of E_{r}=259.3(8) keV, in the middle of the ^{30}P(p,γ)^{31}S Gamow window for peak nova temperatures. This state exhibits isospin mixing with the nearby isobaric analog state at E_{x}=6279.0(6) keV, giving it an unambiguous spin and parity of 3/2^{+} and making it an important l=0 resonance for proton capture on ^{30}P.

Cohort profile of the UK Biobank: diagnosis and characteristics of cerebrovascular disease.

PURPOSE: The UK Biobank is a large-scale biomedical resource, containing sociodemographic and medical information, including data on a previous diagnosis of stroke or transient ischaemic attack (TIA). We described these participants and their medication usage. PARTICIPANTS: We identified participants who either self-reported or were identified from a nurse-led interview, having suffered a stroke or a TIA and compared them against participants without stroke ort TIA. We assessed their risk factor burden (sex, age, deprivation, waist to hip ratio (WHR), hypertension, smoking, alcohol intake, diabetes, physical exercise and oral contraception use (oral contraceptive pill, OCP)) and medication usage. FINDINGS TO DATE: We studied 502,650 people (54.41% women), 6669 (1.23%) participants self-reported a stroke. The nurse-led interview identified 7669 (1.53%) people with stroke and 1781 (0.35%) with TIA. Hypertension, smoking, higher WHR, lower alcohol consumption and diabetes were all more common in people with cerebrovascular disease (p<0.0001 for each). Women with cerebrovascular disease were less likely to have taken the OCP (p=0.0002). People with cerebrovascular disease did more exercise (p=0.03). Antithrombotic medication was taken by 81% of people with stroke (both self-report and nurse-led responders) and 89% with TIA. For self-reported stroke, 63% were taking antithrombotic and cholesterol medications, 54% taking antithrombotic and antihypertensive medications and 46% taking all 3. For the nurse-led interview and TIA, these figures were 65%, 54% and 46%, and 70%, 53% and 45%, respectively. FUTURE PLANS: The UK Biobank provides a large, generalisable and contemporary data source in a young population. The characterisation of the UK Biobank cohort with cerebrovascular disease will form the basis for ongoing research using this data source.

Solidification in soft-core fluids: Disordered solids from fast solidification fronts.

Using dynamical density functional theory we calculate the speed of solidification fronts advancing into a quenched two-dimensional model fluid of soft-core particles. We find that solidification fronts can advance via two different mechanisms, depending on the depth of the quench. For shallow quenches, the front propagation is via a nonlinear mechanism. For deep quenches, front propagation is governed by a linear mechanism and in this regime we are able to determine the front speed via a marginal stability analysis. We find that the density modulations generated behind the advancing front have a characteristic scale that differs from the wavelength of the density modulation in thermodynamic equilibrium, i.e., the spacing between the crystal planes in an equilibrium crystal. This leads to the subsequent development of disorder in the solids that are formed. In a one-component fluid, the particles are able to rearrange to form a well-ordered crystal, with few defects. However, solidification fronts in a binary mixture exhibiting crystalline phases with square and hexagonal ordering generate solids that are unable to rearrange after the passage of the solidification front and a significant amount of disorder remains in the system.

Lower incidence of post-dural puncture headache with spinal catheterization after accidental dural puncture in obstetric patients.

BACKGROUND: Accidental dural puncture (ADP) and post-dural puncture headache (PDPH) are important complications of obstetric regional anesthesia. Inserting the catheter intrathecally after ADP to prevent PDPH has gained popularity. Nonetheless, data on the effect of an intrathecal catheter on PDPH and epidural blood patch (EBP) rates are mixed. Our primary objective was to examine if spinal catheterization reduces the incidence of PDPH after ADP in obstetric patients. METHODS: Anesthetic records of 29,749 regional blocks performed between January 1997 and July 2013 were analyzed retrospectively. In all blocks containing an epidural component, 18-gauge epidural needles were used. All patients who experienced a witnessed ADP or PDPH without ADP were identified. Data from patients with or without a prolonged spinal catheter were compared. RESULTS: There were 128 events of witnessed ADP (0.43%). Following known ADP, 39 women had an epidural catheter placed at a different level and 89 had an intrathecal catheter (20-gauge) for at least 24 h. Sixty-one patients developed PDPH after observed ADP (48%). Prolonged intrathecal catheter placement significantly reduced the incidence of PDPH after ADP to 42% compared with 62% in those who have the catheter re-sited epidurally [odds ratio = 2.3 (95% confidence interval 1.04-4.86); P = 0.04]. CONCLUSIONS: The incidence of ADP, PDPH and blood patching is similar with previously published studies. After witnessed ADP, inserting the epidural catheter intrathecally significantly reduced the incidence of PDPH.

Allopurinol reduces brachial and central blood pressure, and carotid intima-media thickness progression after ischaemic stroke and transient ischaemic attack: a randomised controlled trial.

OBJECTIVE: Central blood pressure (CBP) and carotid intima-media thickness (CIMT) are surrogate measures of cardiovascular risk. Allopurinol reduces serum uric acid and oxidative stress and improves endothelial function and may therefore reduce CBP and CIMT progression. This study sought to ascertain whether allopurinol reduces CBP, arterial stiffness and CIMT progression in patients with ischaemic stroke or transient ischaemic attack (TIA). METHODS: We performed a randomised, double-blind, placebo-controlled study, examining the effect of 1-year treatment with allopurinol (300 mg daily), on change in CBP, arterial stiffness and CIMT progression at 1 year and change in endothelial function and circulating inflammatory markers at 6 months. Patients aged over 18 years with recent ischaemic stroke or TIA were eligible. RESULTS: Eighty participants were recruited, mean age 67.8 years (SD 9.4). Systolic CBP [-6.6 mm Hg (95% CI -13.0 to -0.3), p=0.042] and augmentation index [-4.4% (95% CI -7.9 to -1.0), p=0.013] were each lower following allopurinol treatment compared with placebo at 12 months. Progression in mean common CIMT at 1 year was less in allopurinol-treated patients compared with placebo [between-group difference [-0.097 mm (95% CI -0.175 to -0.019), p=0.015]. No difference was observed for measures of endothelial function. CONCLUSIONS: Allopurinol lowered CBP and reduced CIMT progression at 1 year compared with placebo in patients with recent ischaemic stroke and TIA. This extends the evidence of sustained beneficial effects of allopurinol to these prognostically significant outcomes and to the stroke population, highlighting the potential for reduction in cardiovascular events with this treatment strategy. TRIAL REGISTRATION NUMBER: ISRCTN11970568.

Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats.

BACKGROUND: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed. METHODS: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. RESULTS: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs. CONCLUSIONS: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures.

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.