Prevent Lung Collapse Research Articles

Lung Surfactant Protein B Peptide Mimics Interact with the Human ACE2 Receptor

Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD–ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

The anti-inflammatory and antiviral properties of anionic pulmonary surfactant phospholipids.

The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.

MORPHOLOGICAL CHARACTERISTICS OF LUNGS IN LABORATORY BALB/C MICE

Substantiation. Comparing the course of experimental pathological conditions in mice with human pathology requires deep knowledge of the morphology of the lungs of mice. Although it is advisable to obtain pathomorphological conclusions from an experienced pathologist, in reality, this is not always possible. Therefore, many scientists have to conduct such an analysis by themselves. The aim of the research was to study and describe the normal lung morphology of BALB/c mice for the improved characterization of phenotypes of experimentally induced lesions. Materials and methods. The study included 10 healthy male BALB/c mice, 8 weeks old, housed in individual conditions with unlimited access to food and water. Animals were euthanized by inhalation of ethyl ether. The lung necropsy was performed by tracheal infusion with 10% neutral buffered formalin. The research was carried out on paraffin sections of 3 μm, part of the left lung or one of the right lobes. Sections were stained with hematoxylin-eosin, Schiff-iodic acid, and according to Mallory. Morphology was analyzed using a light microscope. Results. The applied method of euthanasia by inhalation with ethyl ether does not cause agonal changes in the lungs. Fixation by tracheal formalin infusion prevents lung collapse and ensures optimal morphology. For the study of experimental human pathology, it is optimal to examine the left lobe of the lungs, with its longitudinal orientation for obtaining sections, which is of great importance. A blinded way of describing microspecimens does not always provide the best result. Collagen deposits in the outer layer are observed around the large arteries of the lungs; goblet cells are rare, and only in large bronchi. Bronchus-associated lymphoid tissue (BALT) is barely visible in healthy lungs of mice. Natural mouse pathogens can alter the physiology of the host, making it unsuitable for many experimental purposes, and although the number and prevalence of these pathogens have decreased significantly, many of them can be found in laboratory animals and are undesirable in research. Conclusion. Laboratory BALB/c mice may suffer from background diseases, regardless of satisfactory housing conditions, nutrition, and experimental pathology, which should be considered in the design and description of studies.

Increased peak end-expiratory pressure in ventilated preterm lambs changes cerebral microvascular perfusion: direct synchrotron microangiography assessment.

Positive end-expiratory pressure (PEEP) improves oxygenation in mechanically ventilated preterm neonates by preventing lung collapse. However, high PEEP may alter cerebral blood flow secondarily to the increased intrathoracic pressure, predisposing to brain injury. The precise effects of high PEEP on cerebral hemodynamics in the preterm brain are unknown. We aimed to assess the effect of PEEP on microvessels in the preterm brain by using synchrotron radiation (SR) microangiography, which enables in vivo real-time high-resolution imaging of the cerebral vasculature. Preterm lambs (0.8 gestation, n = 4) were delivered via caesarean section, anesthetized, and ventilated. SR microangiography of the right cerebral hemisphere was performed with iodine contrast administered into the right carotid artery during PEEP ventilation of 5 and 10 cmH2O. Carotid blood flow was measured using an ultrasonic flow probe placed around the left carotid artery. An increase of PEEP from 5 to 10 cmH2O increased the diameter of small cerebral vessels (<150 µm) but decreased the diameter of larger cerebral vessels (>500 µm) in all four lambs. Additionally, the higher PEEP increased the cerebral contrast transit time in three of the four lambs. Carotid blood flow increased in two lambs, which also had increased carbon dioxide levels during PEEP 10. Our results suggest that PEEP of 10 cmH2O alters the preterm cerebral hemodynamics, with prolonged cerebral blood flow transit and engorgement of small cerebral microvessels likely due to the increased intrathoracic pressure. These microvascular changes are generally not reflected in global assessment of cerebral blood flow or oxygenation.NEW & NOTEWORTHY An increase of positive end-expiratory pressure (PEEP) from 5 to 10 cmH2O increased the diameter of small cerebral vessels (<150 µm) but decreased the diameter of larger cerebral vessels (>500 µm). This suggests increased intrathoracic pressure due to high PEEP can drive microvessel engorgement in the preterm brain, which may play a role in cerebrovascular injury.

Knowledge The Use of Continous Positive Airway Pressure (CPAP) In Neonatus

CPAP maintains the positive pressure of the baby's airways during spontaneous, simple, and effective breathing. The use of CPAP decreases breathing difficulties, reduces dependence on oxygen, helps repair and maintains lung residual capacity, prevents upper airway obstruction and prevents lung collapse. Good CPAP behaviour reduces the risk or complications that occur in neonates such as nasal injuries, pneumothoracs and gastric distension. The purpose of this study was to determine the relationship of knowledge with CPAP usage behaviour in neonates in the NICU room at X Bekasi Hospital and Depok Y Hospital. Quantitative research methods, descriptive analytic design. A total sampling of 30 nurse respondents worked in the NICU room in X Bekasi Hospital and Depok Y Hospital. Measuring instruments using a questionnaire. Statistical tests with Chi Square, Kendals Tau B and Kendals Tau C. The results of the majority of respondents aged 26-35 years 53.3%, D3 Nursing education 90%, working period between 1-5 years 40%, and those who have attended training 73 , 3%. There was no significant relationship between age (p = 0.136), education (p = 0.897), years of service (p = 0.099), training (p = 0.322), knowledge (p = 0.284) with CPAP usage behaviour in neonates. It is hoped that this research can improve nurses 'knowledge in caring for neonates by using respiratory aids, CPAP by conducting continuous training to improve nurses' behaviour in preventing complications.Keywords: neonates, knowledge, CPAP usage

Ventilation during extracorporeal support : Why and how.

The main target of extracorporeal support is to achieve viable gas exchange, while minimizing the risk of ventilator-induced lung injury, achieved through adecreased mechanical ventilation load on the natural lung. However, during veno-venous extracorporeal membrane oxygenation (ECMO), mechanical ventilation is still necessary in order to prevent lung collapse and/or if extracorporeal blood flow is not sufficient to guarantee adequate gas exchange. In this review, we will summarize the physiology of extracorporeal support and the rationale for continuing mechanical ventilation in this context. Furthermore, we will review the current clinical practice among ECMO centers and their suggestions regarding mechanical ventilator settings. While optimal ventilatory settings are still amatter of debate, the use of astrategy combining low tidal volume and limited inspiratory pressures is accepted worldwide. On the contrary, the choice of applied positive end-expiratory pressure (PEEP) varies between the total rest strategy and open lung strategy. Finally, the use of assisted or spontaneous ventilation will be discussed.

EMC3 coordinates surfactant protein and lipid homeostasis required for respiration.

Adaptation to respiration at birth depends upon the synthesis of pulmonary surfactant, a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle. Herein, we demonstrated that the gene encoding a subunit of the endoplasmic reticulum membrane complex, EMC3, also known as TMEM111 (Emc3/Tmem111), was required for murine pulmonary surfactant synthesis and lung function at birth. Conditional deletion of Emc3 in murine embryonic lung epithelial cells disrupted the synthesis and packaging of surfactant lipids and proteins, impaired the formation of lamellar bodies, and induced the unfolded protein response in alveolar type 2 (AT2) cells. EMC3 was essential for the processing and routing of surfactant proteins, SP-B and SP-C, and the biogenesis of the phospholipid transport protein ABCA3. Transcriptomic, lipidomic, and proteomic analyses demonstrated that EMC3 coordinates the assembly of lipids and proteins in AT2 cells that is necessary for surfactant synthesis and function at birth.

Structural and Functional Insight into Recombinant Lung Surfactant Protein B (rSP-B)

Surfactant associated-protein B (SP-B) is the most essential protein component of lung surfactant; its absence is fatal. Lung surfactant associated proteins, especially SP-B, assist in the organization and rearrangement of the lipid monolayers and multilayers that coat the air-water interface of the alveolus, and so help to reduce the work of breathing and prevent lung collapse. Due to SP-B's unique hydrophobic nature however, the overall three dimensional structure of the protein is not yet determined, therefore the molecular basis for its activity is not clearly understood. Recently, our lab has managed to produce the protein in bacteria using recombinant DNA technology. Recombinant SP-B (rSP-B) is shown to retain the expected alpha helical conformation in different membrane mimetic conditions, over a range of temperatures as observed by far UV circular dichroism (CD) spectroscopy. Dynamic Light Scattering (DLS) of rSP-B suspended in detergent micelles indicates two populations, one with a hydrodynamic size of ∼3 nm and the other at ∼100 nm. Nanoparticle Tracking Analysis (NTA) confirmed the ∼100 nm species to be prominent. Preliminary 1D solution NMR experiments have also been carried out.Moreover, rSP-B also shows promising results in in vitro measures of functionality when tested with a Langmuir-Blodgett trough. Lipid films in the presence of rSP-B are able to promote multilayer formation when compressed to a sufficiently high surface pressure, comparable to that of clinical lung surfactants. What is more, films in the presence of rSP-B demonstrate superior compressibility and film recovery when compared to films compressed without rSP-B.

The Diaphragm Acts as a Brake during Expiration to Prevent Lung Collapse.

The diaphragm is the major inspiratory muscle and is assumed to relax during expiration. However, electrical postinspiratory activity has been observed. Whether there is an expiratory diaphragmatic contraction that preserves lung patency has yet to be explored. We hypothesized the occurrence of an expiratory diaphragmatic contraction directed at stabilizing peripheral airways and preventing or reducing cyclic expiratory lung collapse. Mild acute respiratory distress syndrome was induced in 10 anesthetized, spontaneously breathing pigs. Lung volume was decreased by lowering end-expiratory airway pressure in a stepwise manner. We recorded the diaphragmatic electric activity during expiration, dynamic computed tomographic scans, and respiratory mechanics. In five pigs, the same protocol was repeated during mechanical ventilation after muscle paralysis. Diaphragmatic electric activity during expiration increased by decreasing end-expiratory lung volume during spontaneous breathing. This enhanced the diaphragm muscle force, to a greater extent with lower lung volume, indicating a diaphragmatic electromechanical coupling during spontaneous expiration. In turn, the resulting diaphragmatic contraction delayed and reduced the expiratory collapse and increased lung aeration compared with mechanical ventilation with muscle paralysis and absence of diaphragmatic activity. The diaphragm is an important regulator of expiration. Its expiratory activity seems to preserve lung volume and to protect against lung collapse. The loss of diaphragmatic expiratory contraction during mechanical ventilation and muscle paralysis may be a contributing factor to unsuccessful respiratory support.

The 30-year evolution of airway pressure release ventilation (APRV).

Airway pressure release ventilation (APRV) was first described in 1987 and defined as continuous positive airway pressure (CPAP) with a brief release while allowing the patient to spontaneously breathe throughout the respiratory cycle. The current understanding of the optimal strategy to minimize ventilator-induced lung injury is to “open the lung and keep it open”. APRV should be ideal for this strategy with the prolonged CPAP duration recruiting the lung and the minimal release duration preventing lung collapse. However, APRV is inconsistently defined with significant variation in the settings used in experimental studies and in clinical practice. The goal of this review was to analyze the published literature and determine APRV efficacy as a lung-protective strategy. We reviewed all original articles in which the authors stated that APRV was used. The primary analysis was to correlate APRV settings with physiologic and clinical outcomes. Results showed that there was tremendous variation in settings that were all defined as APRV, particularly CPAP and release phase duration and the parameters used to guide these settings. Thus, it was impossible to assess efficacy of a single strategy since almost none of the APRV settings were identical. Therefore, we divided all APRV studies divided into two basic categories: (1) fixed-setting APRV (F-APRV) in which the release phase is set and left constant; and (2) personalized-APRV (P-APRV) in which the release phase is set based on changes in lung mechanics using the slope of the expiratory flow curve. Results showed that in no study was there a statistically significant worse outcome with APRV, regardless of the settings (F-ARPV or P-APRV). Multiple studies demonstrated that P-APRV stabilizes alveoli and reduces the incidence of acute respiratory distress syndrome (ARDS) in clinically relevant animal models and in trauma patients. In conclusion, over the 30 years since the mode’s inception there have been no strict criteria in defining a mechanical breath as being APRV. P-APRV has shown great promise as a highly lung-protective ventilation strategy.

Failure of noninvasive ventilation in adult patients with acute asthma exacerbation.

Failure of noninvasive ventilation in adult patients with acute asthma exacerbation.

Time required for gas exchange equilibration after a change of positive end-expiratory pressure in acute respiratory distress syndrome

Positive end-expiratory pressure (PEEP) is fundamental to prevent lung collapse in ARDS patients. A common method to titrate PEEP is to perform a PEEP test, recording the variation of cardiorespiratory parameters after a PEEP change [1]. The aim of this study was to evaluate the time-course changes of gas exchanges following a PEEP test.

Visual anatomical lung CT scan assessment of lung recruitability

The computation of lung recruitability in acute respiratory distress syndrome (ARDS) is advocated to set positive end-expiratory pressure (PEEP) for preventing lung collapse. The quantitative lung CT scan, obtained by manual image processing, is the reference method but it is time consuming. The aim of this study was to evaluate the accuracy of a visual anatomical analysis compared with a quantitative lung CT scan analysis in assessing lung recruitability. Fifty sets of two complete lung CT scans of ALI/ARDS patients computing lung recruitment were analyzed. Lung recruitability computed at an airway pressure of 5 and 45 cm H(2)O was defined as the percentage decrease in the collapsed/consolidated lung parenchyma assessed by two expert radiologists using a visual anatomical analysis and as the decrease in not aerated lung regions using a quantitative analysis computed by dedicated software. Lung recruitability was 11.3 % (interquartile range 7.39-16.41) and 15.5 % (interquartile range 8.18-21.43) with the visual anatomical and quantitative analysis, respectively. In the Bland-Altman analysis, the bias and agreement bands between the visual anatomical and quantitative analysis were -2.9 % (-11.8 to +5.9 %). The ROC curve showed that the optimal cutoff values for the visual anatomical analysis in predicting high versus low lung recruitability was 8.9 % (area under the ROC curve 0.9248, 95 % CI 0.8550-0.9946). Considering this cutoff, the sensitivity, specificity, and diagnostic accuracy were 0.96, 0.76, and 0.86, respectively. Visual anatomical analysis can classify patients into those with high and low lung recruitability allowing more intensivists to get access to lung recruitability assessment.

Investigation of IL-6 Effects on SP-A Expression in A549 Lung Cell Line

ABSTRACTInterleukin-6 (IL-6) is a multifunctional cytokine that is important in inflammatory processes, cell differentiation and growth.Pulmonary surfactant is a protein-lipid complex, which reduce surface tension at the alveolar air-liquid interface, thereby preventing lung collapse. Surfactant protein A (SP-A) is the most abundant of four surfactant proteins (SP-A, SP-B, SP-C and SP-D). It binds to alveolar macrophages, stimulates their chemotaxis and serves as an opsonin in the phagocytosis.Low SP-A levels are associated with increased inflammation in the lung. In the absence of infection SP-A inhibited IL-6 production. In infected macrophages, SP-A augmented IL-6 production. In humans, SP-A levels are altered by a number of disease states. In some of them there is increased levels of SP-A in the bronchoalveolar lavage fluid.Here we investigate presence/absence of correlation between increased levels of IL-6 and production of SP-A from A549 lung cell line. Our results suggest that incubation of A549 cells with IL-6 for different time changes significantly the expression levels of SP-A.

Surfactant and its Role in the Upper Respiratory System and Eustachian Tube

Surfactant and its Role in the Upper Respiratory System and Eustachian TubeSurfactant research was originally directed toward lung mechanics, however, with growing information on the biology of the surfactant system it has expanded beyond the borders of basic physiology. The research has become interdisciplinary, not only considering aspects of lungs biology relevant for breathing, but also those aspects relevant for airway defence.Surfactant consists mainly of phospholipids that lower the alveolar surface tension to prevent lung collapse at expiration. They also support mechanical elimination of inhaled pathogens by reducing the viscosity of airway mucus. Approximately 8-10% of surfactant is made up of proteins. Among them, specific proteins SP-A and SP-D play a crucial role in the innate defence system. They belong to collectins family and serve as the first step in immune response to inhaled pathogens. In limited extent, SP-B and SP-C are also involved in immunomodulation.Although numerous studies have focused on the physiological function of surfactant in the lower airways, relatively little is known about its role in the upper respiratory system. Identification of lamellar bodies in ciliated epithelium of the upper airways indicates that surfactant may have a role in normal sinonasal function and pathology. Decreased levels of the

Establishing and Maintaining an FRC at Birth

The transition to life after birth is dependent upon airway liquid clearance by allowing pulmonary gas exchange to begin. This initiates major changes in cardiovascular physiology that allows the infant to successfully adapt to life after birth. As lung aeration is critical for the transition to air-breathing at birth, it is essential to understand the mechanisms involved to develop procedures that facilitate these processes in very preterm infants.We have used phase contrast X-ray imaging to image air entry into lungs at birth and have investigated ventilation procedures that facilitate uniform lung aeration and prevent lung collapse in very preterm rabbits. Images acquired during spontaneous breathing demonstrate that lung aeration is mainly driven by transpulmonary pressure gradients, which provides a new understanding of how aeration can be facilitated in preterm infants. An initial sustained inflation (SI; 20 secs) over comes the long time constant required to move liquid through the airways and fully recruit FRC and tidal volume before the onset of tidal breathing. It is currently unclear if multiple shorter (3 secs) SIs are as effective as a single long SI and, if not, how do we know when the lung has successfully aerated. Furthermore, we have investigated the role of additional factors, such as the rate of expiratory gas flow, on the maintenance of FRC after birth.These studies are clarifying the primary factors regulating airway liquid clearance and lung aeration at birth and provide a new understanding of how this can be facilitated in preterm infants.

Jamming dynamics of stretch-induced surfactant release by alveolar type II cells

Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less surfactant than unstretched cells; however, stretched cells secreted significantly more surfactant than unstretched cells after an extended lag period. We developed a model based on the hypothesis that stretching leads to jamming of surfactant traffic escaping the cell, similar to vehicular traffic jams. In the model, stretch increases surfactant transport from the interior to the exterior of the cell. This transport is mediated by a surface layer with a finite capacity due to the limited number of fusion pores through which secretion occurs. When the amount of surfactant in the surface layer approaches this capacity, interference among lamellar bodies carrying surfactant reduces the rate of secretion, effectively creating a jam. When the stretch stops, the jam takes an extended time to clear, and subsequently the amount of secreted surfactant increases. We solved the model analytically and show that its dynamics are consistent with experimental observations, implying that surfactant secretion is a fundamentally nonlinear process with memory representing collective behavior at the level of single cells. Our results thus highlight the importance of a jamming dynamics in stretch-induced cellular secretory processes.

Reflexes from pulmonary arterial baroreceptors in dogs: interaction with carotid sinus baroreceptors

In contrast to the reflex vasodilatation occurring in response to stimulation of baroreceptors in the aortic arch, carotid sinuses and coronary arteries, stimulation of receptors in the wall of pulmonary arteries results in reflex systemic vasoconstriction. It is rare for interventions to activate only one reflexogenic region, therefore we investigated how these two types of reflexes interact. In anaesthetized dogs connected to cardiopulmonary bypass, reflexogenic areas of the carotid sinuses, aortic arch and coronary arteries and the pulmonary artery were subjected to independently controlled pressures. Systemic perfusion pressure (SPP) measured in the descending aorta (constant flow) provided an index of systemic vascular resistance. In other experiments, sympathetic efferent neural activity was recorded in fibres dissected from the renal nerve (RSNA). Physiological increases in pulmonary arterial pressure (PAP) induced significant increases in SPP (+39.1 ± 10.4 mmHg) and RSNA (+17.6 ± 2.2 impulses s(−1)) whereas increases in carotid sinus pressure (CSP) induced significant decreases in SPP (−42.6 ± 10.8 mmHg) and RSNA (−42.8 ± 18.2 impulses s(−1)) (P < 0.05 for each comparison; paired t test). To examine possible interactions, PAP was changed at different levels of CSP in both studies. With CSP controlled at 124 ± 2 mmHg, the threshold, 'set point' and saturation pressures of the PAP–SPP relationship were higher than those with CSP at 60 ± 1 mmHg; this rightward shift was associated with a significant decrease in the reflex gain. Similarly, increasing CSP produced a rightward shift of the PAP–RSNA relationship, although the effect on reflex gain was inconsistent. Furthermore, the responses to changes in CSP were influenced by setting PAP at different levels; increasing the level of PAP from 5 ± 1 to 33 ± 3 mmHg significantly increased the set point and threshold pressures of the CSP–SPP relationship; the reflex gain was not affected. These results indicate the existence of interaction between pulmonary arterial and carotid sinus baroreceptor reflexes; physiological and pathological states that alter the stimulus to one may alter the reflex responses from the other.

Solid State NMR Studies of Lung Surfactant Protein B Fragment, Mini-B, in Mechanically Oriented Lipid Bilayers

Lung surfactant, a lipid protein complex, prevents lung collapse by lowering the surface tension at the alveolar air-water interface. Surfactant protein B (SP-B) is an essential component of lung surfactant and is indispensable for life. Mini-B is a two-helix fragment of lung surfactant protein SP-B which retains significant biological activity compare to the full-length protein SP-B. Solid-state 2H-NMR was used to characterize mechanically oriented lipid bilayers composed of POPC-d31/POPG or DPPC-d62 doped BLES, with and without associated Mini-B. The spectra were interpreted with the help from orientational order parameter profiles. The results indicated that 1) the order parameters were greater when the number of plates were increased 2) DPPC-d62 in BLES is more ordered than POPC-d31/POPG and 3) Mini-B tends to disrupt the orientation of bilayers and reduce the order in the fraction of the sample that remain oriented. Even in the presence of Mini-B, the lipid bilayers could be well oriented by optimizing plate dimension, amount of sample between the plates and volume of the solvent used to dissolve the organic solvent. These well-oriented samples are being used in further studies of lipid headgroup orientation, as indicated by 31P NMR, as well as for studies of 15N-lebled peptides, to indicate the tilt angle of the peptide's helices within the bilayer.