Inducing Propeller Chirality in Triaryl Boranes with Chiral Amines.

Stabilization of chiral propeller conformations in triaryl compounds is challenging due to generally low racemization barriers. Nonetheless, it was recently found that chiral conformational preferences can be induced to triaryl boranes by incorporating point-chiral alkylether chains to the aryl blades and subsequently locking the structure with ammonia. A four-point interaction, meaning that the cooperative effects of Lewis-adduct formation and three hydrogen bonds, was proposed as stabilizing mechanism. Herein, it was shown that three such strong interactions suffice to introduce a preferential propeller handedness. Although DFT calculations predict no noteworthy preferences for either P- or M-chiral propellers for some of the investigated triarylborane-amine adducts that were prepared with chiral primary amines, vibrational circular dichroism (VCD) spectroscopic characterizations revealed that there is indeed a measurable excess of one propeller handedness. Furthermore, the steric demand of the amine was found to play a key role in the induction process and especially in preventing blade rotations.

Cuffed versus uncuffed tracheal tubes in a rabbit training model for establishing an emergency front-of-neck airway in infants: a prospective trial.

BACKGROUND: There is a paucity of evidence regarding the optimal type of tracheal tube to be advanced over a Frova catheter when performing a 'bougie' emergency front-of-neck airway (eFONA) technique in infants during a 'cannot intubate, cannot oxygenate' situation. METHODS: A prospective non-inferiority trial in a rabbit cadaver surgical tracheotomy model to assess the performance of the eFONA technique with an uncuffed 3.5 mm ID tracheal tube vs a cuffed 3.0 mm ID tracheal tube. Queried outcomes include success rate, performance time, and severe secondary airway injuries among tracheal tube types. RESULTS: In 60 tracheostomies performed by 30 participants, the overall success rate was 98%. Performance time was independent from tracheal tube choice (uncuffed: 61 s [95% confidence interval (CI), 52-76], cuffed: 64 s [95% CI, 55-79]; P = 0.82). No tracheal tube type was preferred in terms of usability by participants. The cuffed tracheal tube required increased force to be advanced over the Frova catheter and was associated with a risk ratio of 2.5 (95% CI, 0.53-11.9; number needed to harm, 10) for severe secondary airway injuries when compared with the uncuffed tracheal tube. CONCLUSION: In performing eFONA in the rabbit cadaver model, an ID 3.5 uncuffed is non-inferior to an ID 3.0 cuffed tracheal tube regarding performance time and preference by the operator. Greater force application to advance the cuffed tube over the Frova catheter and more severe airway injuries may argue for the standardised performance of the eFONA technique with a uncuffed tracheal tube in infants.

Stereochemistry Controls Dihydrogen Bonding Strengths in Chiral Amine Boranes Adducts.

The growing interest in exploiting novel concepts of non-covalent interactions in catalysts and supramolecular chemistry made us revisit a special kind of hydrogen bonding: the dihydrogen bond (DHB), formed between a classical hydrogen bond donor and a hydridic hydrogen as acceptor. Herein, we investigate how the strength of the N-Hδ+ ⋅⋅⋅δ- H-B interaction and hence the DHB-driven self-aggregation of amine-borane adducts is governed by steric effects by comparing the structures and binding enthalpies of various chiral derivatives. For a diastereomeric pair of amine-boranes prepared from a chiral secondary amine, we show that the stereochemistry at the nitrogen has significant influence on the interaction enthalpy. Based on this finding, N-chiral amine boranes can be envisioned to become interesting building blocks in supramolecular chemistry to fine-tune the formation dynamics of assemblies.

Dimensional compatibility of currently available equipment for cricothyroidotomy and adult airway anatomy: an in vitro analysis.

BACKGROUND: There are scant data on the dimensional compatibility of cricothyroidotomy equipment and related airway anatomy. We compared the dimensional design of devices for cricothyroidotomy with anatomical airway data for adult patients. METHODS: For all available cricothyroidotomy equipment the outer diameter was recorded from manufacturer information or, if not available, measured using a sliding calliper. Outer diameters were compared with recently published mean (standard deviation [sd]) values for the height of the cricothyroid membrane obtained from computed tomography, separately for males (7.9 [2.2] mm) and for females (5.9 [1.7] mm). RESULTS: Twenty-one cricothyroidotomy sets (10 uncuffed, 11 cuffed) with 15 differently designed devices were included. Inner diameters of the tubes ranged from 3.5 to 6.0 mm and outer diameters from 5.0 to 11.7 mm. The outer diameter of the 15 different tubes was found to be greater than the mean membrane height of the adult male cricothyroid membrane in eight devices and greater than the mean membrane height for female adults in 10 devices. Considering the lower range of cricothyroid membrane height, 12 tube outer diameters would be too large for male adults and all 15 for female adults in this range. CONCLUSION: The outer diameter of many devices currently marketed for cricothyroidotomy are oversized for adult airway anatomy, particularly for females. For emergency front-of-neck access through the cricothyroid membrane, anatomical data suggest that cricothyroidotomy devices with outer tube diameters of <7 mm for male and <6 mm for female adult patients should be preferred.

Rabbit training model for establishing an emergency front of neck airway in children.

BACKGROUND: A 'cannot intubate, cannot oxygenate' (CICO) situation is rare in paediatric anaesthesia, but can always occur in children under certain emergency situations. There is a paucity of literature on specific procedures for securing an emergency invasive airway in children younger than 6 yr. A modified emergency front of neck access (eFONA) technique using a rabbit cadaver model was developed to teach invasive airway protection in a CICO situation in children. METHODS: After watching an instructional video of our eFONA technique (tracheotomy, intubation with Frova catheter over which a tracheal tube is inserted), 29 anaesthesiologists performed two separate attempts on rabbit cadavers. The primary outcome was the success rate and the performance time overall and in subgroups of trained and untrained participants. RESULTS: The overall success rate across 58 tracheotomies was 95% and the median performance time was 67 s (95% confidence interval [CI], 56-76). Performance time decreased from the first to the second attempt from 72 s (95% CI, 57-81) to 61 s (95% CI, 50-81). Performance time was 59 s (95% CI, 49-79) for untrained participants and 72 s (95% CI, 62-81) for trained participants. Clinical experience and age of the participants was not correlated with performance time, whereas the length of the tracheotomy incision showed a significant correlation (P=0.006). CONCLUSION: This eFONA training model for children facilitates rapid skill acquisition under realistic anatomical conditions to perform an emergency invasive airway in children younger than 2 yr.

Dimensional compatibility and limitations of tracheal intubation through supraglottic airway devices: a mannequin-based in vitro study.

BACKGROUND: Flexible bronchoscope-guided tracheal intubation through supraglottic airway devices (SGAs) is a well-established element of difficult intubation algorithms. Success can be limited by dimensional incompatibilities between tracheal tubes (TTs) and SGAs. METHODS: In this in vitro study, we tested the feasibility of TT passage through SGAs, removal of SGAs over TTs, and the ability to guide the flexible bronchoscope with 13 TT brands (internal diameter, 6.5-8.0 mm) and ten different SGAs (#4 and #5) in an intubation mannequin. RESULTS: We tested 1,040 combinations of SGAs and TTs. Tracheal tube passage failed in 155 (30%) combinations of the five tested first-generation SGAs (117 [46%] with SGA #4, 38 [15%] with SGA #5) and in three (0.6%) combinations of the five tested second-generation SGAs (two [0.8%] with SGA #4 and one [0.4%] with SGA #5). The reason for failed passage of a TT through a first-generation SGA consistently was a too-narrow SGA connector. Removal of the SGA over the TT in the 882 remaining combinations was impossible for all sizes of reinforced TTs, except the Parker Reinforced TT, and was possible for all non-reinforced TTs. Only one combination with SGA #4 and 84 combinations with SGA #5 were not ideal to adequately guide the flexible bronchoscope. CONCLUSION: Clinically relevant combinations of adult-size TTs and SGAs can be incompatible, rendering flexible bronchoscope-guided tracheal intubation through an SGA impossible. Additional limitations exist regarding removal of the SGA and maneuverability of the flexible bronchoscope.

RéSUMé: CONTEXTE: L'intubation endotrachéale guidée par bronchoscope flexible via un dispositif supraglottique (DSG) est un élément établi des algorithmes utilisés pour les intubations difficiles. La réussite de l'intubation peut être limitée par des incompatibilités dimensionnelles entre les tubes endotrachéaux (TET) et les DSG. MéTHODE: Dans cette étude in vitro, nous avons testé la faisabilité de faire passer un TET par un DSG, le retrait du DSG par-dessus le TET et la possibilité de guider un bronchoscope flexible avec 13 marques de TET (diamètre interne, 6,5-8,0 mm) et dix DSG différents (#4 et #5) sur un mannequin d'intubation. RéSULTATS: Nous avons testé 1040 combinaisons de DSG et de TET. Le passage du tube endotrachéal a échoué dans 155 (30 %) combinaisons avec les cinq DSG de première génération testés (117 [46 %] avec DSG #4, 38 [15 %] avec DSG #5) et dans trois (0,6 %) combinaisons avec les cinq DSG de deuxième génération testés (deux [0,8 %] avec DSG #4 et une [0,4 %] avec DSG #5). La raison de l'échec du passage d'un TET à travers un DSG de première génération était systématiquement liée à un connecteur de DSG trop étroit. Dans les 882 combinaisons restantes, le retrait du DSG par-dessus le TET s'est avéré impossible avec toutes les tailles de TET armés, à l'exception du TET armé Parker, et était possible avec tous les TET non armés. Une seule combinaison avec le DSG #4 et 84 combinaisons avec le DSG #5 n'étaient pas idéales pour guider le bronchoscope flexible de manière adéquate. CONCLUSION: Les combinaisons cliniquement pertinentes de TET et de DSG de tailles adultes peuvent être incompatibles, rendant impossible l'intubation endotrachéale guidée par bronchoscope flexible via un DSG. D'autres limites existent en ce qui concerne le retrait du DSG et la maniabilité du bronchoscope flexible.

Inflation volume-balloon diameter and inflation pressure-balloon diameter characteristics of commonly used bronchial blocker balloons for single-lung ventilation in children.

BACKGROUND: Balloon-tipped bronchial blocker catheters are widely used in pediatric thoracic anesthesia to establish single-lung ventilation. In clinical practice, their balloons demonstrate sudden expansion when inflated with air. In addition, there are concerns related to the high inflation pressures required to expand the balloons. METHODS: This in vitro study assessed inflation volume- and inflation pressure-balloon diameter characteristics of the Fogarty arterial embolectomy catheters and Arndt endobronchial blockers. Balloon diameters were photographically assessed during unrestricted volume- and pressure-guided inflation, using air up to the maximum allowed inflation volume as indicated by the manufacturers. Inflation pressures required to open the blocker balloons and inflation pressures needed to expand them to maximum indicated diameter were measured. RESULTS: Volume-guided inflation demonstrated a late acute rise in diameter in Fogarty blocker balloons, whereas in the Arndt endobronchial blocker balloons almost linear inflation volume-to-diameter characteristics were observed. Pressure-guided inflation on the other hand demonstrated low-volume, high-pressure characteristics in the Fogarty blocker balloons, with inflation pressures required to expand the balloons to maximum diameters ranging from (mean (SD)) 636 (75) to 947 (152) cmH2 O. The inflation pressures required to open the Fogarty blocker balloons were even >1000 cmH2 O. Inflation pressures required to expand the 5 F, 7 F, and 9 F Arndt endobronchial blocker balloons to maximum indicated diameter were much lower, namely at 218 (15), 252 (28), and 163 (8) cmH2 O. CONCLUSION: Based on these study findings, the balloons of Fogarty arterial embolectomy catheters represent high-pressure devices and do not permit stepwise controlled bronchial blockage. The Arndt endobronchial blockers have some advantages over the Fogarty blocker devices, but also represent high-pressure equipment and must be used with caution and limited duration. Manufacturers are asked to design pediatric endobronchial blocker catheters with truly high-volume, low-pressure balloons in accordance to age-related pediatric airway dimensions.

Insertion of cuff inflation line into pediatric tracheal tubes related to oral and nasal tracheal intubation depth.

BACKGROUND: In clinical practice, the cuff inflation line of cuffed pediatric tracheal tubes often interferes with securing tracheal tubes. METHODS: The insertion site of the cuff inflation lines and the lengths of four different brands and nine sizes of commonly used cuffed pediatric tracheal tubes were measured and compared in vitro with oral and nasotracheal intubation depths as calculated by different formulas for pediatric patients aged from birth to 16 years. Motoyama's recommendation was used for age-related size selection of cuffed pediatric tracheal tubes. RESULTS: The proportion of the distance from the tracheal tube tip to the insertion site of the cuff inflation line varied considerably between the tracheal tubes (Microcuff: 48.5-60.7%; Parker: 48.7-73.2%; Ruesch: 59.1-77.8%; and Shiley: 46.0-60.3%). Using different formulas for oral or nasotracheal intubation depth, the insertion site of the cuff inflation line was placed within the oral or nasal cavity or within an area 1 cm beyond the teeth or the nostrils in almost all tracheal tubes tested. Positioning the insertion site 2 cm from the proximal end of the tracheal tubes resulted in a cuff line-free tube area of ≥1 cm in all orally and almost in all nasally inserted tracheal tubes, considering maximum recommended tracheal intubation depths. CONCLUSION: The cuff inflation line in almost all commonly used cuffed pediatric tracheal tubes interferes with securing the tracheal tube due to its insertion site into the tracheal tube. This potentially carries the risk of kinking, obstruction, or damage to the cuff inflation line with ensuing failure to deflate or inflate the cuff. The proposed position of the insertion of the cuff inflation line 2 cm from the proximal end of the tracheal tube would ensure a 1-cm-wide cuff line-free circular area beyond the oral or nasal cavity in nearly all assessed tracheal tube sizes.

Dimensional compatibility of rigid ventilating bronchoscopes with pediatric airway anatomy using different recommendations for age-related sizing-A bench study.

BACKGROUND: Appropriate size selection of pediatric rigid bronchoscopes is fundamental to avoidance of airway trauma and to a high success rate with the first intubation attempt. The aim of the present study was to compare the outer diameters of pediatric rigid bronchoscopes with the anatomical data on the pediatric airway. METHODS: Outer diameters (OD) of pediatric rigid ventilating bronchoscopes as given by the manufacturer were compared with published computed tomography internal diameters (ID) of the cricoid outlet, the trachea as well as the left and right proximal mainstem bronchus, using six published recommendations for age-related size selection. The ratios between a specific OD and ID were calculated and given as percentage values (%) for lower and upper age ranges. RESULTS: Nominal sized 2.5, 3, 3.5, 3.7, 4, 4.5, 5, and 6 rigid bronchoscopes were included. In five recommendations, the rigid bronchoscopes' OD was larger than the internal cricoid diameter in the upper age range, whereas in one recommendation the ratio (OD rigid bronchoscope to ID of cricoid) ranged between 77% and 90% for the upper age and between 95% and 109% for the lower age range. In only one of the six recommendations was the rigid bronchoscope OD larger than the tracheal ID. Ratios for the left and right proximal mainstem bronchus ranged from 70% to 146% for the upper and from 78% to 156% for the lower age range, with the highest ratios for the left proximal mainstem bronchus. CONCLUSION: Based on this in vitro study, most of the recommendations analyzed result in a balanced fit of rigid bronchoscopes within the cricoid. Since the left mainstem bronchus is considerably smaller than the cricoid, any insertion on this level will require careful endoscopic guidance to avoid damage to the left mainstem bronchus.

Simulated dimensional compatibility of uncuffed and cuffed tracheal tubes for selective endobronchial intubation in children.

BACKGROUND: Cuffed tracheal tubes have recently been recommended for selective endobronchial intubation to establish single-lung ventilation even in smaller children. This implies that, compared with uncuffed tracheal tubes, the cuffed tracheal tubes selected will be smaller and therefore have a shorter length. We hypothesized that cuffed tracheal tubes might be of insufficient length for selective endobronchial intubation if the tube cuff were fully immersed in the left or right mainstem bronchus. METHODS: The distance from the proximal end of the tracheal tube to the upper border of the cuff in cuffed tracheal tubes and to the upper margin of the Murphy eye in uncuffed tracheal tubes, respectively, was assessed in sizes 3.0-7.0 mm internal diameter. The raw data sets of two previously performed studies obtained from 337 children aged from birth to 16 years, including the distances "teeth to tracheal tube tip" and "tracheal tube tip to carina," were used to calculate age-, weight-, and height-related data for the distance from "teeth to carina." Tracheal tube dimensions were compared with age-related distances from "teeth to carina," applying published recommendations for the selection of uncuffed and cuffed tracheal tubes for selective endobronchial intubation in children. RESULTS: The differences between the length of the age-related tracheal tube and the tracheal tube insertion length required to guarantee full insertion of the tracheal tube cuff or the Murphy eye within the mainstem bronchus ranged from -3.5 to 52.6 mm in cuffed tracheal tubes and from 42.3 to 83.3 mm in uncuffed tracheal tubes. CONCLUSIONS: For many age groups of patients requiring selective endobronchial intubation, the lengths of cuffed tracheal tubes, in contrast to those of uncuffed tracheal tubes, were revealed to be critically short for safe taping outside the oral cavity with the cuff placed completely within the right or left mainstem bronchus.

Chiral Molecular Propellers of Triarylborane Ammonia Adducts.

Chiral molecular propeller conformations have been induced to various triaryl structures including trityl derivatives and triaryl boranes. For borane-amine adducts, such induced propeller chirality has not been reported yet due to the low energy barrier for racemization in common triarylboranes such as B(C6 H5 )3 or B(C6 F5 )3 . Herein, we demonstrate that point chirality in side chains of chiral triarylborane-ammonia adducts, which feature intramolecular hydrogen bonds in addition to the dative N→B bond, can efficiently be transferred to triarylborane propeller chirality. Employing X-ray crystallography and ECD/VCD spectroscopy for structural characterizations, we investigate three examples with different steric demands of the incorporated chiral alkoxy side groups. We elucidate the conformational preferences of the molecular propellers. Furthermore, we show that computationally predicted conformational preferences obtained for the isolated, only implicitly solvated molecules are actually opposite to the experimentally observed ones.

Cuffed pediatric endotracheal tubes-Outer cuff diameters compared to age-related airway dimensions.

BACKGROUND: Reliable sealing of the pediatric airway requires appropriately sized tracheal tube cuffs. The aim of this study was to compare residual cuff diameters of pediatric tracheal tubes with pediatric airway dimensions. METHODS: Cuff diameters of five different brands of locally marketed pediatric cuffed tracheal tubes with internal diameters of 3.0-7.0 mm were measured at a cuff pressure of 20 cm H2 O and compared with cuff diameters indicated by their manufacturers. The latter values were compared to tracheal dimensions using the Motoyama and Khine formulas for cuffed tracheal tube size selection. RESULTS: There is considerable heterogeneity in cuff diameters among pediatric tracheal tube brands, except for two brands from different manufacturers (Halyard and Parker Medical) which were identically designed. Cuffs made from polyurethane revealed fewer differences (91%-118%) between measured and manufacturer-indicated values for outer cuff diameters than did those made from polyvinylchloride (91%-146%). Particularly in smaller sized tracheal tubes, cuffs did not reach 100% of the tracheal lateral diameter, while others were oversized in larger tracheal tubes, independent of the two formulas used for cuffed tracheal tube size selection. Cuff diameters indicated by the manufacturer corresponded to 86%-188% of the median and 68%-157% of the maximum mid-tracheal lateral diameter of the corresponding upper age range. CONCLUSION: Our findings show that many of the cuff diameters of currently marketed tracheal tube brands lack an age-related anatomical rationale. A proposal for age-related anatomically based cuff diameters is provided for both recommendations for cuffed tracheal tube size selection in children.

Dimensional compatibility of balloon-tipped bronchial blockers with the pediatric airway anatomy using different recommendations for age-related size selection.

BACKGROUND: Age-related recommendations for size selection of bronchial blocker devices are based on a few dated anatomical autopsy and radiological studies determining lower airway dimensions in children. These recommendations are based on anterior-posterior internal bronchial diameters, which are smaller than the more relevant lateral internal bronchial diameters. METHODS: Outer diameters of bronchial blocker balloons inflated with the maximum recommended volume of air were compared to age-related lateral internal bronchial diameters of left and right proximal mainstem bronchi using five different recommendations for age-related size selection of bronchial blocker equipment in children published in the literature. RESULTS: The ratio of outer bronchial blocker diameters inflated with the maximal recommended volume of air to the median lateral internal diameters of the proximal mainstem bronchus ranged from 71.4% to 181.8% for the left side and from 61.7% to 162.6% for the right side. There were many settings where the blocker diameters did not reach the median lateral internal diameters of the proximal mainstem bronchus. This was more often observed for the right than for the left side (37 vs 22 settings). CONCLUSION: This analysis demonstrates that irrespective of the recommendation for size selection used, neither the two balloon-tipped vascular catheters included nor the Arndt endobronchial blockers are ideal for lung isolation in children are compared with the age-related relevant dimensions of pediatric airway anatomy. A redesign of bronchial blocker equipment with age-related anatomically based high-volume, low-pressure blocker balloons made from ultrathin membranes and with smaller catheters would be desirable.

Pediatric airway dimensions-A summary and presentation of existing data.

BACKGROUND: Age-related pediatric airway dimension reference values for cricoid, tracheal, and bronchial diameters as well as tracheal and bronchial lengths are essential for distinguishing normal from pathological airway findings and for manufacturing and selecting appropriately sized airway equipment. AIM: The aim of this work was to summarize and present existing pediatric airway dimension data for the larynx, trachea, and main stem bronchi from fetus to adolescence. METHODS: A systematic literature search was carried out using PubMed, Scopus, Embase, and Google Scholar. Publications containing original data on pediatric airway dimensions as mean or median in tabular form and spanning narrow age groups of 1 or 2 years were included in our study. Original data such as diameters, lengths, and cross-sectional areas of trachea, cricoid, left and right main bronchi in fetuses and children were collected and presented as figures. RESULTS: Pediatric airway dimension data were gathered and compiled from 15 studies fulfilling the inclusion criteria. Data were obtained from different measurement methods such as autopsy, chest X-ray, computed tomography, magnetic resonance imaging, rigid and flexible bronchoscopy as well as ultrasound examinations. There was considerable variation among age-related data due to biologic heterogeneity, different presentation of data, different definitions, and various measurement techniques. CONCLUSION: This investigation revealed heterogeneous data on pediatric airway dimensions, making it impossible to compile them into standard reference values for airway dimensions. New studies with structured and standardized measurements and data presentation in large populations of children are required to provide more valid pediatric airway dimension data.

Impact of a Glycolic Acid-Containing pH 4 Water-in-Oil Emulsion on Skin pH.

The skin pH is crucial for physiological skin functions. A decline in stratum corneum acidity, as observed in aged or diseased skin, may negatively affect physiological skin functions. Therefore, glycolic acid-containing water-in-oil (W/O) emulsions adjusted to pH 4 were investigated regarding their effect on normal or increased skin pH. A pH 4 W/O emulsion was applied on three areas with pathologically increased skin surface pH in diabetics (n = 10). Further, a 28-day half-side trial (n = 30) was performed to test the long-term efficacy and safety of a pH 4 W/O emulsion (n = 30). In summary, the application of a pH 4 W/O emulsion reduced the skin pH in healthy, elderly and diabetic subjects, which may improve epidermal barrier functions.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

A Bnr-like formin links actin to the spindle pole body during sporulation in the filamentous fungus Ashbya gossypii.

Formin proteins are nucleators of actin filaments and regulators of the microtubule cytoskeleton. As such, they play important roles in the development of yeast and other fungi. We show here that AgBnr2, a homologue of the ScBnr1 formin from the filamentous fungus Ashbya gossypii, localizes to the spindle pole body (SPB), the fungal analogue of the centrosome of metazoans. This protein plays an important role in the development of the typical needle-shaped spores of A. gossypii, as suggested by several findings. First, downregulation of AgBNR2 causes defects in sporangium formation and a decrease in the total spore number. Second, a fusion of AgBNR2 to GFP that is driven by the native AgBNR2 promoter is only visible in sporangia. Third, AgBnr2 interacts with a AgSpo21, a sporulation-specific component of the SPB. Furthermore, we provide evidence that AgBnr2 might nucleate actin cables, which are connected to SPBs during sporulation. Our findings add to our understanding of fungal sporulation, particularly the formation of spores with a complex, elongated morphology, and provide novel insights into formin function.

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase.

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.

Human leukocyte antigen B27 selects for rare escape mutations that significantly impair hepatitis C virus replication and require compensatory mutations.

UNLABELLED: Human leukocyte antigen B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant, HLA-B27-restricted, HCV-specific, cluster of differentiation (CD)8(+) T-cell epitope, nonstructural protein (NS)5B(2841-2849) (ARMILMTHF), has been shown to be limited by viral fitness costs as well as broad T-cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27-restricted epitope, NS5B(2936-2944) (GRAAICGKY), to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a-infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T-cell receptor contact site, neither of which impaired viral replication capacity, as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27(+) subjects, rare escape mutations arose at the HLA-B27 anchor residue, R(2937) , which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Coexpression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥ 70% of wild-type levels. CONCLUSION: The selection of rare CTL escape mutations in this HLA-B27-restricted epitope dramatically impairs viral replicative fitness, unless properly compensated. These data support a role for the targeting of highly constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens.

Protective effect of human leukocyte antigen B27 in hepatitis C virus infection requires the presence of a genotype-specific immunodominant CD8+ T-cell epitope.

UNLABELLED: Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. CONCLUSION: Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection.