Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1–25 and SP-B8–25

AbstractRaman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B1–25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B1–25 was compared with that of a truncated homodimer (dSP-B8–25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S–S stretching region centered at ∼500 cm−1 indicated a stable, although highly strained disulfide conformation with a χ(CS–SC) dihedral angle of ±10° for the dSP-B1–25 dimer. In contrast, the truncated dimer dSP-B8–25 exhibited a series of disulfide conformations with the χ(CS–SC) dihedral angle taking on values of either ±30° or 85±20°. For conformations with χ(CS–SC) close to the ±90° value, the Raman spectra of the 8–25 truncated dimers exhibited χ(SS–CC) dihedral angles of 90/180° and 20–30°. In the presence of a lipid mixture, both constructs showed a ν(S–S) band at ∼488 cm−1, corresponding to a χ(CS–SC) dihedral angle of ±10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼60–70° to the surface normal, while the β structure had ∼40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation

Thrombocytopenia in neonates and the risk of intraventricular hemorrhage: a retrospective cohort study

<p>Abstract</p> <p>Background</p> <p>The overall prevalence of thrombocytopenia in neonates admitted to neonatal intensive care units ranges from 22 to 35%. There are only a few small studies that outline the relationship between the severity of thrombocytopenia and the risk of bleeding. This makes it difficult to form an evidence-based threshold for platelet transfusions in neonatal patients. The aim of this study was to determine the prevalence of thrombocytopenia in a tertiary neonatal intensive care unit and to study the relation between thrombocytopenia and the risk of intraventricular hemorrhage (IVH).</p> <p>Methods</p> <p>We performed a retrospective cohort study of all patients with thrombocytopenia admitted to our neonatal tertiary care nursery between January 2006 and December 2008. Patients were divided into 4 groups according to the severity of thrombocytopenia: mild (100-149 × 10⁹/L), moderate (50-99 × 10⁹/L), severe (30-49 × 10⁹/L) or very severe (< 30 × 10⁹/L). The primary outcome was IVH ≥ grade 2. Pearson's chi-squared and Fischer's exact tests were used for categorical data. ANOVA, logistic regression analysis and multivariate linear regression were used for comparisons between groups and for confounding factors.</p> <p>Results</p> <p>The prevalence of thrombocytopenia was 27% (422/1569). Risk of IVH ≥ grade 2 was 12% (48/411) in neonates with versus 5% (40/844) in neonates without thrombocytopenia (p < 0.01). After multivariate linear regression analysis, risk of IVH ≥ grade 2 in the subgroups of thrombocytopenic infants was not significantly different (p = 0.3).</p> <p>After logistic regression analysis the difference in mortality rate in neonates with and without thrombocytopenia was not significant (p = 0.4). Similarly, we found no difference in mortality rate in the subgroups of neonates with thrombocytopenia (p = 0.7).</p> <p>Conclusion</p> <p>Although IVH ≥ grade 2 occurs more often in neonates with thrombocytopenia, this relation is independent of the severity of thrombocytopenia. Prospective studies should be conducted to assess the true risk of hemorrhage depending on underlying conditions. Randomized controlled trials are urgently needed to determine a safe lower threshold for platelet transfusions.</p

A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities [version 2; referees: 2 approved]

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

Surfactant protein C peptides with salt-bridges (“ion-locks”) promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein

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 12C-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

Does Parenteral Nutrition Influence Electrolyte and Fluid Balance in Preterm Infants in the First Days after Birth?

New national guidelines recommend more restricted fluid intake and early initiation of total parenteral nutrition (TPN) in very preterm infants. The aim was study the effect of these guidelines on serum sodium and potassium levels and fluid balance in the first three days after birth.Two cohorts of infants <28 weeks gestational age, born at the Leiden University Medical Center in the Netherlands, were compared retrospectively before (2002–2004, late-TPN) and after (2006–2007, early-TPN) introduction of the new Dutch guideline. Outcome measures were serum sodium and potassium levels, diuresis, and changes in body weight in the first three postnatal days.In the first three postnatal days no differences between late-TPN (N = 70) and early-TPN cohort (N = 73) in mean (SD) serum sodium (141.1 (3.8) vs 141.0 (3.7) mmol/l) or potassium (4.3 (0.5) vs 4.3 (0.5) mmol/l) were found, but in the early-TPN cohort diuresis (4.5 (1.6) vs 3.2 (1.4) ml/kg/h) and loss of body weight were decreased (−6.0% (7.7) vs −0.8% (8.0)).Initiation of TPN immediately after birth and restricted fluid intake in very preterm infants do not seem to influence serum sodium and potassium levels in first three postnatal days. Further research is needed to see if a decreased diuresis and loss of body weight in the first days is the result of a delayed postnatal adaptation or better energy balance

Poor Accuracy of Methods Currently Used to Determine Umbilical Catheter Insertion Length

This study compares the methods of Dunn and Shukla in determining the appropriate insertion length of umbilical catheters. In July 2007, we changed our policy for umbilical catheter insertions from the method of Dunn to the method of Shukla. We report our percentage of inaccurate placement of umbilical-vein catheters (UVCs) and umbilical-artery catheters (UACs) before and after the change of policy. In the Dunn-group, 41% (28/69) of UVCs were placed directly in the correct position against 24% (20/84) in the Shukla-group. The position of the catheter-tip of UVCs in the Dunn-group and the Shukla-group was too high in 57% (39/69) and 75% (63/84) of neonates, respectively. UACs in the Dunn-group were placed directly in the correct position in 63% (24/38) compared to the 87% (39/45) of cases in Shukla-group. The position of the catheter-tip of UACs in the Dunn-group and the Shukla-group was too high in 34% (13/38) and 13% (6/45) of neonates, respectively. In conclusion, the Dunn-method is more accurate than the Shukla-method in predicting the insertion length for UVCs, whereas the Shukla-method is more accurate for UACs

Dynamic Surface Activity of a Fully Synthetic Phospholipase-Resistant Lipid/Peptide Lung Surfactant

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.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.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

Aerosol delivery of dry powder synthetic lung surfactant to surfactant-deficient rabbits and preterm lambs on non-invasive respiratory support [version 2; referees: 3 approved]

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

Aerosol delivery of dry powder synthetic lung surfactant to surfactant-deficient rabbits and preterm lambs on non-invasive respiratory support [version 1; referees: 3 approved]

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 active 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