First-in-human use of a new robotic electromagnetic navigation bronchoscopic platform with integrated Tool-in-Lesion Tomosynthesis (TiLT) technology for peripheral pulmonary lesions: The FRONTIER study.

BACKGROUND AND OBJECTIVE: As the presentation of pulmonary nodules increases, the importance of a safe and accurate method of sampling peripheral pulmonary nodules is highlighted. First-generation robotic bronchoscopy has successfully assisted navigation and improved peripheral reach during bronchoscopy. Integrating tool-in-lesion tomosynthesis (TiLT) may further improve yield. METHODS: We performed a first-in-human clinical trial of a new robotic electromagnetic navigation bronchoscopy system with integrated digital tomosynthesis technology (Galaxy System, Noah Medical). Patients with moderate-risk peripheral pulmonary nodules were enrolled in the study. Robotic bronchoscopy was performed using electromagnetic navigation with TiLT-assisted lesion guidance. Non-specific results were followed up until either a clear diagnosis was achieved or repeat radiology at 6 months demonstrated stability. RESULTS: Eighteen patients (19 nodules) were enrolled. The average lesion size was 20 mm, and the average distance from the pleura was 11.6 mm. The target was successfully reached in 100% of nodules, and the biopsy tool was visualized inside the target lesion in all cases. A confirmed specific diagnosis was achieved in 17 nodules, 13 of which were malignant. In one patient, radiological monitoring confirmed a true non-malignant result. This translates to a yield of 89.5% (strict) to 94.7% (intermediate). Complications included one pneumothorax requiring observation only and another requiring an overnight chest drain. There was one case of severe pneumonia following the procedure. CONCLUSION: In this first-in-human study, second-generation robotic bronchoscopy using electromagnetic navigation combined with integrated digital tomosynthesis was feasible with an acceptable safety profile and demonstrated a high diagnostic yield for small peripheral lung nodules.

Tracheobronchial stents: an expanding prospect.

The first dedicated tracheobronchial silicone stent was designed by the French pulmonologist Jean-Paul Dumon. The most common indications for stenting are to minimise extrinsic airway compression from mass effect, maintain airway patency due to intrinsic obstruction or treat significant nonmalignant airway narrowing or fistulae. Silicone stents require rigid bronchoscopy for insertion; however, they are more readily repositioned and removed compared with metallic stents. Metallic stents demonstrate luminal narrowing when loads are applied to their ends, therefore stents should either be reinforced at the ends or exceed the area of stenosis by a minimum of 5 mm. Nitinol, a nickel-titanium metal alloy, is currently the preferred material used for airway stents. Airway stenting provides effective palliation for patients with severe symptomatic obstruction. Drug-eluting and three-dimensional printing of airway stents present promising solutions to the challenges of the physical and anatomical constraints of the tracheobronchial tree. Biodegradable stents could also be a solution for the treatment of nonmalignant airway obstruction.

Long ssRNA undergoes continuous compaction in the presence of polyvalent cations.

In the presence of polyvalent cations, long double-stranded DNA (dsDNA) in dilute solution undergoes a single-molecule, first-order, phase transition ("condensation"), a phenomenon that has been documented and analyzed by many years of experimental and theoretical studies. There has been no systematic effort, however, to determine whether long single-stranded RNA (ssRNA) shows an analogous behavior. In this study, using dynamic light scattering, analytical ultracentrifugation, and gel electrophoresis, we examine the effects of increasing polyvalent cation concentrations on the effective size of long ssRNAs ranging from 3000 to 12,000 nucleotides. Our results indicate that ssRNA does not undergo a discontinuous condensation as does dsDNA but rather a "continuous" decrease in size with increasing polyvalent cation concentration. And, instead of the 10-fold decrease in size shown by long dsDNA, we document a 50% decrease, as demonstrated for a range of lengths and sequences of ssRNA.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

Cryobiopsy for Identification of Usual Interstitial Pneumonia and Other Interstitial Lung Disease Features. Further Lessons from COLDICE, a Prospective Multicenter Clinical Trial.

Rationale: Transbronchial lung cryobiopsy (TBLC) is an emerging technique for interstitial lung disease diagnosis. Good histopathologic agreement between TBLC and surgical lung biopsy (SLB) was demonstrated in the COLDICE (Cryobiopsy versus Open Lung Biopsy in the Diagnosis of Interstitial Lung Disease Alliance) study; however, diagnostic confidence was frequently lower for TBLC than SLB. Objectives: To characterize specific features of TBLC predictive of usual interstitial pneumonia (UIP) in corresponding SLB and to identify clinical indices predictive of biopsy concordance. Methods: The COLDICE study was a prospective, multicenter study investigating diagnostic agreement between TBLC and SLB. The participants underwent both procedures with blinded pathologist analysis of specimens, applying international guideline criteria. The TBLC features predictive of UIP in the paired SLB and predictive features of overall concordance were analyzed. Measurements and Main Results: A total of 65 patients (66.1 ± 9.3 yr; FVC, 84.7 ± 14.2%; DlCO, 63.4 ± 13.8%) participated in the COLDICE study. UIP was identified in 33/65 (50.8%) SLB, and 81.5% were concordant with corresponding TBLC (κ, 0.61; 95% confidence interval [CI], 0.38-0.77). The UIP guideline criteria of "predominantly subpleural or paraseptal fibrosis" was infrequently reported in TBLC (8/33, 24.2%), whereas "patchy fibrosis," "fibroblast foci," and the "absence of alternative diagnostic features" were frequently observed in TBLC. The combination of these three features strongly predicted UIP in paired SLB (odds ratio [OR], 23.4; 95% CI, 6.36-86.1; P < 0.0001). Increased numbers of TBLC samples predicted histopathologic concordance with SLB (OR, 1.8; 95% CI, 1.08-3.01; P = 0.03). The predictors of discordance included older age, family history, and radiologic asymmetry. Conclusions: Subpleural and/or paraseptal fibrosis were not essential for diagnosing UIP in TBLC, provided that other guideline criteria features were present. The diagnostic accuracy of TBLC was strengthened when increased numbers of samples were taken. Clinical trial registered with www.anzctr.org.au (ACTRN12615000718549).

Interventional bronchoscopy for chronic obstructive pulmonary disease: more than a pipe dream.

Endoscopic lung volume reduction (ELVR) is recognised in both national and international expert guidelines as one of the few additive treatments to benefit patients with advanced chronic obstructive pulmonary disease (COPD) who are otherwise receiving optimal medical and supportive care. Despite these recommendations and a growing evidence base, these procedures are not widely offered across Australia and New Zealand, and general practitioner and physician awareness of this therapy can be improved. ELVR aims to mitigate the impact of hyperinflation and gas trapping on dyspnoea and exercise intolerance in COPD. Effective ELVR is of proven benefit in improving symptoms, quality of life, lung function and survival. Several endoscopic techniques to achieve ELVR have been developed, with endobronchial valve placement to collapse a single lobe being the most widely studied and commonly practised. This review describes the physiological rationale underpinning lung volume reduction, highlights the challenges of patient selection, and provides an overview of the evidence for current and investigational endoscopic interventions for COPD.

Inhibition of the stem cell factor 248 isoform attenuates the development of pulmonary remodeling disease.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

The role of fit-for-purpose assays within tiered testing approaches: A case study evaluating prioritized estrogen-active compounds in an in vitro human uterotrophic assay.

Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC50s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment.

The Streptococcus pyogenes Shr protein captures human hemoglobin using two structurally unique binding domains.

In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes, a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. Here, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). These hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture.

Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN2N3) triggers hemin release by distorting Hb's F-helix. Here, we report that IsdHN2N3 promotes hemin release from both the α- and ß-subunits. Using a receptor mutant that only binds to the α-subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400-fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 Nϵ-Fe3+ bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdHN2N3·Hb protein·protein interfaces promote hemin release. A high-affinity receptor·Hb(A-helix) interface contributed ∼95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb's F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.

A pragmatic application of endobronchial ultrasound-guided transbronchial needle aspiration: a single institution experience.

BACKGROUND: Endobronchial ultrasound-guided trans-bronchial needle aspiration (EBUS-TBNA) is minimally invasive technique used for diagnosis and/or staging of benign and malignant pulmonary and non-pulmonary disease. Previous studies have established the utility of EBUS-TBNA in narrowly defined indications and populations. In this pragmatic 'real world' study we have analysed the use of EBUS-TBNA for a variety of clinical presentations and its clinical application in conjunction with other invasive investigations. METHODS: All EBUS-TBNA procedures performed at Sir Charles Gardiner Hospital in 2012-2014 were reviewed retrospectively, using relevant hospital databases. RESULTS: A total of 327 patients underwent 337 EBUS-TBNA procedures. EBUS-TBNA procedures were used to diagnose a wide spectrum of benign and malignant conditions. The main application was in the diagnosis and staging of malignant conditions (70.6%), and in the diagnosis of benign conditions such as sarcoidosis 40 (12.2%), and silicoanthracosis 17 (5.2%). EBUS-TBNA was sufficient to diagnose and stage the disease as a single stand-alone invasive procedure in 191 (59.2%) patients. EBUS-TBNA was the final invasive procedure undertaken in 283 (87.6%) patients. Only 13.3% of non small cell lung cancer (NSCLC) patients who had EBUS-TBNA as a first investigation required multiple procedures compared to 51.1% of all NSCLC patients undergoing EBUS-TBNA. Overall sensitivity, specificity, NPV and diagnostic accuracy for EBUS-TBNA were 89.7, 100, 85.1 and 89.9%, respectively and three minor complications (0.9%) occurred as a result of the procedure. CONCLUSIONS: EBUS-TBNA was undertaken for a wide variety of clinical conditions. Good diagnostic accuracy and safety profiles were demonstrated for the procedure, supporting its application as a first line investigation in the diagnosis and/or staging of a range of malignant and benign conditions. Our study was unique in its documentation of the use of EBUS-TBNA in a real-world setting in conjunction with other invasive modalities. EBUS-TBNA was utilised as a stand alone invasive procedure in more than half of the patients. Importantly, in NSCLC, when EBUS-TBNA was performed as primary diagnostic and staging investigation, less patients underwent subsequent invasive procedures.

Implementing Endobronchial Ultrasound-Guided (EBUS) for Staging and Diagnosis of Lung Cancer: A Cost Analysis.

BACKGROUND Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and guide sheath (EBUS-GS) are gaining popularity for diagnosis and staging of lung cancer compared to CT-guided transthoracic needle aspiration (CT-TTNA), blind fiber-optic bronchoscopy, and mediastinoscopy. This paper aimed to examine predictors of higher costs for diagnosing and staging lung cancer, and to assess the effect of EBUS techniques on hospital cost. MATERIAL AND METHODS Hospital costs for diagnosis and staging of new primary lung cancer patients presenting in 2007-2008 and 2010-2011 were reviewed retrospectively. Multiple linear regression was used to determine relationships with hospital cost. RESULTS We reviewed 560 lung cancer patient records; 100 EBUS procedures were performed on 90 patients. Higher hospital costs were associated with: EBUS-TBNA performed (p<0.0001); increasing inpatient length of stay (p<0.0001); increasing number of other surgical/diagnostic procedures (p<0.0001); whether the date of management decision fell within an inpatient visit (p<0.0001); and if the patient did not have a CT-TTNA, then costs increased as the number of imaging events increased (interaction p<0.0001). Cohort was not significantly related to cost. Location of the procedure (outside vs. inside theater) was a predictor of lower one-day EBUS costs (p<0.0001). Cost modelling revealed potential cost saving of $1506 per EBUS patient if all EBUS procedures were performed outside rather than in the theater ($66,259 per annum). CONCLUSIONS EBUS-TBNA only was an independent predictor of higher cost for diagnosis and staging of lung cancer. Performing EBUS outside compared to in the theater may lower costs for one-day procedures; potential future savings are considerable if more EBUS procedures could be performed outside the operating theater.

Developing a Physiologically-Based Pharmacokinetic Model Knowledgebase in Support of Provisional Model Construction.

Developing physiologically-based pharmacokinetic (PBPK) models for chemicals can be resource-intensive, as neither chemical-specific parameters nor in vivo pharmacokinetic data are easily available for model construction. Previously developed, well-parameterized, and thoroughly-vetted models can be a great resource for the construction of models pertaining to new chemicals. A PBPK knowledgebase was compiled and developed from existing PBPK-related articles and used to develop new models. From 2,039 PBPK-related articles published between 1977 and 2013, 307 unique chemicals were identified for use as the basis of our knowledgebase. Keywords related to species, gender, developmental stages, and organs were analyzed from the articles within the PBPK knowledgebase. A correlation matrix of the 307 chemicals in the PBPK knowledgebase was calculated based on pharmacokinetic-relevant molecular descriptors. Chemicals in the PBPK knowledgebase were ranked based on their correlation toward ethylbenzene and gefitinib. Next, multiple chemicals were selected to represent exact matches, close analogues, or non-analogues of the target case study chemicals. Parameters, equations, or experimental data relevant to existing models for these chemicals and their analogues were used to construct new models, and model predictions were compared to observed values. This compiled knowledgebase provides a chemical structure-based approach for identifying PBPK models relevant to other chemical entities. Using suitable correlation metrics, we demonstrated that models of chemical analogues in the PBPK knowledgebase can guide the construction of PBPK models for other chemicals.

Impact of the introduction of EBUS on time to management decision, complications, and invasive modalities used to diagnose and stage lung cancer: a pragmatic pre-post study.

BACKGROUND: Utilisation of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and guide sheath (EBUS-GS) for diagnosis and staging of lung cancer is gaining popularity, however, its impact on clinical practice is unclear. This study aimed to determine the impact of the introduction of endobronchial ultrasound-guided procedures (EBUS) on time to management decision for lung cancer patients, and on the utilisation of other invasive diagnostic modalities, including CT-guided trans-thoracic needle aspiration (CT-TTNA), bronchoscopy, and mediastinoscopy. METHODS: Hospital records of new primary lung cancer patients presenting in 2007 and 2008 (Pre-EBUS cohort) and in 2010 and 2011 (Post-EBUS cohort) were reviewed retrospectively. RESULTS: The Pre-EBUS cohort included 234 patients. Of the 326 patients in the Post-EBUS cohort, 90 had an EBUS procedure (EBUS-TBNA for 19.0% and EBUS-GS for 10.4% of cases). The number of CT-TTNAs and bronchoscopies decreased following the introduction of EBUS (p = 0.015 and p < 0.001 respectively). Of 162 CT-TTNAs, 59 (36%) resulted in complications compared to 1 complication each for bronchoscopy and EBUS-GS, and no complications from EBUS-TBNA. Fewer complications occurred overall in the Post-EBUS cohort compared to the Pre-EBUS cohort (p = 0.0264). The median time to management decision was 17 days (IQR 24) for the Pre-EBUS and 13 days (IQR 21) for the Post-EBUS cohort (p = 0.07). Within the Post-EBUS cohort, median time to management decision was longer for the EBUS group (n = 90) than the Non-EBUS group (17 days (IQR 29) vs. 10 days (IQR 10), p < 0.001). For half of EBUS-TBNA patients (n = 28, 50.0%) and EBUS-GS patients (n = 14, 50.0 %), EBUS alone provided sufficient diagnostic and/or staging information; these patients had median time to management decision of 10 days. Regression analysis revealed that the number of imaging events, inpatient, and outpatient visits were significant predictors of time to management decision of >28 days; EBUS was not a predictor of time to management decision. CONCLUSIONS: The introduction of EBUS led to fewer CT-TTNAs and bronchoscopies and did not impact on the time to management decision. EBUS-TBNA or EBUS-GS alone provided sufficient information for diagnosis and/or regional staging in half of the lung cancer patients referred for this investigation.

Patterns of sedentary behaviour and physical activity in people following curative intent treatment for non-small cell lung cancer.

This study aimed to compare patterns of sedentary behaviour (SB) and physical activity (PA) in people following curative intent treatment for non-small cell lung cancer (NSCLC) with healthy controls. Participants 6-10 weeks following lobectomy for NSCLC and healthy controls wore two activity monitors for 7 days. Waking hours were divided into time spent in SB (<1.5 metabolic equivalent of tasks (METs)), light intensity PA (LIPA ≥ 1.5 to <3.0METs) and moderate-to-vigorous intensity PA (≥3.0METs). Daily steps were also recorded. Data were available in 20 participants with NSCLC (13 females; 68 ± 10 years) and 20 healthy controls (13 females; 69 ± 5 years). The NSCLC group accumulated a greater percentage of time in SB in uninterrupted bouts ≥30 minutes (49% vs. 42%; p = 0.048). Further, the NSCLC group spent a lower percentage of waking hours in LIPA (21 ± 9% vs. 26 ± 8%; p = 0.04) and accumulated a lower percentage of time in this domain in uninterrupted bouts ≥10 minutes (13% vs. 19%; p = 0.025). The NSCLC group also had a lower daily step count (8863 ± 3737 vs. 11,856 ± 3024 steps/day; p = 0.009). Time spent in moderate-to-vigorous intensity PA was similar in both groups (p = 0.92). People following curative intent treatment for NSCLC spend more time in prolonged bouts of SB at the expense of LIPA.

Excessive centrifugal fields damage high density lipoprotein.

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds.

Formation of amyloid fibers by monomeric light chain variable domains.

Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers.

The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions.

BACKGROUND: The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. RESULTS: NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. CONCLUSIONS: The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD.

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain.