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Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events., (© Wildlife Disease Association 2024.)

Salmonella isolated from dairy farms has a significant effect on animal health and productivity. Different serogroups of Salmonella affect both human and bovine cattle causing illness in both reservoirs. Dairy cows and calves can be silent Salmonella shedders, increasing the possibility of dispensing Salmonella within the farm. The aim of this study was to determine the genomic characteristics of Salmonella isolates from dairy farms and to detect the presence of virulence and antimicrobial resistance genes. A total of 377 samples were collected in a cross-sectional study from calves, periparturient cow feces, and maternity beds in 55 dairy farms from the states of Aguascalientes, Baja California, Chihuahua, Coahuila, Durango, Mexico, Guanajuato, Hidalgo, Jalisco, Queretaro, San Luis Potosi, Tlaxcala, and Zacatecas. Twenty Salmonella isolates were selected as representative strains for whole genome sequencing. The serological classification of the strains was able to assign groups to only 12 isolates, but with only 5 of those being consistent with the genomic serotyping. The most prevalent serovar was Salmonella Montevideo followed by Salmonella Meleagridis. All isolates presented the chromosomal aac(6')-Iaa gene that confers resistance to aminoglycosides. The antibiotic resistance genes qnr B19, qnr A1, sul 2, aph (6)-Id, aph (3)-ld, dfr A1, tet A, tet C, flor 2, sul 1_15, mph (A), aad A2, bla CARB, and qac E were identified. Ten pathogenicity islands were identified, and the most prevalent plasmid was Col(pHAD28). The main source of Salmonella enterica is the maternity areas, where periparturient shedders are contaminants and perpetuate the pathogen within the dairy in manure, sand, and concrete surfaces. This study demonstrated the necessity of implementing One Health control actions to diminish the prevalence of antimicrobial resistant and virulent pathogens including Salmonella.

The three-dimensional structure of the synthetic lung Surfactant Protein B Peptide Super Mini-B was determined using an integrative experimental approach, including mass spectrometry and isotope enhanced Fourier-transform infrared (FTIR) spectroscopy. Mass spectral analysis of the peptide, oxidized by solvent assisted region-specific disulfide formation, confirmed that the correct folding and disulfide pairing could be facilitated using two different oxidative structure-promoting solvent systems. Residue specific analysis by isotope enhanced FTIR indicated that the N-terminal and C-terminal domains have well defined α-helical amino acid sequences. Using these experimentally derived measures of distance constraints and disulfide connectivity, the ensemble was further refined with molecular dynamics to provide a medium resolution, residue-specific structure for the peptide construct in a simulated synthetic lung surfactant lipid multilayer environment. The disulfide connectivity combined with the α-helical elements stabilize the peptide conformationally to form a helical hairpin structure that resembles critical elements of the Saposin protein fold of the predicted full-length Surfactant Protein B structure., Competing Interests: FW, AW, LG and the Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center hold a patent on Super Mini-B (US 8,563,683).

Background: Optimal functionality of synthetic lung surfactant for treatment of respiratory distress syndrome in preterm infants largely depends on the quality and quantity of the surfactant protein B (SP-B) peptide mimic and the lipid mixture. B-YL peptide is a 41-residue sulfur-free SP-B mimic with its cysteine and methionine residues replaced by tyrosine and leucine, respectively, to enhance its oxidation resistance., Aim: Testing the structural and functional stability of the B-YL peptide in synthetic surfactant lipids after long-term storage., Methods: The structural and functional properties of B-YL peptide in surfactant lipids were studied using three production runs of B-YL peptides in synthetic surfactant lipids. Each run was held at 5 °C ambient temperature for three years and analyzed with structural and computational techniques, i.e., MALDI-TOF mass spectrometry, ATR-Fourier Transform Infrared Spectroscopy (ATR-FTIR), secondary homology modeling of a preliminary B-YL structure, and tertiary Molecular Dynamic simulations of B-YL in surfactant lipids, and with functional methods, i.e., captive bubble surfactometry (CBS) and retesting in vivo surface activity in surfactant-deficient young adult rabbits., Results: MALDI-TOF mass spectrometry showed no degradation of the B-YL peptide as a function of stored time. ATR-FTIR studies demonstrated that the B-YL peptide still assumed stable alpha-helical conformations in synthetic surfactant lipids. These structural findings correlated with excellent in vitro surface activity during both quasi-static and dynamic cycling on CBS after three years of cold storage and in vivo surface activity of the aged formulations with improvements in oxygenation and dynamic lung compliance approaching those of the positive control surfactant Curosurf®., Conclusions: The structure of the B-YL peptide and the in vitro and in vivo functions of the B-YL surfactant were each maintained after three years of refrigeration storage., (© 2021. The Author(s).)

Dental enamel is a principal component of teeth 1 , and has evolved to bear large chewing forces, resist mechanical fatigue and withstand wear over decades 2 . Functional impairment and loss of dental enamel, caused by developmental defects or tooth decay (caries), affect health and quality of life, with associated costs to society 3 . Although the past decade has seen progress in our understanding of enamel formation (amelogenesis) and the functional properties of mature enamel, attempts to repair lesions in this material or to synthesize it in vitro have had limited success 4-6 . This is partly due to the highly hierarchical structure of enamel and additional complexities arising from chemical gradients 7-9 . Here we show, using atomic-scale quantitative imaging and correlative spectroscopies, that the nanoscale crystallites of hydroxylapatite (Ca 5 (PO 4 ) 3 (OH)), which are the fundamental building blocks of enamel, comprise two nanometric layers enriched in magnesium flanking a core rich in sodium, fluoride and carbonate ions; this sandwich core is surrounded by a shell with lower concentration of substitutional defects. A mechanical model based on density functional theory calculations and X-ray diffraction data predicts that residual stresses arise because of the chemical gradients, in agreement with preferential dissolution of the crystallite core in acidic media. Furthermore, stresses may affect the mechanical resilience of enamel. The two additional layers of hierarchy suggest a possible new model for biological control over crystal growth during amelogenesis, and hint at implications for the preservation of biomarkers during tooth development.

In recent years, communities and institutions have sought new interventions intended to reduce sugar sweetened beverage (SSB) consumption among children. Among these interventions are "SSB-free zones," where such beverages are not permitted to be consumed on the premises. Insufficient knowledge still exists, however, about the readiness for such restrictive SSB policies within health care institutions. Understanding attitudes toward SSB consumption among adults is necessary to guide an institution-wide policy, where staff and patients serve as role models for parents and their children. We conducted focus groups with health center patients and staff to determine perceptions surrounding health and SSB consumption and to better understand the support and readiness (or lack thereof) for an SSB-free zone intervention prior to its implementation. We found that contextual practices present challenges to breaking personal consumption habits, even if beverages are banned from the worksite. Nevertheless, participants expressed support for SSB-free zones, and recommended more education about the harmful effects of soda and energy drink consumption to help improve acceptability for the policy. We conclude that policies restricting onsite SSB consumption may be more effective when combined with educational information and expressions of understanding that this specific behavior change can be difficult., Competing Interests: The authors have declared that no competing interests exist.

Background : The development of synthetic lung surfactant for preterm infants has focused on peptide analogues of native surfactant proteins B and C (SP-B and SP-C). Non-invasive respiratory support with nasal continuous positive airway pressure (nCPAP) may benefit from synthetic surfactant for aerosol delivery. Methods : A total of three dry powder (DP) surfactants, consisting of phospholipids and the SP-B analogue Super Mini-B (SMB), and one negative control DP surfactant without SMB, were produced with the Acorda Therapeutics ARCUS® Pulmonary Dry Powder Technology. Structure of the DP surfactants was compared with FTIR spectroscopy, in vitro surface activity with captive bubble surfactometry, and in vivo activity in surfactant-deficient adult rabbits and preterm lambs. In the animal experiments, intratracheal (IT) aerosol delivery was compared with surfactant aerosolization during nCPAP support. Surfactant dosage was 100 mg/kg of lipids and aerosolization was performed using a low flow inhaler. Results: FTIR spectra of the three DP surfactants each showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to that previously noted for surface-active SMB in other lipids. The DP surfactants with SMB demonstrated in vitro surface activity <1 mN/m. Oxygenation and lung function increased quickly after IT aerosolization of DP surfactant in both surfactant-deficient rabbits and preterm lambs, similar to improvements seen with clinical surfactant. The response to nCPAP aerosol delivery of DP surfactant was about 50% of IT aerosol delivery, but could be boosted with a second dose in the preterm lambs. Conclusions: Aerosol delivery of DP synthetic surfactant during non-invasive respiratory support with nCPAP significantly improved oxygenation and lung function in surfactant-deficient animals and this response could be enhanced by giving a second dose. Aerosol delivery of DP synthetic lung surfactant has potential for clinical applications., Competing Interests: No competing interests were disclosed.

Millions of people take animal pictures during wildlife interactions, yet the impacts of photographer behaviour and photographic flashes on animals are poorly understood. We investigated the pathomorphological and behavioural impacts of photographer behaviour and photographic flashes on 14 benthic fish species that are important for scuba diving tourism and aquarium displays. We ran a field study to test effects of photography on fish behaviour, and two laboratory studies that tested effects of photographic flashes on seahorse behaviour, and ocular and retinal anatomy. Our study showed that effects of photographic flashes are negligible and do not have stronger impacts than those caused solely by human presence. Photographic flashes did not cause changes in gross ocular and retinal anatomy of seahorses and did not alter feeding success. Physical manipulation of animals by photographing scuba divers, however, elicited strong stress responses. This study provides important new information to help develop efficient management strategies that reduce environmental impacts of wildlife tourism.

Background : Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied 'B-YL', a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Methods: Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. Results: CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent in vitro surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. Conclusions: 'Sulfur-free' B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL's possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS., Competing Interests: No competing interests were disclosed.

Background : Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied 'B-YL', a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Methods: Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. Results: CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent in vitro surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. Conclusions: 'Sulfur-free' B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL's possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS., Competing Interests: No competing interests were disclosed.

Purpose: Clinicians and stewardship programs are challenged with positioning of novel, higher priced antibiotic agents for the treatment of clinical infections. We developed a decision-analytic model to describe costs, including drug, total treatment costs, and health care outcomes, associated with telavancin (TLV) compared with vancomycin (VAN) for patients with Staphylococcus aureus (SA) hospital-acquired bacterial pneumonia (HABP)., Methods: This decision-analytic model assessed the treatment of SA-HABP with TLV versus VAN. Data were obtained from the ATTAIN (Assessment of Telavancin for Treatment of Hospital-Acquired Pneumonia) clinical trials on the following: the probability of clinical cure; probability of nephrotoxicity; and prevalence of polymicrobial infection (30%), methicillin-resistant Staphylococcus aureus (MRSA) (68%), and SA with VAN MIC ≥1 µg/mL (85%). Data on length of stay for cure (10 days), failure (10 additional days), and nephrotoxicity (3.5 days) were based on literature. Cost per treated patient and incremental cost-effectiveness ratio (ICER) per additional cure were calculated for SA-HABP and for monomicrobial SA-HABP. One-way sensitivity analyses were performed., Findings: Patients with SA-HABP were sub-grouped by methicillin susceptibility (n = 140, 32%) or resistance (n = 293, 68%), and occurrence of polymicrobial (n = 128, 30%) vs monomicrobial (n = 305, 70%) infections. Under the base case, hospital cost for patients with HABP treated with TLV was $42,564 and with VAN, it was $42,296. Telavancin was associated with higher drug ($2082) and nephrotoxicity ($467) costs and lower intensive care unit (-$1738) and ventilator (-$114) costs. ICER was $4156 per additional cure. ICER was sensitive to probabilities of cure, length of treatment in cures, intensive care unit cost, TLV cost, and additional length of stay due to failure. For monomicrobial SA-HABP, TLV was associated with a net cost savings of $907 per patient and yielded economic dominance., Implications: Our decision-analytic model suggests that TLV for monomicrobial SA-HABP is associated with higher drug acquisition costs but a favorable ICER relative to VAN, provided that effective antimicrobial stewardship limits therapy to 7 days. Sensitivity analyses suggest a potential economic benefit of TLV treatment with appropriate patient selection. Antimicrobial stewardship programs may be able to reduce total costs through judicious use of novel antimicrobial agents. ClinicalTrials.gov identifiers: NCT00107952 and NCT00124020., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The objectives of this study were to determine whether pediatricians are more likely than other primary care physicians (PCPs) to refer newly diagnosed adolescent and young adult patients with cancer to pediatric oncological specialists, and to assess the physician and patient characteristics that affect patterns of referral., Methods: A cross-sectional vignette survey was mailed to PCPs to examine hypothetical referral decisions as a function of physician characteristics and patient characteristics, including diagnosis, age, gender, race/ethnicity, family support, transportation, insurance, and patient preference for site of care. Pediatrician PCPs and nonpediatrician PCPs (family medicine, internal medicine, and emergency medicine physicians) practicing in North Carolina and in Washington State participated in the study., Results: A total of 406 surveys were completed (35.8% response rate). Sixty percent of pediatric PCPs referred their hypothetical patients with cancer to pediatric specialists (PSs), compared with only 37% of nonpediatric PCPs. Patient age also influenced referral patterns; 89% of 13-year-olds, 74% of 16-year-olds, 25% of 19-year-olds, and only 9% of 22-year-old patients were referred to a PS. Multivariate logistic regression demonstrated that diagnosis and physician practice setting also were associated with referral patterns., Conclusions: Both patient age and PCP specialty were significant predictors of referral patterns in hypothetical vignettes of newly diagnosed adolescent and young adult patients with cancer. Pediatricians were more likely than nonpediatrician PCPs to refer patients to a PS. Referrals to PSs decreased dramatically between ages 16 and 19. Because the site of oncological care can impact outcomes, these data have the potential to inform awareness and education initiatives directed at PCPs., (Copyright © 2017 The Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1-25 and 63-78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2-8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To assess the effectiveness of a dual rapid test compared to a single rapid test for syphilis and HIV screening., Methods: A cluster-randomized open-label clinical trial was performed in 12 public antenatal care (ANC) centers in the cities of Bogotá and Cali, Colombia. Pregnant women who were over 14 years of age at their first antenatal visit and who had not been previously tested for HIV and syphilis during the current pregnancy were included. Pregnant women were randomized to single HIV and single syphilis rapid diagnostic tests (Arm A) or to dual HIV and syphilis rapid diagnostic tests (Arm B). The four main outcomes measured were: (1) acceptability of the test, (2) uptake in testing, (3) treatment on the same day (that is, timely treatment), and (4) treatment at any time for positive rapid test cases. Bivariate and multivariate analyses were calculated to adjust for the clustering effect and the period., Results: A total of 1 048 patients were analyzed in Arm A, and 1 166 in Arm B. Acceptability of the rapid tests was 99.8% in Arm A and 99.6% in Arm B. The prevalence of positive rapid tests was 2.21% for syphilis and 0.36% for HIV. Timely treatment was provided to 20 of 29 patients (69%) in Arm A and 16 of 20 patients (80%) in Arm B (relative risk (RR), 1.10; 95% confidence interval (CI): (1.00 -1.20). Treatment at any time was given to 24 of 29 patients (83%) in Arm A and to 20 of 20 (100%) in Arm B (RR, 1.11; 95% CI: 1.01-1.22)., Conclusions: There were no differences in patient acceptability, testing and timely treatment between dual rapid tests and single rapid tests for HIV and syphilis screening in the ANC centers. Same-day treatment depends also on the interpretation of and confidence in the results by the health providers.

Three water adsorption-desorption mechanisms are common in inorganic materials: chemisorption, which can lead to the modification of the first coordination sphere; simple adsorption, which is reversible; and condensation, which is irreversible. Regardless of the sorption mechanism, all known materials exhibit an isotherm in which the quantity of water adsorbed increases with an increase in relative humidity. Here, we show that carbon-based rods can adsorb water at low humidity and spontaneously expel about half of the adsorbed water when the relative humidity exceeds a 50-80% threshold. The water expulsion is reversible, and is attributed to the interfacial forces between the confined rod surfaces. At wide rod spacings, a monolayer of water can form on the surface of the carbon-based rods, which subsequently leads to condensation in the confined space between adjacent rods. As the relative humidity increases, adjacent rods (confining surfaces) in the bundles are drawn closer together via capillary forces. At high relative humidity, and once the size of the confining surfaces has decreased to a critical length, a surface-induced evaporation phenomenon known as solvent cavitation occurs and water that had condensed inside the confined area is released as a vapour.

Surfactant protein (SP)-B is a 79-residue polypeptide crucial for the biophysical and physiological function of endogenous lung surfactant. SP-B is a member of the Saposin or Saposin-like proteins (SAPLIP) family of proteins that share an overall three-dimensional folding pattern based on secondary structures and disulfide connectivity and exhibit a wide diversity of biological functions. Here we review the synthesis, molecular biophysics and activity of synthetic analogs of Saposin proteins designed to mimic those interactions of the parent proteins with lipids that enhance interfacial activity. Saposin proteins generally interact with target lipids as either monomers or multimers via well-defined amphipathic helices, flexible hinge domains, and insertion sequences. Based on the known 3D-structural motif for the Saposin family, we show how bioengineering techniques may be used to develop minimal peptide constructs that maintain desirable structural properties and activities in biomedical applications. One important application is the molecular design, synthesis and activity of Saposin mimics based on the SP-B structure. Synthetic lung surfactants containing active SP-B analogs may be potentially useful in treating diseases of surfactant deficiency or dysfunction including the neonatal respiratory distress syndrome and acute lung injury/acute respiratory distress syndrome., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Dental enamel has evolved to resist the most grueling conditions of mechanical stress, fatigue, and wear. Adding insult to injury, it is exposed to the frequently corrosive environment of the oral cavity. While its hierarchical structure is unrivaled in its mechanical resilience, heterogeneity in the distribution of magnesium ions and the presence of Mg-substituted amorphous calcium phosphate (Mg-ACP) as an intergranular phase have recently been shown to increase the susceptibility of mouse enamel to acid attack. Herein we investigate the distribution of two important constituents of enamel, residual organic matter and inorganic carbonate. We find that organics, carbonate, and possibly water show distinct distribution patterns in the mouse enamel crystallites, at simple grain boundaries, and in the amorphous interphase at multiple grain boundaries. This has implications for the resistance to acid corrosion, mechanical properties, and the mechanism by which enamel crystals grow during amelogenesis.

Dental enamel, a hierarchical material composed primarily of hydroxylapatite nanowires, is susceptible to degradation by plaque biofilm-derived acids. The solubility of enamel strongly depends on the presence of Mg(2+), F(-), and CO3(2-). However, determining the distribution of these minor ions is challenging. We show—using atom probe tomography, x-ray absorption spectroscopy, and correlative techniques—that in unpigmented rodent enamel, Mg(2+) is predominantly present at grain boundaries as an intergranular phase of Mg-substituted amorphous calcium phosphate (Mg-ACP). In the pigmented enamel, a mixture of ferrihydrite and amorphous iron-calcium phosphate replaces the more soluble Mg-ACP, rendering it both harder and more resistant to acid attack. These results demonstrate the presence of enduring amorphous phases with a dramatic influence on the physical and chemical properties of the mature mineralized tissue., (Copyright © 2015, American Association for the Advancement of Science.)

Metastable precursors are thought to play a major role in the ability of organisms to create mineralized tissues. Of particular interest are the hard and abrasion-resistant teeth formed by chitons, a class of rock-grazing mollusks. The formation of chiton teeth relies on the precipitation of metastable ferrihydrite (Fh) in an organic scaffold as a precursor to magnetite. In vitro synthesis of Fh under physiological conditions has been challenging. Using a combination of X-ray absorption and electron paramagnetic resonance spectroscopy, we show that, prior to Fh formation in the chiton tooth, iron ions are complexed by the organic matrix. In vitro experiments demonstrate that such complexes facilitate the formation of Fh under physiological conditions. These results indicate that acidic molecules may be integral to controlling Fh formation in the chiton tooth. This biological approach to polymorph selection is not limited to specialized proteins and can be expropriated using simple chemistry., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as 'helical adjuvants' to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges ("ion-locks") promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu(-)-Lys(+) into the parent SP-C's. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with 'amyloid-like' properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard (12)C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu(-)-Lys(+) insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu(-)-Lys(+) ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.

Background. Chemical spills are on the rise and inhalation of toxic chemicals may induce chemical acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Although the pathophysiology of ALI/ARDS is well understood, the absence of specific antidotes has limited the effectiveness of therapeutic interventions. Objectives. Surfactant inactivation and formation of free radicals are important pathways in (chemical) ALI. We tested the potential of lipid mixtures with advanced surfactant protein B and C (SP-B and C) mimics to improve oxygenation and lung compliance in rabbits with lavage- and chemical-induced ALI/ARDS. Methods. Ventilated young adult rabbits underwent repeated saline lung lavages or underwent intratracheal instillation of hydrochloric acid to induce ALI/ARDS. After establishment of respiratory failure rabbits were treated with a single intratracheal dose of 100 mg/kg of synthetic surfactant composed of 3% Super Mini-B (S-MB), a SP-B mimic, and/or SP-C33 UCLA, a SP-C mimic, in a lipid mixture (DPPC:POPC:POPG 5:3:2 by weight), the clinical surfactant Infasurf(®), a bovine lung lavage extract with SP-B and C, or synthetic lipids alone. End-points consisted of arterial oxygenation, dynamic lung compliance, and protein and lipid content in bronchoalveolar lavage fluid. Potential mechanism of surfactant action for S-MB and SP-C33 UCLA were investigated with captive bubble surfactometry (CBS) assays. Results. All three surfactant peptide/lipid mixtures and Infasurf equally lowered the minimum surface tension on CBS, and also improved oxygenation and lung compliance. In both animal models, the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA led to better arterial oxygenation and lung compliance than single peptide synthetic surfactants and Infasurf. Synthetic surfactants and Infasurf improved lung function further in lavage- than in chemical-induced respiratory failure, with the difference probably due to greater capillary-alveolar protein leakage and surfactant dysfunction after HCl instillation than following lung lavage. At the end of the duration of the experiments, synthetic surfactants provided more clinical stability in ALI/ARDS than Infasurf, and the protein content of bronchoalveolar lavage fluid was lowest for the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA. Conclusion. Advanced synthetic surfactant with robust SP-B and SP-C mimics is better equipped to tackle surfactant inactivation in chemical ALI than synthetic surfactant with only a single surfactant peptide or animal-derived surfactant.

Nanocrystalline biological apatites constitute the mineral phase of vertebrate bone and teeth. Beyond their central importance to the mechanical function of our skeleton, their extraordinarily large surface acts as the most important ion exchanger for essential and toxic ions in our body. However, the nanoscale structural and chemical complexity of apatite-based mineralized tissues is a formidable challenge to quantitative imaging. For example, even energy-filtered electron microscopy is not suitable for detection of small quantities of low atomic number elements typical for biological materials. Herein we show that laser-pulsed atom probe tomography, a technique that combines subnanometer spatial resolution with unbiased chemical sensitivity, is uniquely suited to the task. Common apatite end members share a number of features, but can clearly be distinguished by their spectrometric fingerprint. This fingerprint and the formation of molecular ions during field evaporation can be explained based on the chemistry of the apatite channel ion. Using end members for reference, we are able to interpret the spectra of bone and dentin samples, and generate the first three-dimensional reconstruction of 1.2 × 10(7) atoms in a dentin sample. The fibrous nature of the collagenous organic matrix in dentin is clearly recognizable in the reconstruction. Surprisingly, some fibers show selectivity in binding for sodium ions over magnesium ions, implying that an additional, chemical level of hierarchy is necessary to describe dentin structure. Furthermore, segregation of inorganic ions or small organic molecules to homophase interfaces (grain boundaries) is not apparent. This has implications for the platelet model for apatite biominerals.

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf®) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.

The aim of this study was to demonstrate convincing evidence that percutaneous breast biopsy may result in displacement of malignant cells that can initiate tumor growth at a separate anatomic site, other than the index lesion. Two patients with malignancy diagnosed by percutaneous breast biopsy were followed up with subsequent imaging. The observation of displaced cells initiating subsequent malignant growth was compared retrospectively with the previous year's outcomes following percutaneous breast biopsy. Two cases of displaced malignant cells resulting in malignant growth at a separate site along the biopsy needle track were demonstrated by imaging. During the preceding year, 1644 biopsies with 298 malignant results were compared with the outcomes of these two patients, resulting in a 0.7% incidence of malignant seeding. No identifiable factors could be identified to predict under what conditions this iatrogenic complication occurs. Although uncommon, percutaneous breast biopsy can result in malignant seeding, and raises issues regarding informed consent and proper subsequent treatment., (© 2011 Wiley Periodicals, Inc.)

Biological organisms possess an unparalleled ability to control the structure and properties of mineralized tissues. They are able, for example, to guide the formation of smoothly curving single crystals or tough, lightweight, self-repairing skeletal elements. In many biominerals, an organic matrix interacts with the mineral as it forms, controls its morphology and polymorph, and is occluded during mineralization. The remarkable functional properties of the resulting composites-such as outstanding fracture toughness and wear resistance-can be attributed to buried organic-inorganic interfaces at multiple hierarchical levels. Analysing and controlling such interfaces at the nanometre length scale is critical also in emerging organic electronic and photovoltaic hybrid materials. However, elucidating the structural and chemical complexity of buried organic-inorganic interfaces presents a challenge to state-of-the-art imaging techniques. Here we show that pulsed-laser atom-probe tomography reveals three-dimensional chemical maps of organic fibres with a diameter of 5-10 nm in the surrounding nano-crystalline magnetite (Fe(3)O(4)) mineral in the tooth of a marine mollusc, the chiton Chaetopleura apiculata. Remarkably, most fibres co-localize with either sodium or magnesium. Furthermore, clustering of these cations in the fibre indicates a structural level of hierarchy previously undetected. Our results demonstrate that in the chiton tooth, individual organic fibres have different chemical compositions, and therefore probably different functional roles in controlling fibre formation and matrix-mineral interactions. Atom-probe tomography is able to detect this chemical/structural heterogeneity by virtue of its high three-dimensional spatial resolution and sensitivity across the periodic table. We anticipate that the quantitative analysis and visualization of nanometre-scale interfaces by laser-pulsed atom-probe tomography will contribute greatly to our understanding not only of biominerals (such as bone, dentine and enamel), but also of synthetic organic-inorganic composites.

Background: Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity., Methodology/results: FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B., Conclusion: Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Insufficiency fractures occur when physiological loads are applied to bone deficient in mechanical resistance. A better understanding of pelvic mechanics and the effect of bone density alterations could lead to improved diagnosis and treatment of insufficiency fractures. This study aimed to develop and validate a subject-specific three-dimensional (3D) finite element (FE) model of a pelvis, to analyse pelvic strains as a function of interior and cortical surface bone density, and to compare high strain regions with common insufficiency fracture sites. The FE model yielded strong agreement between experimental and model strains. By means of the response surface method, changes to cortical surface bone density using the FE model were found to have a 60 per cent greater influence compared with changes in interior bone density. A small interaction was also found to exist between surface and interior bone densities (< 3 per cent), and a non-linear effect of surface bone density on strain was observed. Areas with greater increases in average principal strains with reductions in density in the FE model corresponded to areas prone to insufficiency fracture. Owing to the influence of cortical surface bone density on strain, it may be considered a strong global (non-linear) indicator for insufficiency fracture risk.

Given their high alanine and glycine levels, plaque formation, alpha-helix to beta-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit 'amyloid-like' characteristics, by contrasting its structural and functional properties with those of Abeta(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Abeta(26-42) formed similar networked beta-sheet fibrils, although the FP fibril interactions were weaker. FP and Abeta(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of alpha-helix to beta-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Abeta(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound beta-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an 'amyloid homolog' (or 'amylog').

Problem gamblers account for almost one-third of the industry's total revenue with the adverse effects of problem gambling including significant financial loss, legal and occupational difficulties, family problems, psychological distress and suicide. As such, it is important to understand the influential factors in gambling abstinence and relapse, which will assist in the development of relapse prevention methods in therapeutic treatment regimes. This paper reported the role of a set of seven predictors in distinguishing between abstinent and relapsed gamblers among 75 Gambling Anonymous (GA) members (55 males; 20 females; Mean age 45 years) in Southeast Queensland. The measures taken were meeting Attendance and Participation, Social Support, God Belief, Belief in a Higher Power, Working the 12-steps of Recovery, Gambling Urges and Erroneous Cognitions. Discriminant analysis revealed that the variables separating the two groups were significant, suggesting that GA members achieving abstinence could be distinguished from those who relapsed, with Attendance and Participation, and Social Support contributing the greatest influence on member's ability to abstain from gambling. The findings suggested that GA member's involvement in meetings, and support from family and friends had significant impact on their gambling abstinence. In contrast, increased gambling urges and erroneous cognitions increased the chance of relapse.

Background: This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control., Results: DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A(2) (PLA(2)) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions <1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions <1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions <1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates., Conclusions: These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.

To study the membrane actions of various domains of HIV-1 glycoprotein 41,000 (gp41), synthetic peptides were prepared corresponding to the N-terminal fusion region (FP; gp41 residues 519-541), the nearby N-leucine zipper domain (N-peptides; DP-107; gp41 residues 560-597), the C-leucine zipper domain (C-peptides; DP-178; gp41 residues 645-680), and the viral envelope adjacent domain that partially overlaps DP-178 (Pre-TM; gp41 residues 671-690). With erythrocytes, FP, DP-107, and Pre-TM induced hemolysis and cell aggregation; the order for hemolytic activity was Pre-TM > FP > DP-107, but each was equally effective in aggregating cells at the highest peptide concentrations tested. DP-178 produced neither hemolysis nor aggregation, but efficiently reduced FP-, DP-107-, and Pre-TM-induced membrane actions. Fourier transform infrared spectroscopy indicated that the membrane perturbations of Pre-TM, as well as the ability of DP-178 to block membrane activities of other gp41 domains, are dependent on Pre-TM and DP-178 each maintaining helical conformations and tryptophans at residues 673, 677, and 679. These results suggest that the corresponding N-terminal fusion, N-leucine zipper, and viral membrane-adjacent regions of HIV-1 gp41 may similarly promote key membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the antiviral mechanism of exogenous DP-178 (clinically approved enfuvirtide) may be partially explained by its coordinate inhibition of the fusogenic actions of the FP, DP-107, and Pre-TM regions of gp41.

Lung surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). Its major function in the lung alveolus is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film enriched in surfactant lipids, hence preventing cellular collapse during respiration. Surfactant therapy using bovine or porcine lung surfactant extracts, which contain only polar lipids and native SP-B and SP-C, has dramatically improved the therapeutic outcomes of preterm infants with respiratory distress syndrome (RDS). One important goal of surfactant researchers is to replace animal-derived therapies with fully synthetic preparations based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. Here, we review recent research developments with peptide analogues of SP-B and SP-C, designed using either the known primary sequence and three-dimensional (3D) structure of the native proteins or, alternatively, the known 3D structures of closely homologous proteins. Such SP-B and SP-C mimics offer the possibility of studying the mechanisms of action of the respective native proteins, and may allow the design of optimized surfactant formulations for specific pulmonary diseases (e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS)). These synthetic surfactant preparations may also be a cost-saving therapeutic approach, with better quality control than may be obtained with animal-based treatments., (Copyright (c) 2007 S. Karger AG, Basel.)

Selecting and evaluating appropriate treatments for children with cerebral palsy has been challenging. One difficulty is in the ability to quantify the presence and importance of coexisting motor signs. This study presents quantitative measures developed to assess spasticity and dystonia. Children diagnosed with extrapyramidal or spastic cerebral palsy and matched control children were studied. Spasticity was measured as the slope of the force-velocity relationship from a test where we measured the forces required to passively extend the elbow at different velocities. Dystonia was assessed by measuring "overflow" movements of arm during active movement of the other arm. Measures of dystonia and spasticity did not correlate with one another, but did correlate with their respective clinical measurement tools, the Modified Ashworth scale and the Barry-Albright Dystonia scale. Most children had a combination of both spasticity and dystonia, despite diagnosis. Our measures also related to different aspects of reaching: children with increased dystonia made more curved paths, and children with increased spasticity hit higher peak velocities. These measurements allow us to distinguish between different motor disorders and the degree to which each contributes to reaching performance. Use of quantitative measures should improve selection and evaluation of treatments for childhood motor disorders.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic dimeric SP-B(1-25) (SP-B(1-25)), a peptide based on the N-terminal domain of human SP-B, efficiently mimics the functional properties of SP-B. The authors investigated the optimum lipid composition for SP-B(1-25) by comparing the effects of natural lung lavage lipids (NLL), a synthetic equivalent of NLL (synthetic lavage lipids SLL), and a standard lipid mixture (TL) on the activities of SP-B(1-25). Surfactant preparations were formulated by mixing 2 mol% SP-B(1-25) in NNL, SLL, and TL. Calfactant, a calf lung lavage extract with SP-B and SP-C, was a positive control and lipids without peptide were negative controls. Minimum surface tension measured on a captive bubble surfactometer was similar for the three SP-B(1-25) surfactant preparations and calfactant. The effects on lung function were compared in ventilated, lavaged, surfactant-deficient rats. Oxygenation and lung volumes were consistently higher in rats treated with calfactant and SP-B(1-25) in NLL or SLL than in rats treated with SP-B(1-25) in TL. Fourier transform infrared spectra observed abnormal secondary conformations for SP-B(1-25) in TL as a possible cause for the reduced lung function. Lipid composition plays a crucial role in the in vitro and in vivo functions of SP-B(1-25) in surfactant preparations.

A novel indole dioxygenase (idoA) gene has been cloned from Pseudomonas alcaligenes PA-10, based on its ability to convert indole to indigo. The chromosomally encoded idoA gene exhibits no similarity to previously cloned naphthalene dioxygenases or to aromatic oxygenases from other species at the nucleotide level. Phylogenetic analysis indicates that the idoA gene product is most similar to an acyl-CoA dehydrogenase from Novosphingobium aromaticivorans. The enzyme encoded by the idoA gene is essential for the metabolism of fluoranthene, since a mutant in which the idoA gene has been disrupted looses the ability to degrade this compound. The idoA gene appears to be constitutively expressed in PA-10, but its expression is also subject to regulation following prior exposure to salicylate and to fluoranthene degradative intermediates.

The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1-23) in aqueous and detergent environments. (12)C-Fourier transform infrared (FTIR) spectroscopy indicated greater alpha-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. (12)C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial beta-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with (13)C-carbonyl. (13)C-FTIR results on FP in SDS at low peptide loading indicated alpha-helix (residues 5 to 16) and disordered conformations (residues 1-4). Because earlier (13)C-FTIR analysis of FP in lipid bilayers demonstrated alpha-helix for residues 1-16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while (13)C-FTIR spectra demonstrated antiparallel beta-sheet for FP (residues 1-12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, alpha-helix to beta-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins.

Surfactant protein B (SP-B) is a constituent surfactant protein critical for normal lung function. Monomeric SP-B(1-25) (mSP-B(1-25)), a peptide based on the N-terminal domain of human SP-B, mixed in phospholipids partially restores lung function in surfactant-deficient animals. Because native SP-B is a homodimer, we synthesized and tested dimeric SP-B(1-25) (dSP-B(1-25)). Circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy indicated that the secondary conformation of SP-B(1-25) was not significantly perturbed by dimerization. The effects on lung function were compared to phospholipids and Survanta in models of neonatal respiratory distress syndrome (RDS) and acute RDS (ARDS). Preterm rabbits born at 27 days of gestation received 100 mg surfactant / kg at birth and were ventilated for 1 hour with a tidal volume of 10 mL / kg. Dynamic compliance was monitored every 15 minutes and postmortem pressure-volume curves were measured. Adult rats were lavaged to induce surfactant deficiency, treated with 100 mg surfactant / kg, and ventilated for 2 hours. Lung function was assessed using arterial blood gases and dynamic compliance every 15 minutes and postmortem pressure-volume curves. Lung volumes in both models and oxygenation in the lavaged rats were consistently higher in the dSP-B(1-25) than in the Survanta and mSP-B(1-25) surfactant groups. The data suggest that dSP-B(1-25) is more efficient in restoring lung function in neonatal RDS and ARDS than mSP-B(1-25) surfactant.

The N-terminal domain of HIV-1 glycoprotein 41000 (FP; residues 1--23; AVGIGALFLGFLGAAGSTMGARSCONH(2)) participates in fusion processes underlying virus--cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, (12)C-Fourier transform infrared (FTIR) spectroscopy indicated the following alpha-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes-hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). (12)C-FTIR spectra also showed disordered FP structures in these environments, along with substantial beta-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with (13)C-carbonyl at multiple sites. Combining these (13)C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: alpha-helix (residues 3-16) and random and beta-structures (residues 1-2 and residues 17-23). Additional (13)C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the alpha-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, (13)C-FTIR spectroscopy showed alpha-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel beta-structure. FP at low loading in ghosts probably inserts deeply as an alpha-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, beta-sheet. In future studies, (13)C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.

The use of mammalian lung surfactant extracts has sharply reduced mortality and morbidity from respiratory distress syndrome in premature infants. Synthesis of surfactant protein B and C (SP-B and SP-C) analogues may lead the way to a synthetic surfactant preparation. Dimeric SP-B(1-25) (dSP-B(1-25)) is based on the N-terminal domain of human SP-B and SP-Cfc is a modified human SP-C in which a single phenylalanine is substituted for a palmitoylated cysteine residue in the N-terminal segment (Phe-4 > Cys-4 variant). We tested the effects of synthetic surfactants with 1 or 2% dSP-B(1-25) and 1% SP-Cfc on lung function in surfactant-deficient rats. Four experimental surfactant preparations were prepared by mixing 1% dSP-B(1-25), 2% dSP-B(1-25), 1% dSP-B(1-25) +1% SP-Cfc, and 2% dSP-B(1-25) +1% SP-Cfc with phospholipids (PL). PL and Survanta, a bovine lung extract, were controls. Groups of 8 rats were ventilated, lavaged until surfactant deficiency, and treated with 100 mg/kg surfactant. Arterial blood gas values and dynamic compliance were measured every 15 min and after 2 h of ventilation, the rats were killed and pressure-volume curves performed. Oxygenation improved quickly after instillation of surfactant with synthetic peptides and Survanta. Oxygenation and lung volumes were consistently higher in the 2% than in the 1% dSP-B(1-25) groups. Addition of 1% SP-Cfc to the synthetic surfactants further improved oxygenation and lung volume, but to a lesser extent than increasing the dSP-B(1-25) content from 1 to 2%. These data indicate that improvements in oxygenation and lung volume in lavaged rats are dependent on the concentration of dSP-B(1-25) in the surfactant preparation and that the presence of SP-Cfc has a relative minor effect on these parameters., (Copyright 2002 S. Karger AG, Basel)

Structural and functional studies were performed to assess the membrane actions of peptides based on HIV-1 glycoprotein 41,000 (gp41). Previous site-directed mutagenesis of gp41 has shown that amino acid changes in either the N-terminal fusion or N-leucine zipper region depressed viral infection and syncytium formation, while modifications in the C-leucine zipper domain both increased and decreased HIV fusion. Here, synthetic peptides were prepared corresponding to the N-terminal fusion region (FP-I; gp41 residues 519-541), the nearby N-leucine zipper domain (DP-107; gp41 residues 560-597), and the C-leucine zipper domain (DP-178; gp41 residues 645-680). With erythrocytes, FP-I or DP-107 induced dose-dependent hemolysis and promoted cell aggregation; FP-I was more hemolytic than DP-107, but each was equally effective in aggregating cells. DP-178 produced neither hemolysis nor aggregation, but blocked either FP-I- or DP-107-induced hemolysis and aggregation. Combined with previous nuclear magnetic resonance and Fourier transform infrared spectroscopic results, circular dichroism (CD) spectroscopy showed that the alpha-helicity for these peptides in solution decreased in the order: DP-107 >> DP-178 > FP-I. CD analysis also indicated binding of DP-178 to either DP-107 or FP-I. Consequently, DP-178 may inhibit the membrane actions mediated by either FP-I or DP-107 through direct peptide interactions in solution. These peptide results suggest that the corresponding N-terminal fusion and N-leucine zipper regions participate in HIV infection, by promoting membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the C-leucine zipper domain in "prefusion" HIV may inhibit these membrane activities by interacting with the N-terminal fusion and N-leucine zipper domains in unactivated gp41. Last, exogenous DP-178 may bind to the N-terminal and N-leucine zipper domains of gp41 that become exposed on HIV stimulation, thereby preventing the fusogenic actions of these gp41 regions leading to infection.

Mammalian lung surfactant is a mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D). Its major function is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film highly enriched in dipalmitoyl phosphatidylcholine (DPPC), thereby preventing alveolar collapse during expiration. SP-A and SP-D are large hydrophilic proteins, which play an important role in host defense, whereas the small hydrophobic peptides SP-B and SP-C interact with DPPC to generate and maintain a surface-active film. Surfactant replacement therapy with bovine and porcine lung surfactant extracts, which contain only polar lipids and SP-B and SP-C, has revolutionized the clinical management of premature infants with respiratory distress syndrome. Newer surfactant preparations will probably be based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. The development of peptide analogues of SP-B and SP-C offers the possibility to study their molecular mechanism of action and will allow the design of surfactant formulations for specific pulmonary diseases and better quality control. This review describes the hydrophobic peptide analogues developed thus far and their potential for use in a new generation of synthetic surfactant preparations., (Copyright 2000 Academic Press.)

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.

The development of a percutaneous artificial internal organ system requires a reliable biocompatible connection between the external environment and the inside of the human body. Such is necessary for the success of a permanent left ventricular assist device. However, the search for a satisfactory interface at the epidermal level has proven to be difficult. Carbon has been proposed for this application, but its texture does not typically promote ingrowth from surrounding tissue. We have therefore employed a new processing method to produce a fine trabecularized carbon implant. The method for preparing the implant involves infiltrating low temperature pyrolytic carbon into the surface of a carbon core which is wrapped with carbon fabric. This results in a tightly woven porous structure of carbon (carbon fiber diameter: 35-50 microm, maximal pore size >200 microm) with gradually increasing porosity from 15-75%. We implanted test samples percutaneously in a calf for in vivo histological evaluation. Thirty days after implantation epidermal downgrowth was minimal. Microscopic analysis revealed that a thin fibrous capsule surrounded the implant, and mature connective tissue with accompanying blood vessels filled the pores of the fine trabecularized carbon layer. From these results we suggest that fine trabecularized carbon is ideally suited for a percutaneous device system in a permanent left ventricular assist device.

We have been developing centrifugal pumps for an implantable left ventricular assist device. We manufactured 2 prototype centrifugal pumps (PI, PII). These two have similar designs except for the PII having a volute casing and a large output port. To determine the differences in the hydraulic characteristics between the PI and PII, we carried out in vitro and in vivo experiments. In vitro study showed that the PII had a shallower H-Q curve than that of the PI, and the PII required a pump speed faster than the PI for the same flow rate and pressure head. On the other hand, in vivo study showed that the PII demonstrated a flow pulsatility greater than that of the PI at 1,900 rpm and 8 L/min although no significant change was observed at low pump speeds (< or = 1,500 rpm). This greater pulsatility consisted of a large discharge according to the small differential pressure during the systolic phase and a small discharge according to the large differential pressure during the diastolic phase. In contrast, the PI, having the steeper H-Q curve, showed a small discharge in the systolic phase and a large discharge in the diastolic phase. These results showed that pulsatility synchronized with the native heart beating depended on the slope of the H-Q curve. As a result, the slope of the H-Q curve is important to determine the component of pulsatility synchronized with native cardiac output. Regarding the slope of the H-Q curve, a pump having a volute casing and a large outlet port demonstrates a shallow slope in the H-Q curve. In conclusion, we suggest that a centrifugal pump for use in left ventricular aortic bypass should be designed considering the effect on the native heart pulsatility.