Thermal Stabilization of an Endoglucanase by Cyclization

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins

Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions

Acute fibrinous and organising pneumonia: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p>Organising pneumonia is a distinct histopathological entity characterized by intra-alveolar buds of granulation tissue, called Masson bodies, which mainly comprise of activated fibroblasts and loose connective tissue. This histopathologic pattern has been described in idiopathic cases, characterizing cryptogenic organising pneumonia as well as in the context of pulmonary infection, drug-induced pneumonitis and following lung transplantation. Although distinct as a clinical and pathological entity, community organising pneumonia may present with atypical clinical and pathological features, such as intra-alveolar fillings of fibrin balls and organising tissue that resembles acute respiratory distress syndrome or diffuse alveolar damage. The latter characteristics constitute a recently described anatomoclinical entity called acute fibrinous and organising pneumonia.</p> <p>Case presentation</p> <p>Here, we describe a rare case of acute fibrinous and organising pneumonia, in an otherwise healthy 65-year-old Greek woman who complained of dry cough, fever, weight loss and progressive dyspnoea. She had never been a smoker. Her clinical symptoms showed a rapid deterioration in the two weeks before admission, despite a course of oral antibiotics. After excluding infection and malignancy with routine laboratory tests and flexible bronchoscopy, high resolution computed tomography and video assisted thoracoscopic lung biopsy were performed. Diagnosis was based on radiological features typical of community organising pneumonia coupled with pathologic features characteristic of acute fibrinous and organising pneumonia. The patient was treated with corticosteroids and showed excellent clinical and radiological response three months after treatment initiation.</p> <p>Conclusion</p> <p>Acute fibrinous and organising pneumonia is an extremely rare pathologic entity, often misdiagnosed as typical community organising pneumonia. To our knowledge, this is the seventh case of acute fibrinous and organising pneumonia in the literature, with no identifiable cause or association in a female patient, with no underlying lung disease or known exposures and with an unremarkable previous medical history. We highlight the need for careful review of lung biopsies from patients with clinical and radiologic characteristics typical of community organising pneumonia. Although it remains uncertain whether fibrin alters the favourable prognosis and treatment response of community organising pneumonia, it becomes obvious that a thorough pathologic review, apart from establishing the diagnosis of acute fibrinous and organising pneumonia, may predict a more unfavorable outcome therefore alerting the clinician to administer more aggressive and prolonged therapeutic regimens.</p

Uniform deposition of protein incorporated mineral layer on three-dimensional porous polymer scaffolds

Inorganic–organic hybrid materials designed to facilitate bone tissue regeneration use a calcium phosphate mineral layer to encourage cell adhesion, proliferation, and osteogenic differentiation. Mineral formed on porous materials is often discontinuous through the thickness of the scaffold. This study aimed to uniformly coat the pores of three-dimensional (3D) porous, polymer scaffolds with a bone-like mineral layer in addition to uniformly incorporating a model protein within this mineral layer. A filtration system designed to induce simulated body fluid flow through the interstices of 3D polylactic- co -glycolic acid scaffolds (10-mm diameter × 2-mm thickness) illustrated that a uniform, continuous mineral layer can be precipitated on the pore surfaces of a 3D porous structure within 5 days. MicroCT analysis showed increased mineral volume percent (MV%) (7.86 ± 3.25 MV%, p = 0.029) and continuous mineralization of filtered scaffolds compared with two static control groups (floating, 0.16 ± 0.26 MV% and submerged, 0.20 ± 0.01 MV%). Furthermore, the system was effective in coprecipitating a model protein, bone sialoprotein (BSA), within the mineral layer. A 10-fold increase in BSA incorporation was seen when coprecipitated filtered scaffolds (1308 ± 464 Μg) were compared to a submerged static control group (139 ± 45 Μg), p < 0.001. Confocal microscopy visually confirmed uniform coprecipitation of BSA throughout the thickness of the filtration scaffolds. The designed system enables 3D mineralization through the thickness of porous materials, and provides the option of including coprecipitated biomolecular cues within the mineral layer. This approach of providing a 3D conductive and osteoinductive environment could be conducive to bone tissue regeneration. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2008Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57926/1/30877_ftp.pd

Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation

BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays

Effect of ionic activity products on the structure and composition of mineral self assembled on three-dimensional poly(lactide- co -glycolide) scaffolds

A biomimetic approach involving the self-assembly of mineral within the pores of three-dimensional porous polymer scaffolds is a promising strategy to integrate advantages of inorganic and organic phases into a single material for hard tissue engineering. Such a material enhances the ability of progenitor cells to differentiate down an osteoblast lineage in vitro and in vivo , compared with polymer scaffolds. The mechanisms regulating mineral formation in this one-step process, however, are poorly understood, especially the effects of ionic activity products (IP) of the mineralizing solution and incubation time. The aims of this study were to define the structure and composition of mineral formed within the pores of biodegradable polymer scaffolds as a function of IP and time. Three-dimensional poly(lactide- co -glycolide) scaffolds were fabricated by solvent casting/particulate leaching and incubated for 4–16 days in six variants of simulated body fluid whose IPs were varied by adjusting ionic concentrations. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy demonstrated the formation of carbonated apatite with sub-micrometer sized crystals that grew into spherical globules extending out of the scaffold pore surfaces. As IP increased, more mineral grew on the scaffold pore surfaces, but the apatite became less crystalline and the Ca/P molar ratio decreased from 1.63 ± 0.005 to 1.51 ± 0.002. Since morphology, composition, and structure of mineral are factors that affect cell function, this study demonstrates that the IP of the mineralizing solution is an important modulator of material properties, potentially leading to enhanced control of cell function. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57403/1/31437_ftp.pd

‘Classical' but not ‘other' mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer

‘Classical' mutations in the EGFR tyrosine kinase domain (exons 18, 19 and 21) have been associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with NSCLC. The aim of the current study was to evaluate whether other than the classical G719X, DEL19 and L858R mutations of EGFR confer sensitivity to TKIs. Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded tumour tissue from 86 patients treated with gefitinib. Exons 18, 19 and 21 were amplified and subjected to direct sequencing. Eleven (13%) patients harboured the classical exon's 18, 19 and 21 mutations, while 14 (16%) had ‘other' variants. There was a significantly higher percentage of ‘never-smoker' patients with ‘classical' EGFR mutations (P=0.002). Among patients with ‘classical' mutations 3 patients achieved PR and 7 SD, while in the ‘other' mutations group 10 patients had SD as best response. In the wild-type group, there were 3 patients with PR and 25 with SD. Median TTP was 16, 64 (P=0.002) and 21 weeks and median survival was 36, 78 and 67 weeks for patients with wild-type, ‘classical' and ‘other' EGFR mutations, respectively. The clinical relevance of ‘other' EGFR mutation variants remains uncertain and requires further assessment in a prospective study

Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imaging and Treating Choroidal Neovascularization

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV

AA-Amyloidosis Can Be Transferred by Peripheral Blood Monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils (‘amyloid enhancing factor’) combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis

Ribonucleotide reductase subunits M1 and M2 mRNA expression levels and clinical outcome of lung adenocarcinoma patients treated with docetaxel/gemcitabine

Ribonucleotide reductase subunits M1 (RRM1) and M2 (RRM2) are involved in the metabolism of gemcitabine (2′,2′-difluorodeoxycytidine), which is used for the treatment of nonsmall cell lung cancer. The mRNA expression of RRM1 and RRM2 in tumours from lung adenocarcinoma patients treated with docetaxel/gemcitabine was assessed and the results correlated with clinical outcome. RMM1 and RMM2 mRNA levels were determined by quantitative real-time PCR in primary tumours of previously untreated patients with advanced lung adenocarcinoma who were subsequently treated with docetaxel/gemcitabine. Amplification was successful in 42 (79%) of 53 enrolled patients. Low levels of RRM2 mRNA were associated with response to treatment (P< 0.001). Patients with the lowest expression levels of RRM1 had a significantly longer time to progression (P=0.044) and overall survival (P=0.02) than patients with the highest levels. Patients with low levels of both RRM1 and RRM2 had a significantly higher response rate (60 vs 14.2%; P=0.049), time to progression (9.9 vs 2.3 months; P=0.003) and overall survival (15.4 vs 3.6; P=0.031) than patients with high levels of both RRM1 and RRM2. Ribonucleotide reductase subunit M1 and RRM2 mRNA expression in lung adenocarcinoma tumours is associated with clinical outcome to docetaxel/gemcitabine. Prospective studies are warranted to evaluate the role of these markers in tailoring chemotherapy