A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

Thoracic ultrasound combined with low-dose computed tomography may represent useful screening strategy in highly exposed population in the industrial city of Taranto (Italy).

Objectives: We validated a screening protocol in which thoracic ultrasound (TUS) acts as a first-line complementary imaging technique in selecting patients which may deserve a second-line low-dose high resolution computed tomography (HRCT) scan among a population of asymptomatic high-risk subjects for interstitial lung abnormalities (ILA) and lung cancer. Due to heavy environmental pollution burden, the district Tamburi of Taranto has been chosen as "case study" for this purpose. Methods: From July 2018 to October 2020, 677 patients aged between 45 and 65 year and who had been living in the Tamburi district of Taranto for at least 10 years were included in the study. After demographic, clinical and risk factor exposition data were collected, each participant underwent a complete TUS examination. These subjects were then asked to know if they agreed to perform a second-level examination by low-dose HRCT scan. Results: On a total of 167 subjects (24.7%) who agreed to undergo a second-level HRCT, 85 patients (50.9%) actually showed pleuro-pulmonary abnormalities. Interstitial abnormalities were detected in a total of 36 patients on HRCT scan. In particular, 34 participants presented subpleural ILAs, that were classified in the fibrotic subtype in 7 cases. The remaining 2 patients showed non-subpleural interstitial abnormalities. Subpleural nodules were observed in 46 patients. TUS showed an overall diagnostic accuracy of 88.6% in detecting pleuro-pulmonary abnormalities in comparison with HRCT scan, with a sensitivity of 95.3%, a specificity of 81.7%, a positive predictive value of 84.4% and a negative predictive value of 94.4%. The matched evaluation of specific pulmonary abnormalities on HRTC scan (i.e., interstitial abnormalities or pulmonary nodules) with determinate sonographic findings revealed a reduction in both TUS sensibility and specificity. Focusing TUS evaluation on the assessment of interstitial abnormalities, a thickened pleural line showed a sensitivity of 63.9% and a specificity of 69.5%, hypoechoic striae showed a sensitivity of 38.9% and a specificity of 90.1% and subpleural nodules showed a sensitivity of 58.3% and a specificity of 77.1%. Regarding to the assessment of subpleural nodules, TUS showed a sensitivity of 60.9% and a specificity of 81.0%. However, the combined employment of TUS examination and HRCT scans allowed to identify 34 patients with early subpleural ILA and to detect three suspicious pulmonary nodules (of which two were intraparenchymal and one was a large subpleural mass), which revealed to be lung cancers on further investigations. Conclusion: A first-line TUS examination might aid the identification of subjects highly exposed to environmental pollution, who could benefit of a second-line low-dose HRCT scan to find early interstitial lung diseases as well as lung cancer. Protocol registration code: PLEURO-SCREENING-V1.0_15 Feb, 17.

The Porcine Odorant-Binding Protein as a Probe for an Impedenziometric-Based Detection of Benzene in the Environment.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

Effectiveness and Safety of Transthoracic Ultrasound in Guiding Percutaneous Needle Biopsy in the Lung and Comparison vs. CT Scan in Assessing Morphology of Subpleural Consolidations.

(1) Background: The aim of this study was to conduct a prospective analysis on the diagnostic accuracy of transthoracic ultrasound-guided percutaneous needle biopsy (TUS-PNB) for the histological assessment of peripheral lung lesions and to assess the performance of transthoracic ultrasound (TUS) examination vs. chest CT (gold standard) in the differentiation between malignant and benign peripheral lung lesions. (2) Methods: A total of 961 consecutive patients with subpleural pulmonary lesions were enrolled. All the patients received a CT scan with contrast; 762 patients underwent TUS-PTNB for suspicion of malignancy, and the remaining 199 enrolled patients underwent only TUS examination as a part of routine follow-up for known non-malignant subpleural consolidations. (3) Results: Among the 762 TUS-guided biopsies, there were 627 (82.28%) malignant lesions, 82 (10.76%) benign lesions, and 53 (6.96%) indeterminate lesions. The overall diagnostic accuracy was 93.04%. The rates of pneumothorax not requiring chest-tube insertion and self-limited hemoptysis were 0.79 and 0.26%, respectively. Patients were divided into two groups based on the benign or malignant nature of the subpleural consolidations. On TUS, both malignant and benign lesions showed mostly irregular margins and a hypoechoic pattern, but no differences were assessed in terms of sonographic margins and pattern between the two groups. There was poor agreement between TUS and chest CT in assessing air bronchograms and necrotic areas. The only finding in the detection of which TUS showed superiority compared to chest-CT was pleural effusion. (4) Conclusions: TUS-PNB was confirmed to be an effective and safe diagnostic method for peripheral pulmonary consolidation, but their sonographic pattern did not allow to rule out a malignant nature. A pre-operative evaluation on CT images, combined with the possibility of performing additional immunohistochemical and cytological investigations and the experience of the medical staff, may improve the diagnostic yield of TUS-guided biopsies.

Extracellular nicotinate phosphoribosyltransferase binds Toll like receptor 4 and mediates inflammation.

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation.

Sweet Sensor for the Detection of Aflatoxin M1 in Whole Milk.

Recently, there is an increase in interest to develop user-friendly monitoring devices in healthcare, environmental, and agrofood fields for a fast detection of contaminants. Aflatoxins (AFs) are a group of toxic substances produced by the fungi of species Aspergillus that contaminate cereals and dried fruits. When dairy cows ingest feed contaminated with aflatoxin B1 (AFB1), it is metabolized and transformed in the liver into a carcinogenic form aflatoxin M1 (AFM1), which is eliminated through the milk. In this work, we developed a sensor assay to detect low amounts of AFM1 directly in whole milk. For this purpose, we produced monospecific polyclonal antibody (IgGMS-M1) that was able to bind with high avidity to AFM1. Then, we conjugated the antibody to the invertase enzyme from Saccharomyces cerevisiae. This enzyme is able to convert sucrose into fructose and glucose. After incubation of invertase-conjugated anti-AFM1 antibody with milk containing AFM1, we measured the produced glucose by a glucometer. The produced glucose was then correlated to the amount of AFM1 present in the milk. The obtained results show that the assay is easily customizable as a portable instrument for on-site AFM1 measurements. In addition, the results point out that the assay is very sensitive since it can detect the presence of 27 parts per trillion (ppt) of AFM1 in whole milk, a value lower than the AFM1 quantities in milk and dairy products set by the European Commission (50 ppt).

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility.

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5µg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 µM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9µg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

A Fluorescence Polarization Assay To Detect Steroid Hormone Traces in Milk.

Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17ß-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17ß-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17ß-estradiol derivative. The successful detection of 17ß-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety.

Novel biosensors based on optimized glycine oxidase.

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 µm).

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

Assessment of ultrasound acoustic artifacts in patients with acute dyspnea: a multicenter study.

BACKGROUND: Recent reports indicate that numerical assessment of B-lines during transthoracic ultrasound may aid the differential diagnosis of acute diffuse pleuropulmonary disorders. PURPOSE: To determine whether B-lines are different in normal and diseased lungs and whether they can be used to discriminate between different types of pulmonary disorders in acutely ill patients. MATERIAL AND METHODS: In this multicenter study, transthoracic ultrasonography was performed on 193 patients with acute dyspnea, 193 healthy non-smokers, and 58 patients who had undergone pneumonectomy for lung cancer. Examinations were done with a low-medium frequency (3.5-5.0 MHz) convex probe and a high-frequency (8-12.5 MHz) linear probe. Video recordings were re-examined by a second set of examiners. In each participant, we measured the number of B-lines observed per scan. RESULTS: B-lines counts were higher in dyspnoic patients (means: 3.11 per scan per linear probe scan vs. 1.93 in healthy controls and 1.86 in pneumonectomized patients; P < 0.001 for all); all counts were higher when convex probes were used (5.4 in dyspnoic patients and 2 in healthy controls; P < 0.001 vs. the linear probe). Subgroups of dyspnoic patients defined by cause of dyspnea displayed no significant differences in the number of B-lines. CONCLUSION: Our results demonstrate that there are a significant higher number of B-lines in the lungs of patients with dyspnea compared to healthy subjects and to pneumonectomized patients. Nevertheless, the quantification of B-lines does not make any significant contribution to the differential diagnosis of dyspnea.

Absorption into fluorescence. A method to sense biologically relevant gas molecules.

In this work we present an innovative optical sensing methodology based on the use of biomolecules as molecular gating nano-systems. Here, as an example, we report on the detection of analytes related to climate change. In particular, we focused our attention on the detection of nitric oxide (NO) and oxygen (O2). Our methodology builds on the possibility of modulating the excitation intensity of a fluorescent probe used as a transducer and a sensor molecule whose absorption is strongly affected by the binding of an analyte of interest used as a filter. The two simple conditions that have to be fulfilled for the method to work are: (a) the absorption spectrum of the sensor placed inside the cuvette, and acting as the recognition element for the analyte of interest, should strongly change upon the binding of the analyte and (b) the fluorescence dye transducer should exhibit an excitation band which overlaps with one or more absorption bands of the sensor. The absorption band of the sensor affected by the binding of the specific analyte should overlap with the excitation band of the transducer. The high sensitivity of fluorescence detection combined with the use of proteins as highly selective sensors makes this method a powerful basis for the development of a new generation of analytical assays. Proof-of-principle results showing that cytochrome c peroxidase (CcP) for NO detection and myoglobin (Mb) for O2 detection can be successfully used by exploiting our new methodology are reported. The proposed technology can be easily expanded to the determination of different target analytes.

The archaeal topoisomerase reverse gyrase is a helix-destabilizing protein that unwinds four-way DNA junctions.

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.

Human galectin-3 interacts with two anticancer drugs.

Human galectin-3 (hGal-3) is a mammalian lectin involved in regulation of RNA splicing, apoptosis, cell differentiation, and proliferation. Multimerized extracellular hGal-3 is thought to crosslink cells by binding to glycoproteins and glycosylated cancer antigens on the cell surface or extracellular matrix. Fluorescence spectroscopy and circular dichroism were used to study the interaction of hGal-3 with two anticancer agents: bohemine and Zn porphyrin (ZnTPPS(4)). The dissociation constant (k(D)) for binding of bohemine with hGal-3 was k(D) 0.23+/-0.05 microM. The hyperbolic titration curve indicated the presence of a single bohemine binding site. The binding of ZnTPPS(4) to hGal-3 (with and without lactose) is of high affinity having k(D)=0.18-0.20 microM and is not inhibited by lactose, indicating that ZnTPPS(4) and carbohydrate bind different sites. Circular dichroism spectra of the hGal-3 complexes suggested that the binding of the hydrophobic compounds changed the hGal-3 secondary structure. In summary, we show that two compounds with anticancer activity, bohemine and ZnTPPS(4), have high affinity for hGal-3 at a site that is distinct from its carbohydrate site. Since hGal-3 binds to several carbohydrate cancer antigens, the results suggest that it may have utility in the targeted delivery of drugs for cancer.

Tumor-specific protein human galectin-1 interacts with anticancer agents.

The present work shows a novel binding activity of the tumor specific lectin--recombinant human galectin-1 (hGal-1)--to three porphyrin compounds: (1) Zn-porphyrin (ZnTPPS); (2) Mn-porphyrin and (3) Au-porphyrin. These compounds are widely applied in the photodynamic therapy of cancer (PDT). Our data indicate that hGal-1, similar to some plant lectins, a bacterial lectin from Pseudomonas aeruginosa and an animal lectin from Helix pomatia, possesses dual functions binding to both carbohydrate and non-carbohydrate ligands. The interaction of ZnTPPS with hGal-1 was studied by the specific fluorescence emission of the porphyrin. The protein binding properties to Mn/Au-porphyrins and adenine were measured by intrinsic protein fluorescence quenching. The values determined for the apparent dissociation constants (K(D)) of 0.6-1.5 microM are similar to the K(D) for complexes of concanavalin A and porphyrin, and are indicative of the high affinity of hGal-1 for these porphyrins. In addition, the analysis of the hyperbolic binding curves obtained suggests the presence of one hGal-1 binding site for porphyrins or adenine. Additionally, we found that hGal-1 interacts with the fluorescent probe 2-(p-toluidinyl)naphthalene sulfonic acid (TNS), that was used to identify the hydrophobic regions within hGal-1. Homodimeric hGal-1 has more than one class of binding site for TNS as revealed by the sigmoidal shape of the fluorescence titration curve. hGal-1 can be characterized as a porphyrin-binding protein based on its interactions with the Zn/Mn- and Au-porphyrins, and this indicates that hGal-1 may have potential as a delivery molecule to target systems (e.g., tumor cells) with possible application in photodynamic therapy.

Transthoracic ultrasound in the evaluation of pulmonary fibrosis: our experience.

The purpose of this study was to identify the ultrasonographic features of mild, moderate and severe pulmonary fibrosis. Between December 2005 and November 2007, transthoracic ultrasonography (US) was performed by a single operator with specific training in lung sonography on 84 consecutive patients (51 males and 33 females, aged 46 to 73 y) with pulmonary fibrosis. The obtained data were compared with those from a sample of 162 healthy subjects (78 men and 84 women, aged 18 to 76 y). The disease was idiopathic (biopsy confirmed) in 53/84 cases (63%). In the remaining (all histologically confirmed) cases, it was associated with systemic sclerosis (n = 18), rheumatoid arthritis (n = 4), mixed connective tissue disease (n = 4), Sjogren syndrome (n = 4), polymyositis (n = 2) or primary biliary cirrhosis (n = 1). Disease severity was classified as mild, moderate or severe based on clinical findings and the results of standard chest radiography, high-resolution computed tomography and pulmonary function tests. Pulmonary fibrosis was associated with the following US findings: (1) fragmented, irregular thickening (micro3 mm) of the "pleural line" distributed over the whole surface of the lung, especially in the lower posterior lobe (observed in all 84 patients); (2) subpleural cysts (seen in 57/84 (68%) cases of moderate-severe disease); (3) reduction or absence of the physiological "gliding sign" related to disease severity (observed in 33/84 to 39% cases); and (4) increased number of horizontal (and to a lesser extent vertical) reverberation artifacts (seen in 41 patients with advanced fibrosis, 34% of the total series). All abnormalities were detected in both lungs. Although lung biopsy is still the gold standard for diagnosis of interstitial lung disease, transthoracic ultrasound can document early and late-stage changes associated with this disease.

The tryptophan phosphorescence of porcine and mutant bovine odorant-binding proteins: a probe for the local protein structure and dynamics.

Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs.

The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein.

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.