Impact of COVID-19 pandemic restrictions on pregnancy duration and outcome in Melbourne, Australia.

OBJECTIVE: To investigate the effect of restriction measures implemented to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during the coronavirus disease 2019 (COVID-19) pandemic on pregnancy duration and outcome. METHODS: A before-and-after study was conducted with cohort sampling in three maternity hospitals in Melbourne, Australia, including women who were pregnant when restriction measures were in place during the COVID-19 pandemic (estimated conception date between 1 November 2019 and 29 February 2020) and women who were pregnant before the restrictions (estimated conception date between 1 November 2018 and 28 February 2019). The primary outcome was delivery before 34 weeks' gestation or stillbirth. The main secondary outcome was a composite of adverse perinatal outcomes. Pregnancy outcomes were compared between women exposed to restriction measures and unexposed controls using the χ-square test and modified Poisson regression models, and duration of pregnancy was compared between the groups using survival analysis. RESULTS: In total, 3150 women who were exposed to restriction measures during pregnancy and 3175 unexposed controls were included. Preterm birth before 34 weeks or stillbirth occurred in 95 (3.0%) exposed pregnancies and in 130 (4.1%) controls (risk ratio (RR), 0.74 (95% CI, 0.57-0.96); P = 0.021). Preterm birth before 34 weeks occurred in 2.4% of women in the exposed group and in 3.4% of women in the control group (RR, 0.71 (95% CI, 0.53-0.95); P = 0.022), without evidence of an increase in the rate of stillbirth in the exposed group (0.7% vs 0.9%; RR, 0.83 (95% CI, 0.48-1.44); P = 0.515). Competing-risks regression analysis showed that the effect of the restriction measures on spontaneous preterm birth was stronger and started earlier (subdistribution hazard ratio (HR), 0.81 (95% CI, 0.64-1.03); P = 0.087) than the effect on medically indicated preterm birth (subdistribution HR, 0.89 (95% CI, 0.70-1.12); P = 0.305). The effect was stronger in women with a previous preterm birth (RR, 0.42 (95% CI, 0.21-0.82); P = 0.008) than in parous women without a previous preterm birth (RR, 0.93 (95% CI, 0.63-1.38); P = 0.714) (P for interaction = 0.044). Composite adverse perinatal outcome was less frequent in the exposed group than in controls (all women: 2.1% vs 2.9%; RR, 0.73 (95% CI, 0.54-0.99); P = 0.042); women with a previous preterm birth: 4.5% vs 8.4%; RR, 0.54 (95% CI, 0.25-1.18); P = 0.116). CONCLUSIONS: Restriction measures implemented to mitigate SARS-CoV-2 transmission during the COVID-19 pandemic were associated with a reduced rate of preterm birth before 34 weeks. This reduction was mainly due to a lower rate of spontaneous prematurity. The effect was more substantial in women with a previous preterm birth and was not associated with an increased stillbirth rate. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Placental gas exchange during amniotic carbon dioxide insufflation in sheep.

OBJECTIVE: Insufflation of the amniotic cavity with carbon dioxide (CO2 ) is used clinically to improve visibility during complex fetoscopic surgery. Insufflation with heated, humidified CO2 has recently been shown to reduce fetal hypercapnia and acidosis in sheep, compared with use of cold and dry CO2 , but the underlying mechanisms are unclear. The aim of this study was to investigate whether differences in placental CO2 and oxygen (O2 ) exchange during insufflation with heated and humidified vs cold and dry CO2 could explain these findings. METHODS: Thirteen fetal lambs at 105 days of gestation (term, 146 days) were exteriorized partially, via a midline laparotomy and hysterotomy, and instrumented with an umbilical artery catheter, an umbilical vein catheter and a common umbilical vein flow probe. Arterial and venous catheters and flow probes were also inserted into the maternal uterine circulation. Six ewes were insufflated with cold, dry CO2 (22°C; 0-5% humidity) and seven with heated, humidified CO2 (40°C; 95-100% humidity) at 15 mmHg for 180 min. Blood-flow recordings and paired arterial and venous blood gases were sampled from uterine and umbilical vessels. Rates of placental CO2 and O2 exchange were calculated. RESULTS: After 180 min of insufflation, fetal survival was 33% (2/6) using cold, dry CO2 and 71% (5/7) using heated, humidified CO2 . By 120 min, fetuses insufflated with heated, humidified CO2 had lower arterial CO2 levels and higher arterial pH compared to those insufflated with cold, dry gas. Insufflation decreased significantly placental gas exchange in both groups, as measured by rates of both (i) fetal CO2 clearance and O2 uptake and (ii) maternal O2 delivery and CO2 uptake from the fetal compartment. CONCLUSIONS: Lower arterial CO2 and higher pH levels in fetuses insufflated with heated and humidified, compared to cold and dry, CO2 could not be explained by differences in placental gas exchange. Instead, heated and humidified insufflation appeared to reduce fetal CO2 absorption from the uterus, supporting its use in preference to cold, dry CO2 . © 2019 International Society of Ultrasound in Obstetrics and Gynecology.

Antenatal sildenafil treatment improves neonatal pulmonary hemodynamics and gas exchange in lambs with diaphragmatic hernia.

OBJECTIVES: Infants with congenital diaphragmatic hernia (CDH) are predisposed to pulmonary hypertension after birth, owing to lung hypoplasia that impairs fetal pulmonary vascular development. Antenatal sildenafil treatment attenuates abnormal pulmonary vascular and alveolar development in rabbit and rodent CDH models, but whether this translates to functional improvements after birth remains unknown. We aimed to evaluate the effect of antenatal sildenafil on neonatal pulmonary hemodynamics and lung function in lambs with diaphragmatic hernia (DH). METHODS: DH was surgically induced at approximately 80 days' gestation in 16 lamb fetuses (term in lambs is approximately 147 days). From 105 days' gestation, ewes received either sildenafil (0.21 mg/kg/h intravenously) or saline infusion until delivery (n = 8 fetuses in each group). At approximately 138 days' gestation, all lambs were instrumented and then delivered via Cesarean section. The lambs were ventilated for 120 min with continuous recording of physiological (pulmonary and carotid artery blood flow and pressure; cerebral oxygenation) and ventilatory parameters, and regular assessment of arterial blood gas tensions. Only lambs that survived until delivery and with a confirmed diaphragmatic defect at postmortem examination were included in the analysis; these comprised six DH-sildenafil lambs and six DH-saline control lambs. RESULTS: Lung-to-body-weight ratio (0.016 ± 0.001 vs 0.013 ± 0.001; P = 0.06) and dynamic lung compliance (0.8 ± 0.2 vs 0.7 ± 0.2 mL/cmH2 O; P = 0.72) were similar in DH-sildenafil lambs and controls. Pulmonary vascular resistance decreased following lung aeration to a greater degree in DH-sildenafil lambs, and was 4-fold lower by 120 min after cord clamping than in controls (0.6 ± 0.1 vs 2.2 ± 0.6 mmHg/(mL/min); P = 0.002). Pulmonary arterial pressure was also lower (46 ± 2 vs 59 ± 2 mmHg; P = 0.048) and pulmonary blood flow higher (25 ± 3 vs 8 ± 2 mL/min/kg; P = 0.02) in DH-sildenafil than in DH-saline lambs at 120 min. Throughout the 120-min ventilation period, the partial pressure of arterial carbon dioxide tended to be lower in DH-sildenafil lambs than in controls (63 ± 8 vs 87 ± 8 mmHg; P = 0.057), and there was no significant difference in partial pressure of arterial oxygen between the two groups. CONCLUSIONS: Sustained maternal antenatal sildenafil infusion reduced pulmonary arterial pressure and increased pulmonary blood flow in DH lambs for the first 120 min after birth. These findings of improved pulmonary vascular function are consistent with improved pulmonary vascular structure seen in two previous animal models. The data support the rationale for a clinical trial investigating the effect of antenatal sildenafil in reducing the risk of neonatal pulmonary hypertension in infants with CDH. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Using medico-legal claims for quality improvement in maternity care: application and revision of an NHSLA coding taxonomy.

OBJECTIVE: To validate the NHSLA maternity claims taxonomy at the level of a single maternity service and assess its ability to direct quality improvement. DESIGN: Qualitative descriptive study. SETTING: Medico-legal claims between 1 January 2000 and 31 December 2016 from a maternity service in metropolitan Melbourne, Australia. POPULATION: All obstetric claims and incident notifications occurring within the date range were included for analysis. METHODS: De-identified claims and notifications data were derived from the files of the insurer of Victorian public health services. Data included claim date, incident date and summary, and claim cost. All reported issues were coded using the NHSLA taxonomy and the lead issue identified. MAIN OUTCOME MEASURES: Rate of claims and notifications, relative frequency of issues, a revised taxonomy. RESULTS: A combined total of 265 claims and incidents were reported during the 6 years. Of these 59 were excluded, leaving 198 medico-legal events for analysis (1.66 events/1000 births). The costs for all claims was $46.7 million. The most common claim issues were related to management of labour (n = 63, $17.7 million), cardiotocographic interpretation (n = 43, $24.4 million), and stillbirth (n = 35, $656,750). The original NHSLA classification was not sufficiently detailed to inform care improvement programmes. A revised taxonomy and coding flowchart is presented. CONCLUSIONS: Systematic analysis of obstetric medico-legal claims data can potentially be used to inform quality and safety improvement. TWEETABLE ABSTRACT: New taxonomy to target health improvement from maternity claims based on NHSLA Ten Years of Maternity Claims.

Physiological effects of partial amniotic carbon dioxide insufflation with cold, dry vs heated, humidified gas in a sheep model.

OBJECTIVE: Partial amniotic carbon dioxide (CO2 ) insufflation (PACI) is used to improve visualization and facilitate complex fetoscopic surgery. However, there are concerns about fetal hypercapnic acidosis and postoperative fetal membrane inflammation. We assessed whether using heated and humidified, rather than cold and dry, CO2 might reduce the impact of PACI on the fetus and fetal membranes in sheep. METHODS: Twelve fetal lambs of 105 days' gestational age (term = 145 days) were exteriorized partially, via a midline laparotomy and hysterotomy, and arterial catheters and flow probes were inserted surgically. The 10 surviving fetuses were returned to the uterus, which was then closed and insufflated with cold, dry (22 °C at 0-5% humidity, n = 5) or heated, humidified (40 °C at 100% humidity, n = 5) CO2 at 15 mmHg for 180 min. Fetal membranes were collected immediately after insufflation for histological analysis. Physiological data and membrane leukocyte counts, suggestive of membrane inflammation, were compared between the two groups. RESULTS: After 180 min of insufflation, fetal survival was 0% in the group which underwent PACI with cold, dry CO2 , and 60% (n = 3) in the group which received heated, humidified gas. While all insufflated fetuses became progressively hypercapnic (PaCO2  > 68 mmHg), this was considerably less pronounced in those in which heated, humidified gas was used: after 120 min of insufflation, compared with those receiving cold, dry gas (n = 3), fetuses undergoing heated, humidified PACI (n = 5) had lower arterial partial pressure of CO2 (mean ± standard error of the mean, 82.7 ± 9.1 mmHg for heated, humidified CO2 vs 170.5 ± 28.5 for cold, dry CO2 during PACI, P < 0.01), lower lactate levels (1.4 ± 0.4 vs 8.5 ± 0.9 mmol/L, P < 0.01) and higher pH (pH, 7.10 ± 0.04 vs 6.75 ± 0.04, P < 0.01). There was also a non-significant trend for fetal carotid artery pressure to be higher following PACI with heated, humidified compared with cold, dry CO2 (30.5 ± 1.3 vs 8.7 ± 5.5 mmHg, P = 0.22). Additionally, the median (interquartile range) number of leukocytes in the chorion was significantly lower in the group undergoing PACI with heated, humidified CO2 compared with the group receiving cold, dry CO2 (0.7 × 10-5 (0.5 × 10-5 ) vs 3.2 × 10-5 (1.8 × 10-5 ) cells per square micron, P = 0.02). CONCLUSIONS: PACI with cold, dry CO2 causes hypercapnia, acidosis, hypotension and fetal membrane inflammation in fetal sheep, raising potential concerns for its use in humans. It seems that using heated, humidified CO2 for insufflation partially mitigates these effects and this may be a suitable alternative for reducing the risk of fetal acid-base disturbances during, and fetal membrane inflammation following, complex fetoscopic surgery. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Lipoxin A4 activates ALX/FPR2 receptor to regulate conjunctival goblet cell secretion.

Conjunctival goblet cells play a major role in maintaining the mucus layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis. Resolution of inflammation is mediated by proresolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i), and identify signaling pathways activated by LXA4. ALX/FPR2 (formyl peptide receptor2) was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and extracellular regulated kinase 1/2 (ERK 1/2) activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared with its initial response. LXA4 activated phospholipases C, D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin-dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.

Interaction of IFN-γ with cholinergic agonists to modulate rat and human goblet cell function.

Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein, we determined whether interferon gamma (IFN-γ), a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca(2+)] ([Ca(2+)](i)), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca(2+)](i) in rat and human goblet cells and prevented the increase in [Ca(2+)](i) caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca(2+)](i). This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca(2+)(i) reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca(2+)](i). IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.

Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism.

CONTEXT: Prior studies showed that Axl /Tyro3 null mice have delayed first estrus and abnormal cyclicity due to developmental defects in GnRH neuron migration and survival. OBJECTIVE: The objective of the study was to test whether the absence of Axl would alter reproductive function in mice and that mutations in AXL are present in patients with Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). DESIGN AND SETTING: The sexual maturation of Axl null mice was examined. The coding region of AXL was sequenced in 104 unrelated, carefully phenotyped KS or nIHH subjects. Frequency of mutations was compared with other causes of GnRH deficiency. Functional assays were performed on the detected mutations. RESULTS: Axl null mice demonstrated delay in first estrus and the interval between vaginal opening and first estrus. Three missense AXL mutations (p.L50F, p.S202C, and p.Q361P) and one intronic variant 6 bp upstream from the start of exon 5 (c.586-6 C>T) were identified in two KS and 2 two nIHH subjects. Comparison of the frequencies of AXL mutations with other putative causes of idiopathic hypogonadotropic hypogonadism confirmed they are rare variants. Testing of the c.586-6 C>T mutation revealed no abnormal splicing. Surface plasmon resonance analysis of the p.L50F, p.S202C, and p.Q361P mutations showed no altered Gas6 ligand binding. In contrast, GT1-7 GnRH neuronal cells expressing p.S202C or p.Q361P demonstrated defective ligand dependent receptor processing and importantly aberrant neuronal migration. In addition, the p.Q361P showed defective ligand independent chemotaxis. CONCLUSIONS: Functional consequences of AXL sequence variants in patients with idiopathic hypogonadotropic hypogonadism support the importance of AXL and the Tyro3, Axl, Mer (TAM) family in reproductive development.

Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.

Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca(2+)] ([Ca(2+)]i) preventing H1 receptor-mediated responses. In human and rat conjunctival goblet cells, RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca(2+)]i and activation of extracellular regulated-protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.

Correlations in intermediate energy two-proton removal reactions.

We report final-state-exclusive measurements of the light charged fragments in coincidence with (26)Ne residual nuclei following the direct two-proton removal from a neutron-rich (28)Mg secondary beam. A Dalitz-plot analysis and comparisons with simulations show that a majority of the triple-coincidence events with two protons display phase-space correlations consistent with the (two-body) kinematics of a spatially correlated pair-removal mechanism. The fraction of such correlated events, 56(12)%, is consistent with the fraction of the calculated cross section, 64%, arising from spin S=0 two-proton configurations in the entrance-channel (shell-model) (28)Mg ground state wave function. This result promises access to an additional and more specific probe of the spin and spatial correlations of valence nucleon pairs in exotic nuclei produced as fast secondary beams.

Acoustic indicators for targeted detection of stored product and urban insect pests by inexpensive infrared, acoustic, and vibrational detection of movement.

Crawling and scraping activity of three stored-product pests, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), and Stegobium paniceum (L.) (Coleoptera: Anobiidae), and two urban pests, Blattella germanica (L.) (Blattodea: Blattellidae) and Cimex lectularius L. (Hemiptera: Cimicidae), were monitored individually by infrared sensors, microphones, and a piezoelectric sensor in a small arena to evaluate effects of insect locomotory behavior and size on the ability of an inexpensively constructed instrument to detect insects and distinguish among different species. Adults of all species could be detected when crawling or scraping. The smallest insects in the study, first-fourth-instar C. lectularius nymphs, could not be detected easily when crawling, but could be detected when scraping. Sound and vibration sensors detected brief, 3-10-ms impulses from all tested species, often grouped in distinctive trains (bursts), typical of impulses in previous acoustic detection experiments. To consider the potential for targeting or focusing detection on particular species of interest, indicators were developed to assess the likelihood of detection of C. lectularius. Statistically significant differences were found between C. lectularius and other species in distributions of three measured variables: infrared signal durations, sound impulse-burst durations, and sound pressure levels (energy) of impulses that best matched an averaged spectrum (profile) of scraping behavior. Thus, there is potential that signals collected by an inexpensive, polymodal-sensor instrument could be used in automated trapping systems to detect a targeted species, 0.1 mg or larger, in environments where servicing of traps is difficult or when timeliness of trapping information is important.

Activin A as a marker of intrauterine infection in women with preterm prelabour rupture of membranes.

OBJECTIVE: To assess maternal serum activin A, an early phase response protein in systemic infection, as an early marker of intrauterine infection in women with preterm prelabour rupture of membranes (PPROM). STUDY DESIGN: A prospective cohort study of women with singleton pregnancies complicated by PPROM at 24 to 34 weeks' gestation. Serum was collected for activin A and cytokine measurements. Activin A was measured using commercial enzyme-linked immunosorbent assay. Cytokines were measured using commercial multiplex assay. Pregnancy outcomes including infection were determined by case-record review. RESULT: Eighteen women with PPROM were studied, with seven developing intrauterine infection. Serum activin A in women with and without infection did not differ. Peripheral white cell count, interleukin (IL)-6 and IL-10 were higher (P=0.03, 0.05 and 0.009, respectively) and IL-7 lower (P=0.04) 72 h before delivery in women with infection. CONCLUSION: Activin A is not a clinically useful marker of intrauterine infection in women with PPROM.

Colour vision testing for diabetic retinopathy: a systematic review of diagnostic accuracy and economic evaluation.

OBJECTIVE: To determine the diagnostic performance and cost-effectiveness of colour vision testing (CVT) to identify and monitor the progression of diabetic retinopathy (DR). DATA SOURCES: Major electronic databases including MEDLINE, EMBASE, Cumulative Index to Nursing and Allied Health Literature, and Cochrane Database of Systematic Reviews were searched from inception to September 2008. REVIEW METHODS: A systematic review of the evidence was carried out according to standard methods. An online survey of National Screening Programme for Diabetic Retinopathy (NSPDR) clinical leads and programme managers assessed the diagnostic tools used routinely by local centres and their views on future research priorities. A decision tree and Markov model was developed to estimate the incremental costs and effects of adding CVT to the current NSPDR. RESULTS: In total, 25 studies on CVT met the inclusion criteria for the review, including 18 presenting 2 x 2 diagnostic accuracy data. The quality of studies and reporting was generally poor. Automated or computerised CVTs reported variable sensitivities (63-97%) and specificities (71-95%). One study reported good diagnostic accuracy estimates for computerised CVT plus retinal photography for detection of sight-threatening DR, but it included few cases of retinopathy in total. Results for pseudoisochromatic plates, anomaloscopes and colour arrangement tests were largely inadequate for DR screening, with Youden indices (sensitivity + specificity - 100%) close to zero. No studies were located that addressed patient preferences relating to CVT for DR. Retinal photography is universally employed as the primary method for retinal screening by centres responding to the online survey; none used CVT. The review of the economic evaluation literature found no previous studies describing the cost and effects of any type of CVT. Our economic evaluation suggested that adding CVT to the current national screening programme could be cost-effective if it adequately increases sensitivity and is relatively inexpensive. The deterministic base-case analysis indicated that the cost per quality-adjusted life-year gained may be 6364 pounds and 12,432 pounds for type 1 and type 2 diabetes respectively. However, probabilistic sensitivity analysis highlighted the substantial probability that CVT is not diagnostically accurate enough to be either an effective or a cost-effective addition to current screening methods. The results of the economic model should be treated with caution as the model is based on only one small study. CONCLUSIONS: There is insufficient evidence to support the use of CVT alone, or in combination with retinal photography, as a method for screening for retinopathy in patients with diabetes. Better quality diagnostic accuracy studies directly comparing the incremental value of CVT in addition to retinal photography are needed before drawing conclusions on cost-effectiveness. The most frequently cited preference for future research was the use of optical coherence tomography for the detection of clinically significant macular oedema.

A comparative study of high-resolution cone beam computed tomography and charge-coupled device sensors for detecting caries.

OBJECTIVES: Conventional radiographic imaging of teeth underestimates the presence of caries. The objective of this study was to compare the efficacy of high-resolution cone beam CT (CBCT) images and conventional charge-coupled device (CCD) images for detecting proximal and occlusal caries. METHODS: Non-restored, extracted human permanent premolar and molar teeth were mounted and then imaged with a 3DX Accuitomo and a CCD. We selected 92 occlusal and 100 proximal surfaces for raters to score. Of these, 36 and 25, respectively, had lesions extending into dentin. Using a five-step confidence scale, eight practising dentists evaluated the images for the presence of caries in dentin using both modalities. Actual presence and extent of caries was established with microCT imaging. RESULTS: For proximal surface lesions extending into dentin, the average sensitivity score using 3DX images (0.61) was almost twice that of CCD images (0.33) and the difference was significant. The specificity values for both systems were high and not significantly different from each other. For occlusal surfaces, raters detected significantly more lesions in the enamel or dentin when using the 3DX images than when using CCD images. However, the raters also had significantly lower average specificity scores for the 3DX images compared with the CCD images for lesions at both depths. CONCLUSIONS: Practising dentists were able to improve their detection of proximal-surface caries extending into the dentin, but not occlusal caries, using 3DX high-resolution cone beam CT images compared with CCD images.

Studies of the minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes.

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour.

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.

Lateral mobility of proteins in liquid membranes revisited.

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D proportional, variant 1/R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.

The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides.

The addition of salts, specifically sodium perchlorate (NaClO4), to mobile phases at acidic pH as ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) has been generally overlooked. To demonstrate the potential of NaClO4 as an effective anionic ion-pairing reagent, we applied RP-HPLC in the presence of 0-100 mM sodium chloride (NaCl), sodium trifluoroacetate (NaTFA) or NaClO4 to two mixtures of synthetic 18-residue peptides: a mixture of peptides with the same net positive charge (+4) and a mixture of four peptides of +1, +2, +3 and +4 net charge. Interestingly, the effect of increasing NaClO4 concentration on increasing peptide retention times and selectivity changes was more dramatic than that of either NaCl or NaTFA, with the order of increasing anion effectiveness being Cl- << TFA- < C104-. Such effects were more marked when salt addition was applied to eluents containing 10 mM phosphoric acid (H3PO4) compared to 10 mM trifluoroacetic acid (TFA) due to the lesser starting anion hydrophobicity of the former mobile phase (containing the phosphate ion) compared to the latter (containing the TFA- ion).

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography.

Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.