Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function.

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1ß and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

Assembly and dynamics of the autophagy-initiating Atg1 complex.

The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17-Atg31-Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen-deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼ 100-nM affinity. The Atg1-Atg13 complex in turn binds as a unit to the Atg17-Atg31-Atg29 scaffold with ∼ 10-µM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.

Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal-lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn(2+). One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn(2+) and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 µM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (~20 µM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (~20 µM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH.

AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to the lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view of better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, yields a substantial loss of catalytic activity without any significant change in the thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM; however, the precise mechanism and its significance are not known. On the basis of a number of biochemical and biophysical analyses, we are able to propose a model for activation according to which activation of AMSH is allowed by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence that supports a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate the transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity.

Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (associated molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent down-regulation of activated cell surface receptors via the endolysosomal degradative pathway. Therefore, small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently, there are no known selective inhibitors of AMSH or high-throughput compatible assays for their identification. We report the development and optimization of a novel fluorescence resonance energy transfer (FRET)-based add-and-read AMSH DUB assay in a 384-well format. In this format, the optimal temperature for a high-throughput screen (HTS) was determined to be 30°C, the assay tolerates 5% dimethyl sulfoxide (DMSO), and it has a Z-score of 0.71, indicating HTS compatibility. The assay was used to show that AMSH selectively cleaves Lys63-linked diubiquitin over Lys48- and Lys11-linked diubiquitin. The IC50 value of the nonspecific small molecule DUB inhibitor N-ethylmaleimide was 16.2±3.2 µM and can be used as a qualitative positive control for the screen. We conclude that this assay is high-throughput compatible and can be used to identify novel small molecule inhibitors of AMSH.

Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 A from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal beta-hairpin at the distal site-a surface-exposed hydrophobic crevice 17 A away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 A of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK).

UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson's disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the X-ray structure of UCHL1 co-crystallized with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resolution. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin's C-terminal tail, thereby occupying the P1' (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of molecular contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.

Effect of an indwelling pleural catheter vs chest tube and talc pleurodesis for relieving dyspnea in patients with malignant pleural effusion: the TIME2 randomized controlled trial.

CONTEXT: Malignant pleural effusion causes disabling dyspnea in patients with a short life expectancy. Palliation is achieved by fluid drainage, but the most effective first-line method has not been determined. OBJECTIVE: To determine whether indwelling pleural catheters (IPCs) are more effective than chest tube and talc slurry pleurodesis (talc) at relieving dyspnea. DESIGN: Unblinded randomized controlled trial (Second Therapeutic Intervention in Malignant Effusion Trial [TIME2]) comparing IPC and talc (1:1) for which 106 patients with malignant pleural effusion who had not previously undergone pleurodesis were recruited from 143 patients who were treated at 7 UK hospitals. Patients were screened from April 2007-February 2011 and were followed up for a year. INTERVENTION: Indwelling pleural catheters were inserted on an outpatient basis, followed by initial large volume drainage, education, and subsequent home drainage. The talc group were admitted for chest tube insertion and talc for slurry pleurodesis. MAIN OUTCOME MEASURE: Patients completed daily 100-mm line visual analog scale (VAS) of dyspnea over 42 days after undergoing the intervention (0 mm represents no dyspnea and 100 mm represents maximum dyspnea; 10 mm represents minimum clinically significant difference). Mean difference was analyzed using a mixed-effects linear regression model adjusted for minimization variables. RESULTS: Dyspnea improved in both groups, with no significant difference in the first 42 days with a mean VAS dyspnea score of 24.7 in the IPC group (95% CI, 19.3-30.1 mm) and 24.4 mm (95% CI, 19.4-29.4 mm) in the talc group, with a difference of 0.16 mm (95% CI, −6.82 to 7.15; P = .96). There was a statistically significant improvement in dyspnea in the IPC group at 6 months, with a mean difference in VAS score between the IPC group and the talc group of −14.0 mm (95% CI, −25.2 to −2.8 mm; P = .01). Length of initial hospitalization was significantly shorter in the IPC group with a median of 0 days (interquartile range [IQR], 0-1 day) and 4 days (IQR, 2-6 days) for the talc group, with a difference of −3.5 days (95% CI, −4.8 to −1.5 days; P < .001). There was no significant difference in quality of life. Twelve patients (22%) in the talc group required further pleural procedures compared with 3 (6%) in the IPC group (odds ratio [OR], 0.21; 95% CI, 0.04-0.86; P = .03). Twenty-one of the 52 patients in the catheter group experienced adverse events vs 7 of 54 in the talc group (OR, 4.70; 95% CI, 1.75-12.60; P = .002). CONCLUSION: Among patients with malignant pleural effusion and no previous pleurodesis, there was no significant difference between IPCs and talc pleurodesis at relieving patient-reported dyspnea. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN87514420.

Structure of the core human NADPH oxidase NOX2.

NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O2•-), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Blood culture bottle culture of pleural fluid in pleural infection.

BACKGROUND: Pleural infection is common, and has a >30% major morbidity and mortality-particularly when infection is caused by Gram-negative, Staphylococcus aureus or mixed aerobic pathogens. Standard pleural fluid culture is negative in ∼40% of cases. Culturing pleural fluid in blood culture bottles may increase microbial yield, and is cheap and easy to perform. OBJECTIVES: To determine whether inoculating pleural fluid into blood culture bottles increases the culture positivity of pleural infection over standard laboratory culture, and to assess the optimum volume of inoculum to introduce. METHODS: 62 patients with pleural infection were enrolled. Pairs of aerobic and anaerobic blood culture bottles were inoculated at the bedside with 2, 5 or 10 ml of pleural fluid, and two pleural fluid specimens were sent for standard culture. Pleural fluid from nine control patients was cultured to test for 'false-positive' results. RESULTS: The addition of blood culture bottle culture to standard culture increased the proportion of patients with identifiable pathogens by 20.8% (20/53 (37.7%) to 31/53 (58.5%) (difference 20.8%, 95% CI difference 8.9% to 20.8%, p<0.001)). The second standard culture did not similarly improve the culture positivity (19/49 (38.8%) to 22/49 (44.9%) (difference 6.1%, 95% CI difference -2.5% to 6.1%, p=0.08)). The culture inoculum volume did not influence bacterial isolation frequency. The control fluids were culture negative. CONCLUSIONS: Blood culture bottle culture of infected pleural fluid increases microbial yield when used in addition to standard culture. This technique should be part of routine care.

Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Mannose-binding lectin genotypes: lack of association with susceptibility to thoracic empyema.

BACKGROUND: The role of the innate immune protein mannose-binding lectin (MBL) in host defence against severe respiratory infection remains controversial. Thoracic empyema is a suppurative lung infection that arises as a major complication of pneumonia and is associated with a significant mortality. Although the pathogenesis of thoracic empyema is poorly understood, genetic susceptibility loci for this condition have recently been identified. The possible role of MBL genotypic deficiency in susceptibility to thoracic empyema has not previously been reported. METHODS: To investigate this further we compared the frequencies of the six functional MBL polymorphisms in 170 European individuals with thoracic empyema and 225 healthy control individuals. RESULTS: No overall association was observed between MBL genotypic deficiency and susceptibility to thoracic empyema (2 x 2 Chi square = 0.02, P = 0.87). Furthermore, no association was seen between MBL deficiency and susceptibility to the Gram-positive or pneumococcal empyema subgroups. MBL genotypic deficiency did not associate with progression to death or requirement for surgery. CONCLUSIONS: Our results suggest that MBL genotypic deficiency does not associate with susceptibility to thoracic empyema in humans.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

U.K. Controlled trial of intrapleural streptokinase for pleural infection.

BACKGROUND: Intrapleural fibrinolytic agents are used in the drainage of infected pleural-fluid collections. This use is based on small trials that did not have the statistical power to evaluate accurately important clinical outcomes, including safety. We conducted a trial to clarify the therapeutic role of intrapleural streptokinase. METHODS: In this double-blind trial, 454 patients with pleural infection (defined by the presence of purulent pleural fluid or pleural fluid with a pH below 7.2 with signs of infection or by proven bacterial invasion of the pleural space) were randomly assigned to receive either intrapleural streptokinase (250,000 IU twice daily for three days) or placebo. Patients received antibiotics and underwent chest-tube drainage, surgery, and other treatment as part of routine care. The number of patients in the two groups who had died or needed surgical drainage at three months was compared (the primary end point); secondary end points were the rates of death and of surgery (analyzed separately), the radiographic outcome, and the length of the hospital stay. RESULTS: The groups were well matched at baseline. Among the 427 patients who received streptokinase or placebo, there was no significant difference between the groups in the proportion of patients who died or needed surgery (with streptokinase: 64 of 206 patients [31 percent]; with placebo: 60 of 221 [27 percent]; relative risk, 1.14 [95 percent confidence interval, 0.85 to 1.54; P=0.43), a result that excluded a clinically significant benefit of streptokinase. There was no benefit to streptokinase in terms of mortality, rate of surgery, radiographic outcomes, or length of the hospital stay. Serious adverse events (chest pain, fever, or allergy) were more common with streptokinase (7 percent, vs. 3 percent with placebo; relative risk, 2.49 [95 percent confidence interval, 0.98 to 6.36]; P=0.08). CONCLUSIONS: The intrapleural administration of streptokinase does not improve mortality, the rate of surgery, or the length of the hospital stay among patients with pleural infection.

The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion.

Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1DD that selectively perturbs the function of the Atg13-free state. Atg1DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.