Peak Cough Flow Fails to Detect Upper Airway Collapse During Negative Pressure Titration for Cough-Assist

ObjectiveTo study the ability of peak cough flow (PCF) and effective cough volume, defined as the volume exsufflated >3 L/s, to detect upper airway collapse during mechanical insufflation-exsufflation (MI-E) titration in neuromuscular patients.DesignProspective observational study.SettingRehabilitation hospital.ParticipantsPatients (N=27) with neuromuscular disease causing significant impairment of chest wall and/or diaphragmatic movement.InterventionsThe lowest insufflation pressure producing the highest inspiratory capacity was used. Exsufflation pressure was decreased from ?20 cm H₂O to ?60/?70 cm H₂O, in 10-cm H₂O decrements, until upper airway collapse was detected using the reference standard of flow-volume curve analysis (after PCF, abrupt flattening or flow decrease vs previous less negative exsufflation pressure).Main Outcome MeasuresPCF and effective cough volume profiles during expiration with MI-E.ResultsUpper airway collapse occurred in 10 patients during titration. Effective cough volume increased with decreasing expiratory pressure then decreased upon upper airway collapse occurrence. PCF continued to increase after upper airway collapse occurrence. In 5 other patients, upper airway collapse occurred at the initial ?20 cm H₂O exsufflation pressure, and during titration, PCF increased and effective cough volume remained unchanged at <200 mL. PCF had 0% sensitivity for upper airway collapse, whereas effective cough volume had 100% sensitivity and specificity.ConclusionOf 27 patients, 15 experienced upper airway collapse during MI-E titration. Upper airway collapse was associated with an effective cough volume decrease or plateau and with increasing PCF. Accordingly, effective cough volume, but not PCF, can detect upper airway collapse.     

In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Powerful selection technologies have made in vitro evolution of protein binders more efficient and paved the way for the use of tailor-made antibodies in therapy. After initial selections of antibody candidates with desired specificity, lead antibodies are typically improved by affinity maturation in multiple rounds of randomization and selection (1) to reach the subnanomolar affinities ideally required for targeting soluble ligands (2–4). This is usually attempted by introduction of point substitutions, either at random positions across the entire V-gene (5, 6) or in the complementary-determining regions (CDRs; e.g., by CDR walking mutagenesis) (7).In Nature, diversification of the primary antibody repertoire occurs by several mechanisms that generate variation in the regions forming the antigen-binding site, the CDRs, including considerable length variation (8–11) that is initially introduced by recombination of V(D)J gene segments. Length variations are concentrated in the CDR3 region (12), at the junctions of the joined segments, where additional diversity is produced by N- or P-nucleotide additions that can further extend the CDR3. The length of the CDRs considerably affects the topography of the combining site, as different shapes brought about by extension or shortening can form pockets, grooves, or fill space (13, 14).Following B cell stimulation by the antigen, further diversification of the antigen-binding interface is generated through somatic hypermutation (SHM) (15), involving mainly point mutagenesis that preferentially targets hotspots in the CDRs (16, 17). This process is initiated through deamination of cytosine to uracil by activation-induced cytidine deaminase (AID), leading to uracil:guanine mismatches (16). Upon removal of these uracil bases by base excision-repair enzymes, error-prone DNA polymerases are then recruited to fill in the gaps and introduce mutations around the position of the deaminated cytosines. Interestingly, up to 6% of the mutations generated by SHM are insertions and deletions (InDels) (18), which occur due to misalignment of repeated DNA sequences (19, 20). Thus, insertions occur by duplication, while deletions are brought about by removal of repeated sequences (21, 22).A small percentage of antibodies selected by in vivo SHM contain InDels in the CDRs 1 and 2 (1.6 to 6.5%) (21–24), while junctional diversity by N- or P-nucleotide additions in the CDR3 confounds the analysis of SHM-derived InDels, leading to an underestimation of the total percentage of affinity-improving InDels. In vitro-directed evolution has been unsuitable for introduction of InDels at random positions into an antibody gene, because of restrictions in the diversity of InDels that could be introduced (i.e., insertions by duplication in in vitro SHM) (22, 25). Rational (26) or computational (27) strategies have been successful at introducing InDels in a few, carefully chosen positions instead of random sampling. In contrast, an unusually high percentage of InDels with a functional role among in vivo affinity matured broadly neutralizing antibodies (bnAbs) to HIV-1 (28–30): ∼40% of the reported anti–HIV-1 bnAbs contain InDels that accumulate during in vivo SHM (28). Based on the frequent occurrence of InDels among multispecific, cross-reactive antibodies, one could infer that they provide a molecular solution for recognizing multiple targets by providing an altered interface (enlarged or tightened), possibly even involving conformational diversity (31). The accumulation of InDels in bnAbs has been attributed to extensive in vivo SHM, so that even positions that are rarely modified by SHM are also altered (17, 28).Insertions in the V-genes occur only by duplication of adjacent sequences (21, 22), so that the actual sequence diversity of the resulting insertions is limited because they repeat existing modules. To introduce more diversity in the inserted sequences, point mutations are required in subsequent rounds of SHM. However, since the CDRs can tolerate considerable length variation, it is likely that the antibody fold can accommodate a larger number of affinity-enhancing InDels compared to those observed in antibodies affinity-matured by SHM.Affinity gains by introduction of InDels have indeed been recognized (22, 25, 26, 32, 33) in in vitro-directed evolution, but often were by-products of campaigns focused on point mutations and not elicited systematically (32, 33). Only in mammalian cell surface display does the action of AID lead to InDels, just as AID brings about InDels in SHM in vivo (22, 25). In a seminal study by Bowers et al. (22), overexpression of AID enabled in vitro SHM of 53 antibodies against 21 antigens to identify InDels in multiple regions likely to improve binding, in particular to variable heavy domain (V_H) and variable light domain (V_L) CDR1, where 9 of 53 antibodies contained InDels. Despite the comprehensive nature of this study, AID-enabled insertions mirrored in vivo SHM and were therefore limited to direct duplication of adjacent sequences, not allowing the full exploration of length and sequence diversity in the insertions, and the low frequency of incorporation of in-frame InDels by AID (<0.1%) limited the combinatorial diversity explored. Finally, InDels have been introduced rationally based on structural analysis and natural length variation (26, 27). Taken together, only limited diversity of InDels in terms of length, position, and insert sequence across the variable domains has been explored thus far.Here we address this omission and explore libraries with in-frame InDels of different lengths and high diversity of inserted sequences at random positions across the entire antibody variable regions (Fig. 1). We applied a new transposon-based mutagenesis approach, dubbed TRIAD (transposition-based random insertion and deletion mutagenesis) (34) that introduces short in-frame insertions and deletions randomly across a gene (in sequences of steps following transposition that excise the transposon, religate the plasmid, and insert designed cassettes) (SI Appendix, Figs. S1 and S2). TRIAD was used here to build libraries with InDels at random positions across an entire single-chain variable fragment (scFv) gene. The antibody chosen for this campaign was the anti–IL-13 antibody BAK1 (35), a derivative of which, tralokinumab, is under clinical investigation for asthma (36). In addition, we built libraries that explore diversity in the different lengths of insertions in a semirandom approach, insertional-scanning mutagenesis (InScaM). These InDel libraries were starting points for antibody affinity evolution in vitro, leading to insertions in two loops that, together with two previously known point mutations, brought about a 256-fold affinity improvement. The observation of alternative routes to affinity maturation validate our strategy and suggest that InDel mutagenesis can complement existing approaches.Open in a separate windowFig. 1.Overview of the affinity maturation of the antibody BAK1 by transposon-based TRIAD and subsequent insertional scanning mutagenesis. TRIAD (Left) was applied to make libraries with deletions of one to three amino acids (step 1a) or single amino acid insertions (step 1b) at random positions across the scFv gene. These libraries were recombined (step 2) and four rounds of ribosome display selections for improved affinity to IL-13 were carried out by panning (step 3). The best binder was carrying an insertion in the V_L FWR3 (BAK1-INS1). Scanning (Right) was used to guide the design of libraries with different lengths of insertions at targeted positions. A fraction of the insertion library generated in step 1b (5,632 variants) was screened by HTRF to identify variants with insertions that retained binding to IL-13 (step 4). Based on sequencing analysis, regions able to tolerate single amino acid insertions were identified (Fig. 4) and the V_L CDR3 was chosen for targeted insertional mutagenesis. Libraries with zero to five amino acid insertions in targeted positions in the V_L CDR3 were constructed (step 5), followed by four rounds of phage display selections for improved affinity to IL-13 (step 6).     

Manual aiming in healthy aging: does proprioceptive acuity make the difference?

The present study examines whether non-active older adults are more dependent on visual information when executing aiming movements and whether age-related declines in proprioception play a mediating role herein. Young (N = 40) and older adults (N = 38) were divided into physically active and non-active subgroups based on self-reported sports participation levels. In experiment 1, participants executed wrist-aiming movements with and without visual feedback. In experiment 2, passive proprioceptive acuity was assessed using wrist motion detection and position matching tests. Results showed similar aiming accuracy across age groups both with and without visual feedback, but older adults exhibited longer movement times, prolonged homing-in phase, and made more corrective submovements. Passive proprioceptive acuity was significantly affected by physical activity level and age, with participants in the active group scoring better than their non-active peers. However, these declines did not predict performance changes on the aiming task. Taken together, our observations suggest that decline in proprioceptive acuity did not predict performance changes on the aiming task and older adults were able to compensate for their decreased motion and position sense when allowed sufficient time. In line with these observations, we proposed that older adults are able to compensate for their decline in proprioception by increasing their reliance on predictive models.     

Beta cyclodextrins bind,stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (β-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of β-CD. Importantly, β-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of β-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of β-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.The retinal pigment epithelium (RPE), strategically situated between the neural retina and the choroid blood vessels, is essential for photoreceptor (PR) function. It recycles vitamin A, which is required for the visual cycle and clears debris generated by the circadian shedding of PR outer segments (1, 2). Each RPE cell phagocytoses and digests the material produced by 30–50 overlying PRs, which shed 10% of their mass daily. The intense and continual phagocytic activity of RPE cells results in the progressive accumulation of indigestible products or “lipofuscin” in their lysosomal compartment (3, 4). Unlike lipofuscins found in other body tissues, which are composed mainly of protein, RPE lipofuscin consists predominantly of lipid-bisretinoids and only 2% protein (5). Lipofuscin bisretinoids (LBs) are vitamin A-derived side products of the visual cycle. Light converts 11-cis-retinal (11CR), the visual pigment chromophore, into all-trans-retinal (ATR), which is immediately flipped by the ATP-binding cassette transporter 4 (Abca4) transporter from the lumen of the outer segment discs to the cytoplasm, where it is reduced to inert all-trans-retinol by retinol dehydrogenase 8 (Rdh8), in mice (6, 7). Small fractions of 11CR and ATR are converted into N-retinylidine-N-ethanolamine (A2E) and other less abundant bisretinoids, which once accumulated in the lysosomes of RPE cells are refractory to all known lysosomal hydrolases (8, 9). The concept that LB accumulation causes retinal degeneration is supported by in vitro and in vivo data that show that excessive LBs are toxic for cultured RPE cells (10, 11), that photoreceptors overlying A2E-laden RPE are more prone to degeneration (12) and that excessive accumulation of LBs in Stargardt’s disease precedes macular degeneration (13). Mice carrying null mutations in Abca4 and Rdh8 develop blindness, basal laminar deposits, and focal accumulations of extracellular debris between the RPE and the Bruch membrane (drusen) (6).Here we report that a family of modified cyclic oligosaccharides, beta cyclodextrins (β-CDs), formed by seven d-glucose units, can encapsulate the hydrophobic arms of A2E within their nonpolar cavity, protect A2E from oxidation, and remove A2E from RPE cells. Our data demonstrate a direct correlation between the ability of β-CDs to perform these protective functions and their affinity for A2E.     

Genetic variability of VEGF pathway genes in six randomized phase III trials assessing the addition of bevacizumab to standard therapy

Background

Despite extensive translational research, no validated biomarkers predictive of bevacizumab treatment outcome have been identified.

Methods

We performed a meta-analysis of individual patient data from six randomized phase III trials in colorectal, pancreatic, lung, renal, breast, and gastric cancer to explore the potential relationships between 195 common genetic variants in the vascular endothelial growth factor (VEGF) pathway and bevacizumab treatment outcome.

Results

The analysis included 1,402 patients (716 bevacizumab-treated and 686 placebo-treated). Twenty variants were associated (P < 0.05) with progression-free survival (PFS) in bevacizumab-treated patients. Of these, 4 variants in EPAS1 survived correction for multiple testing (q < 0.05). Genotype-by-treatment interaction tests revealed that, across these 20 variants, 3 variants in VEGF-C (rs12510099), EPAS1 (rs4953344), and IL8RA (rs2234671) were potentially predictive (P < 0.05), but not resistant to multiple testing (q > 0.05). A weak genotype-by-treatment interaction effect was also observed for rs699946 in VEGF-A, whereas Bayesian genewise analysis revealed that genetic variability in VHL was associated with PFS in the bevacizumab arm (q < 0.05). Variants in VEGF-A, EPAS1, and VHL were located in expression quantitative loci derived from lymphoblastoid cell lines, indicating that they affect the expression levels of their respective gene.

Conclusions

This large genetic analysis suggests that variants in VEGF-A, EPAS1, IL8RA, VHL, and VEGF-C have potential value in predicting bevacizumab treatment outcome across tumor types. Although these associations did not survive correction for multiple testing in a genotype-by-interaction analysis, they are among the strongest predictive effects reported to date for genetic variants and bevacizumab efficacy.     

Does footwear affect articular cartilage volume change after a prolonged run?

The aim of this study was to investigate knee intra-articular cartilage volume changes after a prolonged running bout in three footwear conditions. Twelve participants performed 75-minute running bouts in the three footwear conditions. Before and after each running bout, magnetic resonance imaging (MRI) scans were obtained using a high-resolution 3.0 Tesla MRI. Three-dimensional reconstruction of the cartilage plates of the patella, the femur, and the tibia was created to quantify cartilage volume change due to the 75-minute running bout. Three-dimensional biomechanical data were also collected using an integrated motion capture and force treadmill system. There were no statistically significant differences among shoe conditions for all anatomical regions. However, significant cartilage volume reductions at all anatomical sites were observed after the 75-minute running bout in each footwear condition. These data suggest that the intra-articular knee cartilage undergoes a significant reduction in cartilage volume during a prolonged run that may indicate an increase in joint loading. There was a considerable variation in cartilage volume between participants across footwear conditions indicating an individual cartilage volume response to footwear. An individualistic approach to footwear recommendations may help in minimizing this change in cartilage.     

Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, we combine the efficiency of light microscopy for revealing cellular functions using fluorescent probes with the high resolution of electron microscopy to localize and characterize COs in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I and II COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.Eukaryotic sexual reproduction involves meiosis, a specialized cell division in which DNA duplication in a diploid cell is followed by two cell divisions to produce four haploid cells. The first division, Meiosis I, involves crossing over and chiasmata formation between each pair of homologous chromosomes, thereby ensuring separation of the homologs and formation of two haploid cells, each with one complete set of replicated chromosomes. The second division, Meiosis II, is a mitosis-like division in which the two sister chromatids separate to yield four haploid cells that directly or indirectly form gametes. Because these four products are genetically unique due to crossing over and independent segregation of homologous chromosomes during Meiosis I, meiosis plays an important role in creating genetic diversity in sexually reproducing organisms.Crossing over during meiosis is tightly controlled so each pair of homologs has at least one “obligate” crossover (CO) that ensures balanced reductional segregation, but the presence of a CO reduces the likelihood of another CO in its vicinity, a phenomenon referred to as CO interference (1, 2). Significant progress has been made recently in illuminating the molecular events of meiotic recombination and the control of crossing over (3–8). The initiating event of meiotic recombination in most organisms is formation of numerous DNA double-strand breaks (DSBs). Homolog-dependent repair of a DSB may follow any one of at least three pathways: (i) non-CO that may result in a short gene conversion; (ii) CO with interference (class I COs, produced by pathway P1); or (iii) CO without interference (class II COs, produced by pathway P2) (6, 7, 9). The interfering CO pathway involves the resolution of double Holliday junctions, which requires many proteins including the ZMM group (ZIP1-4, MSH4-5, MER3) and the MutL homolog 1 (MLH1)/MLH3 complex (6, 10). The noninterfering CO pathway depends primarily on the Mus81/Mms4 endonuclease complex in budding yeast (MUS81/EME1 complex in plants and animals) (5–7, 11–14).Meiotic COs occur in association with two cytological structures, synaptonemal complexes (SCs) that link each pair of homologous chromosomes throughout their lengths during pachytene and late recombination nodules (RNs) that are ellipsoidal structures on SCs (15). Every SC has at least one RN, each RN marks a CO site, and most RNs contain MLH1 protein (16–19). RNs are too small (50-100 nm) to be resolved using light microscopy (LM), but they can be readily visualized by transmission electron microscopy (EM), particularly in 2D spreads of SCs (18). Antibodies to MLH1 protein have been used as immunofluorescent probes to map class I COs on SCs (e.g., refs. 19 and 20). Pathway 2 (P2), which was revealed using mutants of the P1 pathway, produces class II COs, and these class II COs showed no interference in the marker intervals studied (21–23). The P1 pathway produces the majority of COs, and the P2 pathway accounts for ∼5–30% of COs (8, 11, 21). CO distributions have been effectively modeled by assuming that class II COs are independent from class I COs (24). However, class II COs have not been independently mapped on chromosomes (12), and little is known about the properties of each pathway or whether they interact in wild-type organisms.Here, we describe an advanced approach that uses SC spreads from wild-type tomato (Solanum lycopersicum, 2n = 2x = 24) to directly identify the pathway of origin for each CO in individual meiotic nuclei. For this, we superimposed the immunofluorescent LM image of an SC spread showing MLH1 foci (class I COs) onto an EM image of the same SC spread showing RN locations (all COs). RNs that coincide with MLH1 foci (MLH1-positive RNs) mark class I COs, and RNs that do not coincide with MLH1 foci (MLH1-negative RNs) are considered to mark class II COs. Because EM is time-consuming, this approach takes advantage of both the relative speed of LM and the high resolution of EM, allowing us to analyze RNs on 1882 tomato SCs.