Rhinoconjunctivitis severity induced by cat exposure influences early and late asthmatic responses: Evidence from an environmental exposure chamber.

BACKGROUND: The impact of allergic rhinoconjunctivitis on the early (EAR) and late asthmatic response (LAR) has yet to be assessed during optimal allergen exposure conditions. OBJECTIVE: We aimed to assess predictive factors of the EAR and LAR and to evaluate the relation between rhinitis, conjunctivitis and asthma induced by cat allergen exposure in an environmental exposure chamber (EEC). METHODS: Data from two cohort studies involving asthmatic patients with cat allergy who performed a cat allergen exposure challenge in ALYATEC EEC were analysed. Spirometry, visual analogue scale (VAS) for asthma, VAS for rhinitis, Total Nasal Symptoms Score, Total Ocular Symptoms Score (TOSS), Rhinoconjunctivitis Total Symptoms Score and Abelson score were used to assess asthma, rhinitis and conjunctivitis during and after exposure. RESULTS: An EAR occurred in 65.1% of patients, 32.1% of whom had a LAR. The diameter of the prick test to cat allergens and non-specific bronchial hypersensitivity level were independent risk factors for EAR (p < .05). No independent risk factors for LAR were identified. Rhinoconjunctivitis severity during exposure correlated with the asthma VAS during EAR and LAR (p < .05). Allergen exposure time needed to trigger an EAR correlated with the Abelson score during exposure (p < .05). The asthma VAS and TOSS during exposure correlated with faster LAR occurrence (p < .05). CONCLUSION: Prick test size and non-specific bronchial hypersensitivity level were confirmed as independent predictive factors of EAR during allergen exposure in an EEC. This study demonstrated the relation between the severity of rhinitis, conjunctivitis and asthma induced by allergen exposure for both EAR and LAR.

Spectral Phasor Applied to Spectrally-Resolved Single Molecule Localization Microscopy.

Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.

Outcomes of total elbow arthroplasty in the treatment of distal humeral fractures in the elderly: A retrospective cohort comparison between primary arthroplasty and arthroplasty secondary to failed internal fixation.

BACKGROUND: The primary objective of this study was to compare the clinical outcomes of total elbow arthroplasty as the index procedure in the treatment of traumatic distal humerus fractures, with secondary total elbow arthroplasty following failed internal fixation. The secondary objectives were to compare the complication rates and the radiographic results in the two groups. Our hypothesis was that the clinical results of total elbow arthroplasty performed secondarily to failed internal fixation were comparable to primary total elbow arthroplasty in the treatment of distal humerus fractures in the elderly population. METHODS: We conducted a retrospective cohort comparison study, including 60 patients with a median age of 80 years (71-85), who either underwent a primary total elbow arthroplasty (group 1; 45 patients), or secondary total elbow arthroplasty following failed internal fixation (group 2; 15 patients), in the treatment of a post-traumatic supra and intercondylar fracture of the distal humerus, between January 2004 and January 2021. The clinical examination, including MEPS score and triceps proficiency test, complication rates and the need for re-operation were noted. The average clinical and radiographic follow-up was 40.8 months (24-120). RESULTS: The clinical results of the two groups were comparable when looking at the MEPS score (90.00 [85.00, 100.00] p= 0.486). With regards to complications, there were 2 surgical site infections in group 1 and 3 in group 2 (p=0.099), 1 case of mechanical loosening of the humeral component in group 1 and 1 in group 2 (p= 0.448), and 1 patient with triceps insufficiency in group 1. CONCLUSION: Secondary total elbow arthroplasty following failed internal fixation has shown good functional results, and a complication rate comparable to that of index total elbow arthroplasty in the treatment of articular fractures of the distal humerus in the elderly.

Complication Rates Are Not Higher After Outpatient Versus Inpatient Fast-Track Total Knee Arthroplasty: A Propensity-Matched Prospective Comparative Study.

BACKGROUND: This prospective study aimed to compare the complication rates and clinical outcomes of propensity-matched patients who received fast-track total knee arthroplasty (FT TKA) in outpatient versus inpatient settings. METHODS: Patients (n = 629) who received FT TKA at various outpatient (n = 176) and inpatient (n = 462) surgery rates were prospectively followed until 90 days after surgery. The decision between inpatient versus outpatient FT TKA was made on a case-by-case basis, depending on consultation between the surgeon and patient. Complications were collected to distinguish between intraoperative complications, complications with no readmission, complications with readmission, and complications with reoperation. Propensity scores based on age, sex, body mass index, and the American Society of Anesthesiologists score were used to match outpatient to inpatient FT TKA. A cumulative incidence function was computed by taking the time to diagnose any postoperative complication in the first 90 days as the end point. RESULTS: Propensity score matching (1:2 ratio) for comparison resulted in 173 outpatient FT TKAs and 316 inpatient FT TKAs. No significant differences were observed between outpatient versus inpatient FT TKA for intraoperative complication rates (2% in both groups). At 90-day follow-up, no significant differences were observed between outpatient versus inpatient FT TKA for total complications with no readmission (8.0 versus 7.9%), complications with readmission but no reoperation (1.1 versus 0.6%), and complications with reoperation (4.0 versus 4.4%). A comparison of postoperative complication diagnosis time using the cumulative incidence function revealed no significant differences between outpatient versus inpatient FT TKA. CONCLUSIONS: The present study revealed that there were no differences in 90-day postoperative complication rates between outpatient and inpatient FT TKA and that there were also no differences in rates of intraoperative complications, readmissions, or reoperations. These findings may encourage hesitant surgeons to move toward outpatient TKA pathways, as there is no greater risk of early postoperative complications that could be more difficult to manage after discharge. LEVEL OF EVIDENCE: Level II.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

Advancing Spectrally-Resolved Single Molecule Localization Microscopy with Deep Learning.

Spectrally-resolved single molecule localization microscopy (srSMLM) is a recent technique enriching single molecule localization microscopy with the simultaneous recording of spectra of the single emitters. srSMLM resolution is limited by the number of photons collected per emitters. Sharing a photon budget to record the localization and the spectroscopic information results in a loss of spatial and spectral resolution-or forces the sacrifice of one at the expense of the other. Here, srUnet-a deep-learning Unet-based image processing routine trained to increase the spectral and spatial signals to compensate for the resolution loss inherent in additionally recording the spectral component is reported. Both localization and spectral precision are improved by srUnet-particularly for the low-emitting species. srUnet increases the fraction of localization whose signal can be both spatially and spectrally characterized. It preserves spectral shifts and the linearity of the dispersion of light. It strongly facilitates wavelength assignment in multicolor experiments. srUnet is a simple post-processing add-on boosting srSMLM performance close to conventional SMLM with the potential to turn srSMLM into the new standard for multicolor single molecule imaging.

In cellulo FRET-FLIM and single molecule tracking reveal the supra-molecular organization of the pyoverdine bio-synthetic enzymes in Pseudomonas aeruginosa.

The bio-synthesis of pyoverdine (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of non-ribosomal peptide synthetases (NRPSs). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesized by co-expressed accessory enzymes. It is generally proposed that different enzymes of a secondary metabolic pathway assemble into large supra-molecular complexes. However, evidence for the assembly of sequential enzymes in the cellular context is sparse. Here, we used in cellulo single-molecule tracking and Förster resonance energy transfer measured by fluorescence lifetime microscopy (FRET-FLIM) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed that PvdA is physically interacting with PvdJ, PvdI, PvdL, and PvdD, the four NRPS involved in the PVD pathway in Pseudomonas aeruginosa PAO1. The binding modes of PvdA were strikingly different according to the NRPS it is interacting with, suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS. Our data provide evidence for strongly organized multi-enzymatic complexes responsible for the bio-synthesis of PVD and illustrate how binding sites have evolved to finely control the co-localization of sequential enzymes and promote metabolic pathway efficiency.

MRI dedicated to the emergency department for diplopia or dizziness: a cost-effectiveness analysis.

OBJECTIVES: The purpose of this study was to compare the costs and organizational benefits of diagnostic workup without and with MRI dedicated to the ED. METHODS: We conducted a prospective observational uncontrolled before-after study in one ED of a university hospital in France from July 1, 2018, and January 3, 2020. We included all consecutive patients presenting with dizziness or diplopia. The main outcomes were the clinical decision time of ED physicians and the total costs for each strategy. Outcomes were compared using propensity score with inverse probability weighting in the 2 arms and an incremental cost-effectiveness ratio (ICER) was calculated. RESULTS: Among the 199 patients during the "before" period (average age: 60.4 years ± 17.6): 112 men (57%), and 181 during the "after" period (average age, 54.8 years ± 18.5): 107 men (59%), the average costs were €2701 (95% CI 1918; 3704) and €2389 (95% CI: €1627; 3280) per patient, respectively. The average time to clinical decision was 9.8 h (95% CI: 8.9 10.7) in the group "before" and 7.7 h (95% CI: 7.1; 8.4) in the group "after" (ICER: €151 saved for a reduction of 1 h in clinical decision time). The probabilistic sensitivity analysis estimated a 71% chance that the MRI dedicated to ED was dominant (less costly and more effective). CONCLUSION: Easy access to MRI in the ED for posterior circulation stroke-like symptoms must be considered a relevant approach to help physicians for an appropriate and rapid diagnostic with reduction of costs. TRIAL REGISTRATION: NCT03660852 KEY POINTS: • A dedicated MRI in the ED for diplopia or dizziness may be considered an efficient strategy improving diagnostic performance, reducing physicians' decision time, and decreasing hospital costs. • This strategy supports clinical decision-making with early treatment and management of patients with posterior circulation-like symptoms in the ED. • There is 71% chance that the MRI dedicated to ED was dominant (less costly and more effective) compared with a strategy without dedicated MRI.

Predictive factors of amoxicillin immediate hypersensitivity and validation of PEN-FAST clinical decision rule.

BACKGROUND: The challenge of delabeling amoxicillin allergy is an important issue for patients and clinicians, especially when anaphylaxis is reported. A recent study has proposed a clinical decision rule, PEN-FAST, to identify low-risk penicillin allergies. OBJECTIVE: To validate the PEN-FAST clinical decision rule in a population with high risk of suspected immediate amoxicillin allergy and to identify clinical predictive factors of amoxicillin immediate hypersensitivity. METHODS: We retrospectively analyzed medical records of patients with a suspected immediate amoxicillin allergy who carried out an allergologic evaluation by a specialist in the Allergy Unit of Strasbourg University Hospital from 2015 to 2020. RESULTS: A total of 142 adult patients (88 women [62.0%]; median age, 52 [interquartile range, 40.3-62.0] years) were analyzed. Most of them reported anaphylaxis (68.8%). Internal validation of PEN-FAST score revealed a good discrimination with area under the curve of 0.86 (95% confidence interval, 0.79-0.92). A cutoff of less than 3 points for PEN-FAST was used to classify 29 from 142 patients at low risk of allergy, of whom only 2 (6.9%) received positive results of allergy testing. The negative predictive value for successful delabeling was 0.93 (95% confidence interval, 0.77-0.99). Predictive clinical features for immediate amoxicillin hypersensitivity were time since reaction (P < .001), time elapsed between drug intake and first symptom (P < .001), severity grade reaction (P < .001), and treatment or hospitalization required (P < .001). CONCLUSION: PEN-FAST has been validated to identify low-risk penicillin allergies in our European cohort of patients mainly reporting anaphylaxis. This is the first reported external validation of a penicillin allergy clinical decision rule internationally.

Accuracy and technical limits of percutaneous pedicle screw placement in the thoracolumbar spine.

PURPOSE: The two-dimensional fluoroscopic method of percutaneous pedicle screw instrumentation has been clinically described as reliable method in the caudal thoracic and lumbosacral spine. Its accuracy has not been clearly reported in the cranial thoracic spine. The aim of this in vitro study was to investigate percutaneous pedicle screw placement accuracy according to pedicle dimensions and vertebral levels. METHODS: Six fresh-frozen human specimens were instrumented with 216 screws from T1 to S1. Pedicle isthmus widths, heights, transversal pedicles and screws were measured on computed tomography. Pedicle cortex violation ≥ 2 mm was defined as screw malposition. RESULTS: The narrowest pedicles were at T3-T5. A large variability between transversal pedicle axes and percutaneous pedicle screw was present, depending on the spinal level. Screw malposition rates were 36.1% in the cranial thoracic spine (T1-T6), 16.7% in the caudal thoracic spine (T7-T12), and 6.9% in the lumbosacral spine (L1-S1). The risk for screw malposition was significantly higher at cranial thoracic levels compared to caudal thoracic (p = 0.006) and lumbosacral (p < 0.0001) levels. Cortex violation ≥ 2 mm was constantly present if the pedicle width was < 4.8 mm. CONCLUSION: Percutaneous pedicle screw placement appears safe in the caudal thoracic and lumbosacral spine. The two-dimensional fluoroscopic method has a limited reliability above T7 because of smaller pedicle dimensions, difficulties in visualizing radiographic pedicle landmarks and kyphosis.

An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization.

Siderophores are iron-chelating molecules produced by bacteria to access iron, a key nutrient. These compounds have highly diverse chemical structures, with various chelating groups. They are released by bacteria into their environment to scavenge iron and bring it back into the cells. The biosynthesis of siderophores requires complex enzymatic processes and expression of the enzymes involved is very finely regulated by iron availability and diverse transcriptional regulators. Recent data have also highlighted the organization of the enzymes involved in siderophore biosynthesis into siderosomes, multi-enzymatic complexes involved in siderophore synthesis. An understanding of siderophore biosynthesis is of great importance, as these compounds have many potential biotechnological applications because of their metal-chelating properties and their key role in bacterial growth and virulence. This review focuses on the biosynthesis of siderophores produced by fluorescent Pseudomonads, bacteria capable of colonizing a large variety of ecological niches. They are characterized by the production of chromopeptide siderophores, called pyoverdines, which give the typical green colour characteristic of fluorescent pseudomonad cultures. Secondary siderophores are also produced by these strains and can have highly diverse structures (such as pyochelins, pseudomonine, yersiniabactin, corrugatin, achromobactin and quinolobactin).

Are high- and low-molecular-weight sensitizing agents associated with different clinical phenotypes of occupational asthma?

BACKGROUND: High-molecular-weight (HMW) proteins and low-molecular-weight (LMW) chemicals can cause occupational asthma (OA) although few studies have thoroughly compared the clinical, physiological, and inflammatory patterns associated with these different types of agents. The aim of this study was to determine whether OA induced by HMW and LMW agents shows distinct phenotypic profiles. METHODS: Clinical and functional characteristics, and markers of airway inflammation were analyzed in an international, multicenter, retrospective cohort of subjects with OA ascertained by a positive inhalation challenge response to HMW (n = 544) and LMW (n = 635) agents. RESULTS: Multivariate logistic regression analysis showed significant associations between OA caused by HMW agents and work-related rhinitis (OR [95% CI]: 4.79 [3.28-7.12]), conjunctivitis (2.13 [1.52-2.98]), atopy (1.49 [1.09-2.05]), and early asthmatic reactions (2.86 [1.98-4.16]). By contrast, OA due to LMW agents was associated with chest tightness at work (2.22 [1.59-3.03]), daily sputum (1.69 [1.19-2.38]), and late asthmatic reactions (1.52 [1.09-2.08]). Furthermore, OA caused by HMW agents showed a higher risk of airflow limitation (1.76 [1.07-2.91]), whereas OA due to LMW agents exhibited a higher risk of severe exacerbations (1.32 [1.01-1.69]). There were no differences between the two types of agents in the baseline sputum inflammatory profiles, but OA caused by HMW agents showed higher baseline blood eosinophilia and a greater postchallenge increase in fractional nitric oxide. CONCLUSION: This large cohort study describes distinct phenotypic profiles in OA caused by HMW and LMW agents. There is a need to further explore differences in underlying pathophysiological pathways and outcome after environmental interventions.

Cluster analysis of indoor environmental factors associated with symptoms of mite allergy.

BACKGROUND: The clinical efficacy of controlling environmental allergens as a component of allergic asthma treatment remains controversial. Multifaceted allergen reductions appeared to be the most efficient methods. However, they require home visits with indoor technicians. OBJECTIVE: To examine the characteristics of indoor environments that might be related to symptoms of children and adult patients with mite allergic rhinitis and/or asthma. METHODS: We included 315 patients allergic to house dust mites with rhinitis and/or asthma who had been visited at home by 2 medical indoor environment counselors (MIECs) from the Strasbourg University Hospital between January 2007 and June 2015. In a cluster analysis, we analyzed 42 characteristics of respiratory symptoms, dwelling characteristics, and indoor pollutants in this population. RESULTS: Three clusters were defined among the patients. Cluster 1 included 55 patients, all with rhinitis, 32% with asthma, and all living in an urban area. Clusters 2 and 3 included 86 and 174 patients, respectively. The important factors in these 2 clusters were asthma incidence and exposure to different indoor pollutants, such as indoor perfumes, cleaning products, and tobacco smoke. CONCLUSION: Our results underlined the variability of indoor environments and the importance of MIEC home visits to investigate individual patient environments and propose an appropriate avoidance management plan. Our results showed that sensitization to mite and exposure to indoor chemical pollutants were associated with severe asthma.

FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein.

During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).

Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription.

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure-activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44-61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44-61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.

A new, faster, and safe nasal provocation test method for diagnosing mite allergic rhinitis.

BACKGROUND: Diagnosing house dust mite (HDM) allergic rhinitis is difficult. The nasal provocation test (NPT) has been shown to be the most pertinent, but several methods are available. According to guidelines, the NPT requires a skin end-point titration and an objective measurement of nasal patency. Hence, NPT is time consuming and its use is limited. OBJECTIVE: To evaluate the sensitivity, specificity, and safety of a new, more rapid, and simple alternative NPT (NPT-R) to HDM. METHODS: Eighty-eight patients with from rhinitis (49 allergic to HDM and 39 controls with and without atopy) were included. Allergic rhinitis to HDM was confirmed by a "classic" NPT based on the Lebel score and rhinomanometry. After a period of 4 weeks, NPT-R was performed and only the clinical score was measured. RESULTS: The study population was young (mean ± SD, 27.7 ± 8.5 years old), composed mostly of women (61 vs 27 men), and 24% reported asthma. The sensitivity and specificity of NPT-R were 83.7% and 100%, respectively. The correlation between the NPTs was statistically significant (0.833, P < .0001, n = 88) and the 2 NPTs were completely safe. Performing NPT-R was more rapid (mean ± SD, 22 ± 8 minutes) than the classic NPT (97 ± 20 minutes). CONCLUSION: The NPT-R is safe and easier and faster than the classic NPT. This new method appears to be a very useful tool in the diagnosis of HDM allergic rhinitis when the diagnosis is uncertain or before initiating immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT01485523.

Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.