Structural aspects of postnatal lung development - alveolar formation and growth

The human lung is born with a fraction of the adult complement of alveoli. The postnatal stages of human lung development comprise an alveolar stage, a stage of microvascular maturation, and very likely a stage of late alveolarization. The characteristic structural features of the alveolar stage are well known; they are very alike in human and rat lungs. The bases for alveolar formation are represented by immature inter-airspace walls with two capillary layers with a central sheet of connective tissue. Interalveolar septa are formed by folding up of one of the two capillary layers. In the alveolar stage, alveolar formation occurs rapidly and is typically very conspicuous in both species; it has therefore been termed 'bulk alveolarization'. During and after alveolarization the septa with double capillary networks are restructured to the mature form with a single network. This happens in the stage of microvascular maturation. After these steps the lung proceeds to a phase of growth during which capillary growth by intussusception plays an important role in supporting gas exchange. In view of reports that alveoli are added after the stage of microvascular maturation, the question arises whether the present concept of alveolar formation needs revision. On the basis of morphological and experimental findings we can state that mature lungs contain all the features needed for 'late alveolarization' by the classical septation process. Because of the high plasticity of the lung tissues, late alveolarization or some forms of compensatory alveolar formation may be considered for the human lung.

Maintenance of end-expiratory recruitment with increased respiratory rate after saline-lavage lung injury

Cyclical recruitment of atelectasis with each breath is thought to contribute to ventilator-associated lung injury. Extrinsic positive end-expiratory pressure (PEEPe) can maintain alveolar recruitment at end exhalation, but PEEPe depresses cardiac output and increases overdistension. Short exhalation times can also maintain end-expiratory recruitment, but if the mechanism of this recruitment is generation of intrinsic PEEP (PEEPi), there would be little advantage compared with PEEPe. In seven New Zealand White rabbits, we compared recruitment from increased respiratory rate (RR) to recruitment from increased PEEPe after saline lavage. Rabbits were ventilated in pressure control mode with a fraction of inspired O(2) (Fi(O(2))) of 1.0, inspiratory-to-expiratory ratio of 2:1, and plateau pressure of 28 cmH(2)O, and either 1) high RR (24) and low PEEPe (3.5) or 2) low RR (7) and high PEEPe (14). We assessed cyclical lung recruitment with a fast arterial Po(2) probe, and we assessed average recruitment with blood gas data. We measured PEEPi, cardiac output, and mixed venous saturation at each ventilator setting. Recruitment achieved by increased RR and short exhalation time was nearly equivalent to recruitment achieved by increased PEEPe. The short exhalation time at increased RR, however, did not generate PEEPi. Cardiac output was increased on average 13% in the high RR group compared with the high PEEPe group (P < 0.001), and mixed venous saturation was consistently greater in the high RR group (P < 0.001). Prevention of end-expiratory derecruitment without increased end-expiratory pressure suggests that another mechanism, distinct from intrinsic PEEP, plays a role in the dynamic behavior of atelectasis.

The effect of cementing technique on structural fixation of pegged glenoid components in total shoulder arthroplasty

Although loosening of cemented glenoid components is one of the major complications of total shoulder arthroplasty, there is little information about factors affecting initial fixation of these components in the scapular neck. This study was performed to assess the characteristics of structural fixation of pegged glenoid components, if inserted with two different recommended cementing techniques. Six fresh-frozen shoulder specimens and two types of glenoid components were used. The glenoids were prepared according to the instructions and with the instrumentation of the manufacturer. In 3 specimens, the bone cement was inserted into the peg receiving holes (n = 12) and applied to the back surface of the glenoid component with a syringe. In the other 3 specimens, the cement was inserted into the holes (n = 15) by use of pure finger pressure: no cement was applied on the backside of the component. Micro-computed tomography scans with a resolution of 36 microm showed an intact cement mantle around all 12 pegs (100%) when a syringe was used. An incomplete cement plug was found in 7 of 15 pegs (47%) when the finger-pressure technique was used. Cement penetration into the cancellous bone was deeper in osteopenic bone. Application of bone cement on the backside of the glenoid prosthesis improved seating by filling out small spaces between bone and polyethylene resulting from irregularities after reaming or local cement extrusion from a drill hole. The fixation of a pegged glenoid component is better if the holes are filled with cement under pressure by use of a syringe and if cement is applied to the back of the glenoid component than if cement is inserted with pure finger pressure and no cement is applied to the back surface of the component.

Long-term outcome after structural failure of rotator cuff repairs

BACKGROUND: In a previous study, twenty consecutive patients with a rerupture of the rotator cuff, as documented with magnetic resonance imaging, were found to have significantly less pain and better function and strength, compared with the preoperative state, at 3.2 years postoperatively. It was the purpose of this study to determine the clinical and structural outcomes of these reruptures in the same twenty patients after a longer period of follow-up. METHODS: At a mean of 7.6 years postoperatively, the twenty patients were reexamined clinically and with standard radiographs and magnetic resonance imaging with use of the same clinical, radiographic, and magnetic resonance imaging criteria as were utilized in the review at 3.2 years. The mean age at the time of final follow-up was sixty-six years. RESULTS: Nineteen of the twenty patients continued to be either very satisfied or satisfied with the outcome. The relative Constant score averaged 88% and was not significantly different from the score at 3.2 years, which averaged 83%. The mean scores for pain, function, and strength also had not changed significantly. Overall, the twenty reruptures had not increased in size, and eight of them had healed structurally at the time of the 7.6-year follow-up. Seven of these eight reruptures had been of the supraspinatus tendon only, and seven had been smaller than 400 mm(2) at 3.2 years. Twelve reruptures persisted, and five were larger than the preoperative tear. Fatty infiltration of the infraspinatus muscle progressed significantly (p = 0.015) and the acromiohumeral distance decreased significantly (p = 0.006) between the two follow-up periods. Neither fatty infiltration of the supraspinatus and subscapularis muscles nor glenohumeral osteoarthritis progressed significantly. CONCLUSIONS: At an average of 7.6 years, the clinical outcomes after structural failure of rotator cuff repairs remained significantly improved over the preoperative state in terms of pain, function, strength, and patient satisfaction. Overall, the reruptures that had been present at 3.2 years did not increase in size. We also found that reruptures of the supraspinatus that had been smaller than 400 mm(2) had the potential to heal.

Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.

Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

GH mutant (R77C) in a pedigree presenting with the delay of growth and pubertal development: structural analysis of the mutant and evaluation of the biological activity

A heterozygous missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C), which was previously reported to have some GH antagonistic effect, was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SDS) at the age of 6 years. His mother and grandfather were also carrying the same mutation, but did not differ in adult height from the other unaffected family members. Hormonal examination in all affected subjects revealed increased basal GH, low IGF-I concentrations, and subnormal IGF-I response in generation test leading to the diagnosis of partial GH insensitivity. However, GH receptor gene (GHR) sequencing demonstrated no abnormalities. As other family members carrying the GH-R77C form showed similar alterations at the hormonal level, but presented with normal final height, no GH therapy was given to the boy, but he was followed through his pubertal development which was delayed. At the age of 20 years he reached his final height, which was normal within his parental target height. Functional characterization of the GH-R77C, assessed through activation of Jak2/Stat5 pathway, revealed no differences in the bioactivity between wild-type-GH (wt-GH) and GH-R77C. Detailed structural analysis indicated that the structure of GH-R77C, in terms of disulfide bond formation, is almost identical to that of the wt-GH despite the introduced mutation (Cys77). Previous studies from our group demonstrated a reduced capability of GH-R77C to induce GHR/GH-binding protein (GHBP) gene transcription rate when compared with wt-GH. Therefore, reduced GHR/GHBP expression might well be the possible cause for the partial GH insensitivity found in our patients. In addition, this group of patients deserve further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity. This might be responsible for the delay of growth and pubertal development. Finally, we clearly demonstrate that GH-R77C is not invariably associated with short stature, but that great care needs to be taken in ascribing growth failure to various heterozygous mutations affecting the GH-IGF axis and that careful functional studies are mandatory.

Regional structural characteristics of bovine periodontal ligament samples and their suitability for biomechanical tests

Mechanical testing of the periodontal ligament requires a practical experimental model. Bovine teeth are advantageous in terms of size and availability, but information is lacking as to the anatomy and histology of their periodontium. The aim of this study, therefore, was to characterize the anatomy and histology of the attachment apparatus in fully erupted bovine mandibular first molars. A total of 13 teeth were processed for the production of undecalcified ground sections and decalcified semi-thin sections, for NaOH maceration, and for polarized light microscopy. Histomorphometric measurements relevant to the mechanical behavior of the periodontal ligament included width, number, size and area fraction of blood vessels and fractal analysis of the two hard-soft tissue interfaces. The histological and histomorphometric analyses were performed at four different root depths and at six circumferential locations around the distal and mesial roots. The variety of techniques applied provided a comprehensive view of the tissue architecture of the bovine periodontal ligament. Marked regional variations were observed in width, surface geometry of the two bordering hard tissues (cementum and alveolar bone), structural organization of the principal periodontal ligament connective tissue fibers, size, number and numerical density of blood vessels in the periodontal ligament. No predictable pattern was observed, except for a statistically significant increase in the area fraction of blood vessels from apical to coronal. The periodontal ligament width was up to three times wider in bovine teeth than in human teeth. The fractal analyses were in agreement with the histological observations showing frequent signs of remodeling activity in the alveolar bone - a finding which may be related to the magnitude and direction of occlusal forces in ruminants. Although samples from the apical root portion are not suitable for biomechanical testing, all other levels in the buccal and lingual aspects of the mesial and distal roots may be considered. The bucco-mesial aspect of the distal root appears to be the most suitable location.

Severe structural damage of the seemingly non-diseased infrarenal aortic aneurysm neck

OBJECTIVE: The success of open and endovascular repair of abdominal aortic aneurysms (AAA) is hampered by postoperative dilatation of the anatomical neck of the AAA, which is used for graft attachment. The purpose of this study was to determine whether the macroscopically non-diseased infrarenal aortic neck of AAA is histologically and biochemically altered at the time of operative repair. METHODS: We harvested full-thickness aortic wall samples as longitudinal stripes spanning from AAA neck to aneurysmal sac in 22 consecutive patients undergoing open surgical AAA repair. Control tissue was obtained from five organ donors and five deceased subjects undergoing autopsy without evidence of aneurysmal disease. We assessed aortic media thickness, number of intact elastic lamellar units, media destruction, and neovascularization grade and performed immunohistochemistry for matrix metalloproteinase (MMP)-9 and phosphorylated c-Jun N-terminal kinase (p-JNK). MMP-9 and p-JNK protein expressions were quantified using Western Blots. RESULTS: The median thickness of the aortic media was 1150 mum in control tissue (range, 1000-1300), 510 mum in aortic necks (250-900), and 200 mum in aortic sacs (50-500, P from nonparametric test for trend <.001). The number of intact elastic lamellar units was 33 in controls (range, 33-55), 12 in aortic necks (0-31) and three in aortic sacs (0-10, P < .001). The expression of MMP-9 and p-JNK as assessed by Western Blots (P = .007 and .061, respectively) and zymography (P for trend <.001) were up regulated in both the AAA neck and sac compared with controls. Except for p-JNK expression, differences between tissues were similar after the adjustment for age, gender, and type of sampling. CONCLUSION: The seemingly non-diseased infrarenal AAA neck in patients with AAA undergoing surgical repair shows histological signs of destruction and upregulation of potential drug targets.

Structural capacity of steel tubular cast-in-place piling

Steel tubular cast-in-place pilings are used throughout the country for many different project types. These piles are a closed-end pipe with varying wall thicknesses and outer diameters, that are driven to depth and then the core is filled with concrete. These piles are typically used for smaller bridges, or secondary structures. Mostly the piling is designed based on a resistance based method which is a function of the soil properties of which the pile is driven through, however there is a structural capacity of these members that is considered to be the upper bound on the loading of the member. This structural capacity is given by the AASHTO LRFD (2010), with two methods. These two methods are based on a composite or non-composite section. Many state agencies and corporations use the non-composite equation because it is requires much less computation and is known to be conservative. However with the trends of the time, more and more structural elements are being investigated to determine ways to better understand the mechanics of the members, which could lead to more efficient and safer designs. In this project, a set of these piling are investigated. The way the cross section reacts to several different loading conditions, along with a more detailed observation of the material properties is considered as part of this research. The evaluation consisted of testing stub sections of pile with varying sizes (10-¾”, 12-¾”), wall thicknesses (0.375”, 0.5”), and testing methods (whole compression, composite compression, push through, core sampling). These stub sections were chosen as they would represent a similar bracing length to many different soils. In addition, a finite element model was developed using ANSYS to predict the strains from the testing of the pile cross sections. This model was able to simulate the strains from most of the loading conditions and sizes that were tested. The bond between the steel shell and the concrete core, along with the concrete strength through the depth of the cross section were some of the material properties of these sections that were investigated.

Structural characterization of water-soluble atmospheric organic matter by ultrahigh-resolution mass spectrometry

Isolated water-soluble analytes extracted from fog water collected during a radiation fog event near Fresno, CA were analyzed using collision induced dissociation and ultrahigh-resolution mass spectrometry. Tandem mass analysis was performed on scan ranges between 100-400 u to characterize the structures of nitrogen and/or sulfur containing species. CHNO, CHOS, and CHNOS compounds were targeted specifically because of the high number of oxygen atoms contained in their molecular formulas. The presence of 22 neutral losses corresponding to fragment ions was evaluated for each of the 1308 precursors. Priority neutral losses represent specific polar functional groups (H2O, CO2, CH3OH, HNO3, SO3, etc., and several combinations of these). Additional neutral losses represent non-specific functional groups (CO, CH2O, C3H8, etc.) Five distinct monoterpene derived organonitrates, organosulfates, and nitroxy-organosulfates were observed in this study, including C10H16O7S, C10H17NO7S, C10H17 NO8S, C10H17NO9S, and C10H17NO10S. Nitrophenols and linear alkyl benzene sulfonates were present in high abundance. Liquid chromatography/mass spectrometery methodology was developed to isolate and quantify nitrophenols based on their fragmentation behavior.

Secondary breast reconstruction with deepithelialized free flaps from the lower abdomen for intractable capsular contracture and maintenance of breast volume

Although surgical techniques and the quality of mammary prostheses have been improved significantly in recent years, capsular contracture attendant on prosthetic mammary reconstruction remains a major flaw. Although rarely, some patients are confronted with recurrent and intractable capsular contractures with resultant breast deformity, even after multiple attempts at capsulectomies and implant exchange. Patients with recurrent capsular contracture often do not want replacement with a new prosthesis, but desire the maintenance of their breast volume with a safe alternative. In an attempt to maintain breast volume and to improve the aesthetic appearance, secondary breast reconstruction using bilateral deepithelialized free flaps from the lower abdomen was performed in a series of seven patients. Three bilateral muscle-sparing TRAM flaps, two bilateral DIEP flaps, one bilateral SIEA flap, one unilateral SIEA flap, and one unilateral DIEP flap (a total number of 14 flaps) were used following implant removal, total capsulectomy, and prophylactic subcutaneous mastectomy. The early postoperative course was uneventful, and all flaps survived completely with no complications. There were no donor-site problems, except in one patient (case 5), who had partial skin necrosis of the abdominal flap. The long-term results (mean follow-up: 4.8 years) demonstrated an aesthetically satisfactory appearance of the breasts, with no major donor-site problems. Several advantages, as well as drawbacks, are highlighted with this technique.

Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.