Proteins from halophilic organisms, which live in extreme saline conditions, have evolved to remain folded at very high ionic strengths. The surfaces of halophilic proteins show a biased amino acid ...composition with a high prevalence of aspartic and glutamic acids, a low frequency of lysine, and a high occurrence of amino acids with a low hydrophobic character. Using extensive mutational studies on the protein surfaces, we show that it is possible to decrease the salt dependence of a typical halophilic protein to the level of a mesophilic form and engineer a protein from a mesophilic organism into an obligate halophilic form. NMR studies demonstrate complete preservation of the three-dimensional structure of extreme mutants and confirm that salt dependency is conferred exclusively by surface residues. In spite of the statistically established fact that most halophilic proteins are strongly acidic, analysis of a very large number of mutants showed that the effect of salt on protein stability is largely independent of the total protein charge. Conversely, we quantitatively demonstrate that halophilicity is directly related to a decrease in the accessible surface area.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein N-glycosylation stands out for its intrinsic and functionally related heterogeneity. Despite its biomedical interest, Glycoprofile analysis still remains a major scientific challenge. Here, ...we present an NMR-based strategy to delineate the N-glycan composition in intact glycoproteins and under physiological conditions. The employed methodology allowed dissecting the glycan pattern of the IgE high-affinity receptor (FcεRIα) expressed in human HEK 293 cells, identifying the presence and relative abundance of specific glycan epitopes. Chemical shifts and differences in the signal line-broadening between the native and the unfolded states were integrated to build a structural model of FcεRIα that was able to identify intramolecular interactions between high-mannose N-glycans and the protein surface. In turn, complex type N-glycans reflect a large solvent accessibility, suggesting a functional role as interaction sites for receptors. The interaction between intact FcεRIα and the lectin hGal3, also studied here, confirms this hypothesis and opens new avenues for the detection of specific N-glycan epitopes and for the studies of glycoprotein–receptor interactions mediated by N-glycans.
Patients with major forms of acute hepatic porphyria present acute neurological attacks with overproduction of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA). Even if ALA is considered the ...most likely agent inducing the acute symptoms, the mechanism of its accumulation has not been experimentally demonstrated. In the most frequent form, acute intermittent porphyria (AIP), inherited gene mutations induce a deficiency in PBG deaminase; thus, accumulation of the substrate PBG is biochemically obligated but not that of ALA. A similar scenario is observed in other forms of acute hepatic porphyria (i.e., porphyria variegate, VP) in which PBG deaminase is inhibited by metabolic intermediates. Here, we have investigated the molecular basis of δ-aminolevulinate accumulation using in vitro fluxomics monitored by NMR spectroscopy and other biophysical techniques. Our results show that porphobilinogen, the natural product of δ-aminolevulinate deaminase, effectively inhibits its anabolic enzyme at abnormally low concentrations. Structurally, this high affinity can be explained by the interactions that porphobilinogen generates with the active site, most of them shared with the substrate. Enzymatically, our flux analysis of an altered heme pathway demonstrates that a minimum accumulation of porphobilinogen will immediately trigger the accumulation of δ-aminolevulinate, a long-lasting observation in patients suffering from acute porphyrias.
Introduction
Endobronchial tuberculosis (EBTB) can lead to bronchopulmonary complications when diagnosis is delayed. Bronchoscopic treatment in children can be challenging due to small airway size. ...We report our experience treating children with EBTB.
Methods
Retrospective study (2014−2020) of patients diagnosed with EBTB. Flexible bronchoscopy (FB) was performed in patients with previous diagnosis of pulmonary tuberculosis (PTB), after respiratory/radiological worsening was observed in spite of medical treatment. Treatment consisted in oral corticotherapy in all patients, and interventional bronchoscopy in selected cases. Our aim is to describe the endoscopic findings, interventional bronchoscopy alternatives, and outcome.
Results
Of 45 patients with PTB, 13 (28.9%, 7 M/6 F) were diagnosed with EBTB, with a mean age of 3.9 years (0.4−12.8). Four bronchoscopic patterns were observed. Endobronchial granuloma (N:9; 69.2%): Excision with rigid bronchoscopy was achieved in five (1−5 procedures per patient), while corticotherapy alone was preferred in the remaining four due to small size/distal location of the granuloma. Caseum obstruction (2; 15.4%): dense mucous molds were removed with flexible/rigid bronchoscopy (6 and 8 procedures, respectively). Bronchial stenosis (1; 7.7%): two balloon dilatations with mitomycin‐C application were performed. Extrinsic compression (1; 7.7%): oral corticotherapy alone was initiated. One patient developed bronchoscopy‐related complications (pneumothorax requiring thoracic tube 48 h). With a medium follow‐up of 4.6 years (1.8−7.6), three patients developed bronchiectasis while the remaining 10 improved clinically and radiologically.
Conclusion
Bronchoscopic findings in EBTB include granuloma, stenosis, caseum obstruction and external compression. In selected cases, interventional bronchoscopy can minimize long‐term bronchopulmonary complications.
More than 100 mutations in the gene encoding fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1), a metabolic disorder characterized by elevated blood levels of tyrosine. ...Some of these mutations are known to decrease FAH catalytic activity, but the mechanisms of FAH mutation–induced pathogenicity remain poorly understood. Here, using diffusion ordered NMR spectroscopy, cryo-EM, and CD analyses, along with site-directed mutagenesis, enzymatic assays, and molecular dynamics simulations, we investigated the putative role of thermodynamic and kinetic stability in WT FAH and a representative set of 19 missense mutations identified in individuals with HT1. We found that at physiological temperatures and concentrations, WT FAH is in equilibrium between a catalytically active dimer and a monomeric species, with the latter being inactive and prone to oligomerization and aggregation. We also found that the majority of the deleterious mutations reduce the kinetic stability of the enzyme and always accelerate the FAH aggregation pathway. Depending mainly on the position of the amino acid in the structure, pathogenic mutations either reduced the dimer population or decreased the energy barrier that separates the monomer from the aggregate. The mechanistic insights reported here pave the way for the development of pharmacological chaperones that target FAH to tackle the severe disease HT1.
Thoracic surgery in children with coronavirus disease-19 (COVID-19) pulmonary disease is rare, as very limited virus-related lung lesions require intervention. However, some patients may suffer from ...other pulmonary abnormalities that can be worsened by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and they may consequently require lung surgery. COVID-19 affects the indications, surgical procedure, and postsurgical care of these patients.
We present a case of a 14-year-old girl with COVID-19 pulmonary disease and persistent air leak due to right apical bullae that required resection. Clinical, surgical, and safety implications are discussed. The role of thoracic minimally invasive surgery under COVID-19 conditions is also analyzed.
The thoracoscopic procedure was scheduled earlier than normally expected. The surgery was performed in a COVID-19 reserved theatre with neutral pressure and only the necessary personnel was allowed inside. The use of the required personal protective equipment was supervised by an expert nurse before and after the intervention.
The surgeons used a three-port technique to resect the bullae with an endostapler and no mechanical pleural abrasion was added to the procedure. Electrocautery and CO
insufflation were avoided, and a chest drain with a closed-circuit aspiration system was installed before removing the ports. The child was discharged home 3 days later after the removal of the chest drain.
COVID-19 has an impact on the standard indications, surgical strategies and postoperative care of some conditions requiring intervention. Extra safety measures are needed in the operating room to limit the chance of transmission. Minimally invasive surgery for thoracic surgery remains safe if the current safety guidelines are followed closely.
Cardiac compression in pectus excavatum remains difficult to evaluate. We describe the findings with intraoperative transesophageal echocardiography during pectus excavatum correction in pediatric ...patients.
We studied right heart changes during surgical correction of pectus excavatum by transesophageal echocardiograph. Four-D echo was associated to assess morphology of the tricuspid annulus.
Twenty patients were included, mean age 13.5 (+/− 2.9). Mean preoperative Haller Index was 6.3 (+/− 2.63) and mean Correction Index 47.63% (+/− 12.4%). Preoperative transthoracic echocardiography at rest showed mild right heart compression in 6. Correction was gained by Nuss technique in 19, and Taulinoplasty in one. Initial transesophageal echocardiography showed compression of the right heart and deformation of the tricuspid annulus in all. During the sternal elevation, diameters of right atrium, ventricle and tricuspid annulus significantly improved: mean augmentation of right ventricle was 5.78 mm (+/− 3.56 p < 0.05), right atrium 6.64 mm (+/− 5.55 p < 0.05) and tricuspid annulus 6.02 mm (+/− 3.29 p < 0.05). The morphology of the tricuspid annulus in 4D normalized.
Preoperative transthoracic echocardiography at rest underestimates right chamber compression in pediatric patients with pectus excavatum. Surgical correction improves diameters of the right ventricle, right atrium and tricuspid annulus and normalizes the morphology of the tricuspid annulus (4D).
Level III.
Pectus Arcuatum (PA) is a very rare anterior chest wall deformity resulting from premature obliteration of the sternomanubrial junction. Unlike other chest wall deformities, surgical correction ...continues to be the only treatment option. We report our initial experience using the hybrid technique and evaluate the results.
We reviewed all the cases of PA that underwent a hybrid procedure between January 2020 and June 2022.
We combined the Nuss procedure and the modified-Ravitch procedure. All patients underwent either percutaneous or thoracoscopic cryoanalgesia to improve postoperative pain management. The Nuss procedure was performed first. A single bar was inserted under thoracoscopic vision.
Sternochondroplasty was performed through a 3-4-centimeter-long mid-sternal skin incision. After dissecting the sternal insertions of both pectoralis major muscles, bilateral subperichondrial chondrectomies were carried out and a wedge-shaped transverse sternal osteotomy was performed using an oscillating saw, preserving the posterior lamina. Sternal realignment was achieved by securing opposite sides of the osteotomy with two vertical titanium plates.
Three patients with PA were subjected to the hybrid procedure between January 2019 and June 2022. The mean age was 15 years (14-16). No intraoperative complications were encountered. The mean operative time was 200 minutes. Postoperative radiography showed optimal sternal realignment and stabilization. Excellent esthetic results were obtained with high patient satisfaction in all cases. The overall hospital stay ranged from four to five days in all cases with optimal pain control. The mean follow-up was 26.6 months (6-38), recurrence was not noted after bar removal.
The simultaneous correction of the sternal depression, with the insertion of a single pectus bar, during the hybrid procedure enables surgeons to perform a less invasive sternochondroplasty and a single sternal wedge-osteotomy through a smaller and more esthetic mid-sternal skin incision, whilst maintaining chest-wall stability. The hybrid technique is a novel procedure that has been carried out successfully in the 3 cases reported in our study, with excellent esthetic results. To our knowledge cryoanalgesia has not been previously employed to better the postoperative pain management of patients undergoing surgical correction for PA.
Esophagobronchial or esophagopulmonary fistulas are complications after esophageal atresia/tracheoesophageal fistula (EA/TEF) repair. Postoperative scarring and recurrent infections render surgical ...treatment a potentially hazardous undertaking.
This report describes the use of infrared (IR) technology to locate and safely dissect a esophagobronchial fistula in a 11-year-old female with a history of EA/TEF. The patient, who had initially undergone primary repair via thoracotomy, was diagnosed with a late tracheoesophageal fistula (TEF) recurrence at the age of 6 after several pulmonary infections and was subjected to a left thoracoscopy with successful closure of the TEF. Despite both surgical procedures, she presented ongoing respiratory symptoms for several years and was referred to our center as an 11-year-old, where further examinations revealed the presence of a esophagobronchial fistula involving the right upper bronchus. After an unsuccessful attempt at endoscopic closure, surgical treatment via right thoracoscopy was planned. Initially, an endoscopic examination was performed, to catheterize the fistula tract in the mid-thoracic esophagus with the infrared probe. Subsequently, right thoracoscopy was performed in the prone position using four 5 mm trocars. Guided by the IR signal, the fistula tract was easily identified and dissected. Fistula closure was achieved using a 5 mm stapler. The staple line was reinforced with a pleural flap and a right thoracic tube was inserted. The patient was discharged after a four-day hospital stay, without complications. The patient remains asymptomatic to date and has returned to her native Ukraine.
Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3-6 M. To remain folded and active, their ...constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K(+) triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl.
PDF
