The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Interrogating Aptamer Chemical Space Through Modified Nucleotide Substitution Facilitated by Enzymatic DNA Synthesis.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids.

Dengue virus NS1 protein conveys pro-inflammatory signals by docking onto high-density lipoproteins.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

Why Don't Adolescents Wear Their Brace? A Prospective Study Investigating Psychosocial Characteristics That Predict Scoliosis Brace Wear.

BACKGROUND: Although bracing for adolescent idiopathic scoliosis can prevent curve progression and reduce the risk for future surgery, children frequently do not wear their braces as prescribed. The purpose of this study is to investigate how a broad array of psychosocial characteristics predict future compliance with scoliosis brace wear. METHODS: This was a single institution, prospective cohort study. All adolescents prescribed a first-time brace for adolescent idiopathic scoliosis were eligible. Patients and their parents completed a separate series of questionnaires that assessed baseline psychosocial characteristics across 6 domains: (1) brace-specific attitudes; (2) body image and self-esteem; (3) school performance and social relationships; (4) psychological health; (5) family functioning; and (6) demographics and scoliosis-specific details (242 total questions across 12 validated questionnaires). Objective brace compliance was collected using temperature-sensitive monitors. Defining compliance as percentage of brace prescription completed, comparative analyses were performed to identify baseline psychosocial characteristics that were associated with future wear. A composite measure (Bracing Fidelity Follow-Up Scale [BFFS]) of the 12 most predictive individual questions across all domains (both parent and adolescent) was constructed to help assess which adolescents were at highest risk of failure to wear their brace. Total BFFS score for each parent-adolescent dyad who completed all the included surveys was then determined by awarding one point for each factor that positively influenced future brace wear (maximum 12 points), and a correlation was calculated between total score and percent adherence to prescribed brace wear. RESULTS: A total of 41 patients were included. On average, patients with high self-esteem, above average peer relationships and poor brace-specific attitudes had lower brace compliance, although patients with increased loneliness and parental religiousness had higher compliance. Body image, socioeconomic status, family dynamics, and school performance had no significant relationship with brace use. Total score on the Bracing Fidelity Follow-Up Scale (BFFS) was significantly associated with improved brace wear (r=0.687, P <0.001). Those with a score of 6 or above (n=15/33 [45%], median compliance 96%) were more reliable users (15/15 with compliance >75%), and those with a score of 5 or less (n=18/33 [55%], median compliance 50%) had less consistent brace wear (9/18 with compliance <50%). CONCLUSION: This prospective study identifies numerous baseline psychosocial factors that are associated with future compliance with scoliosis brace wear. Although in need of further validation before widespread clinical application, the novel BFF scale offers a potential opportunity to partially discriminate between compliant and noncompliant scoliosis brace users such that supportive resources (eg, supportive counseling, peer-support groups, additional provider-based education, etc.) can be targeted to those patients most likely to benefit. LEVEL OF EVIDENCE: II.

Disparate Impact: How Does Social Deprivation Affect Patient-reported Outcomes After ACL Reconstruction in Children and Adolescents?

BACKGROUND: Research has indicated that lower socioeconomic status is associated with delays in the treatment of anterior cruciate ligament (ACL) injuries; however, there is a paucity of literature evaluating its association with patient-reported outcomes (PROs). Using the Area Deprivation Index (ADI), a validated proxy for socioeconomic status, the study aimed to determine how relative socioeconomic disadvantage is related to PROs after primary ACL reconstruction (ACLR) in pediatric patients. METHODS: This retrospective cohort study included all patients 18 years old or above who underwent primary ACLR at an academic institution between 2018 and 2021. Exclusion criteria included multiligament injury, congenital ACL absence, and absent outcomes data. The minimum follow-up was 6 months. A Patient-reported Outcomes Measurement Information System (PROMIS) 50 Pediatric self-report questionnaire was completed at postoperative visits, and domain scores for pain, physical function/mobility, fatigue, anxiety, depression, and peer relationships were generated. The National ADI percentile was calculated using the patients' addresses. Patients were divided into quartiles (low, moderate, moderate-severe, and severe ADI), and comparative analyses were performed to determine the relationship between ADI and PROMIS. RESULTS: A total of 413 patients were identified, including 49% (n=207), 33% (n=139), 11% (n=48), and 7% (n=30) from the low, moderate, moderate-severe, and severe deprivation areas, respectively. As compared with those in the low-deprivation quartile, patients in the severe deprivation quartile had delayed time to the first clinic visit (11 vs. 16.5 d, P=0.044) and surgery (51 vs. 80 d, P=0.004). There were no differences in the number of additional procedures required at index surgery. All quartiles had progressive improvements in physical function/mobility and pain scores throughout recovery, but at 9 months, there was significantly more pain in the severe deprivation cohort, despite no difference in self-reported physical function and mobility. Those with severe socioeconomic disadvantage had worse psychosocial outcomes, including significantly increased depression, fatigue, and anxiety and decreased peer relationship scores. CONCLUSIONS: Although there were no differences in preoperative PROMIS scores, pediatric patients living in areas with higher levels of socioeconomic deprivation/disadvantage had worse psychosocial PROs after ACLR. LEVEL OF EVIDENCE: Level III-retrospective comparative study.

The Epidemiology of Pediatric Basketball Injuries Presenting to US Emergency Departments: 2011-2020.

OBJECTIVES: The purpose of this study is to describe the national epidemiology of basketball-related injuries in children and adolescents presenting to US emergency departments (EDs) from 2011 to 2020 and to quantify the effect of the COVID-19 pandemic. METHODS: The National Electronic Injury Surveillance System database was queried for cases of injury in persons aged 0 to 19 years related to product code 1205 (basketball and related equipment) presenting from January 1, 2011 to December 31, 2020. National injury estimates were calculated using National Electronic Injury Surveillance System-recommended weights and strata. The US Census data were used to determine the incidence of injury by age group and by sex. To quantify the effect of COVID-19, an interrupted time series analysis was performed using March 1, 2020 as the interrupting time point. The pre-COVID-19 trend was used to estimate the difference in injuries attributable to the COVID-19 pandemic. RESULTS: From 2011 to 2020, an estimated 3,210,953 (95% confidence interval = 2,655,812-3,788,094) visits were made to US EDs for basketball-related injuries in those aged younger than 20 years, corresponding to a mean annual incidence of 391 injuries per 100,000 population. The mean age of injury was 14.4 years (95% confidence interval = 14.3-14.5). Boys were more often injured than girls (76% vs 24% of all injuries, respectively). The foot was the most injured body part, accounting for 24% of injuries. Strains or sprains were the most common injury type (38% of injuries). During the COVID-19 pandemic, there were 155,638 fewer injuries than were expected based on pre-COVID-19 trends. During COVID-19, there were no significant differences in the proportions of injury types, body parts involved, sex, or age. CONCLUSIONS: Basketball remains a frequent cause of injury, especially in adolescents. The COVID-19 pandemic profoundly reduced the frequency of basketball-related injuries, but did not affect the type and body location of injuries presenting to the ED.

Structural basis of potent Zika-dengue virus antibody cross-neutralization.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Protein intrinsic viscosity determination with the Viscosizer TD instrument: reaching beyond the initially expected applications.

Intrinsic viscosity is a key hydrodynamic parameter to understand molecular structure and hydration, as well as intramolecular interactions. Commercially available instruments measure intrinsic viscosity by recording the macromolecular mobility in a capillary. These instruments monitor Taylor dispersion using an absorbance or fluorescence detector. By design, these instruments behave like U-tube viscometers. To our knowledge, there are no studies to date showing that the Viscosizer TD instrument (Malvern-Panalytical) is able to measure the intrinsic viscosity of macromolecules. In this study, we then performed our assays on the Poly(ethylene oxide) polymer (PEO), used classically as a standard for viscometry measurements and on three model proteins: the bovine serum albumin (BSA), the bevacizumab monoclonal antibody, and the RTX Repeat Domain (RD) of the adenylate cyclase toxin of Bordetella pertussis (CyaA). The presence of P20 in the samples is critical to get reliable results. The data obtained with our in-house protocol show a strong correlation with intrinsic viscosity values obtained using conventional techniques. However, with respect to them, our measurements could be performed at relatively low concentrations, between 2 and 5 mg/ml, using only 7 µL per injection. Altogether, our results show that the Viscosizer TD instrument is able to measure intrinsic viscosities in a straightforward manner. This simple and innovative approach should give a new boost to intrinsic viscosity measurements and should reignite the interest of biophysicists, immunologists, structural biologists and other researchers for this key physicochemical parameter.

Macromolecular interactions in vitro, comparing classical and novel approaches.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

An extracellular Leptospira interrogans leucine-rich repeat protein binds human E- and VE-cadherins.

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.

Does Advanced Imaging Have a Role in Detecting Local Recurrence of Soft-tissue Sarcoma?

BACKGROUND: Early detection of soft-tissue sarcoma recurrences may decrease the morbidity of reoperation and improve oncologic outcomes. The benefit of imaging compared with clinical surveillance for detecting local recurrences remains controversial, as prior studies have varied in terms of inclusion criteria, factors analyzed, and outcomes reported. QUESTIONS/PURPOSES: (1) What proportion of local recurrences were detected by surveillance imaging compared with clinical signs and symptoms? (2) Were local recurrences detected by imaging smaller than those detected by clinical surveillance? (3) Were relevant tumor, patient, or operative characteristics associated with clinically occult local recurrence? METHODS: Over a 20-year period ending in 2018, we treated 545 patients for soft-tissue sarcoma. During that period, we recommended that patients receive a surgical excision as well as radiation therapy based on current clinical guidelines. Of those we treated, 9% (51 of 545) were excluded for having a low-grade liposarcoma, and 4% (21 of 545) were excluded for being metastatic at the time of presentation. Of the remaining patients, 22% (107 of 473) were lost to follow-up before 2 years but were not known to have died. There were a remaining 366 patients for analysis in this retrospective study of electronic medical records from a single center. Patients routinely underwent advanced imaging and clinical follow-up at intervals based on currently available guidelines for sarcoma surveillance. We recommended that patients with high-grade sarcomas be followed every 3 months until 2 years, then every 6 months until 3 years, then annually thereafter. In contrast, we recommended that patients with low-grade sarcomas be followed every 6 months until 2 years, then annually thereafter. In addition, patients were encouraged to return for evaluation if they noted a new mass or other symptoms. In general, patients with high-grade sarcomas received postoperative radiation therapy unless they underwent amputation, while intermediate- and low-grade sarcomas were radiated according to clinical concern for local recurrence, as determined by the multidisciplinary sarcoma team. Seventeen percent (61 of 366) of patients developed or presented with a local recurrence. Of the local recurrences detected by surveillance imaging, 17 were detected by MRI, three were detected by position emission tomography, and one was detected by CT scan. The proportion of local recurrences first identified by advanced imaging versus clinical detection (physical examination, self-detection, or symptomatic presentation) were compared. Logistic regression with a Wald chi-square test was performed to evaluate if tumor, patient, or operative characteristics are associated with clinical versus imaging detection of local recurrences. RESULTS: A higher proportion of local recurrences were detected by clinical signs and symptoms than by routine imaging (66% (40 of 61) versus 34% (21 of 61), binomial proportion 0.66 [95% CI 0.55 to 0.77]; p = 0.007). With the numbers available, there was no difference in the tumor size detected by clinical signs and symptoms compared with surveillance imaging. The median (interquartile range) largest tumor dimension was 3.9 cm (2.5 to 7.8) for clinical surveillance versus 4.5 cm (2.7 to 6.2) for imaging surveillance (p = 0.98). We were unable to identify any associated factors, alone or in combination, with detection by physical exam, including patient age, tumor size, tumor depth, tumor location, operative closure type, or radiation status. Characteristics such as larger tumors, more superficial tumors, low BMI, the absence of a flap reconstruction or radiation treatment, were not associated with a greater likelihood of detection by physical examination. CONCLUSIONS: We found that although a high proportion of local recurrences were detected by clinical signs and symptoms, approximately one-third were detected by imaging. Although not all patients may benefit equally from routine imaging, we were unable to identify any patient, tumor, or operative characteristics to define a subgroup of patients that are more or less likely benefit from this surveillance technique. These findings support current surveillance guidelines that recommend the use of advanced imaging; however, other factors may also warrant consideration. Futher insight could be gained by studying surveillance imaging in terms of optimal frequency, cost-effectiveness, and psychosocial implications for patients. LEVEL OF EVIDENCE: Level III, diagnostic study.

The stress sigma factor of RNA polymerase RpoS/σS is a solvent-exposed open molecule in solution.

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

IgG subclasses determine pathways of anaphylaxis in mice.

BACKGROUND: Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. OBJECTIVE: We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependent passive systemic anaphylaxis. METHODS: Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. RESULTS: Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. CONCLUSION: We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions in vivo.

The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm.

LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling.

Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination.