Time-dependent nanomechanics of cartilage

In this study, atomic force microscopy-based dynamic oscillatory and force-relaxation indentation was employed to quantify the time-dependent nanomechanics of native (untreated) and proteoglycan (PG)-depleted cartilage disks, including indentation modulus E_ind, force-relaxation time constant τ, magnitude of dynamic complex modulus |E^∗|, phase angle δ between force and indentation depth, storage modulus E′, and loss modulus E″. At ∼2 nm dynamic deformation amplitude, |E^∗| increased significantly with frequency from 0.22 ± 0.02 MPa (1 Hz) to 0.77 ± 0.10 MPa (316 Hz), accompanied by an increase in δ (energy dissipation). At this length scale, the energy dissipation mechanisms were deconvoluted: the dynamic frequency dependence was primarily governed by the fluid-flow-induced poroelasticity, whereas the long-time force relaxation reflected flow-independent viscoelasticity. After PG depletion, the change in the frequency response of |E^∗| and δ was consistent with an increase in cartilage local hydraulic permeability. Although untreated disks showed only slight dynamic amplitude-dependent behavior, PG-depleted disks showed great amplitude-enhanced energy dissipation, possibly due to additional viscoelastic mechanisms. Hence, in addition to functioning as a primary determinant of cartilage compressive stiffness and hydraulic permeability, the presence of aggrecan minimized the amplitude dependence of |E^∗| at nanometer-scale deformation.     

Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes

The success of cell-based tissue engineering approaches in restoring biological function will be facilitated by a comprehensive fundamental knowledge of the temporal evolution of the structure and properties of the newly synthesized matrix. Here, we quantify the dynamic oscillatory mechanical behavior of the engineered matrix associated with individual chondrocytes cultured in vitro for up to 28 days in alginate scaffolds. The magnitude of the complex modulus (|E*|) and phase shift (δ) were measured in culture medium using Atomic Force Microscopy (AFM)-based nanoindentation in response to an imposed oscillatory deformation (amplitude ～5 nm) as a function of frequency (f=1–316 Hz), probe tip geometry (2.5 μm radius sphere and 50 nm radius square pyramid), and in the absence and presence of growth factors (GF, insulin growth factor-1, IGF-1, and osteogenic protein-1, OP-1). |E*| for all conditions increased nonlinearly with frequency dependence approximately f^1/2 and ranged between ～1 and 25 kPa. This result, along with theoretical calculations of the characteristic poroelastic relaxation frequency, f_p, (～50–90 Hz) suggested that this time-dependent behavior was governed primarily by fluid flow-dependent poroelasticity, rather than flow-independent viscoelastic processes associated with the solid matrix. |E*(f)| increased, (f) decreased, and the hydraulic permeability, k, decreased with time in culture and with growth factor treatment. This trend of a more elastic-like response was thought to be associated with increased macromolecular biosynthesis, density, and a more mature matrix structure/organization.     

Structure-Function Relations and Rigidity Percolation in the Shear Properties of Articular Cartilage

Among mammalian soft tissues, articular cartilage is particularly interesting because it can endure a lifetime of daily mechanical loading despite having minimal regenerative capacity. This remarkable resilience may be due to the depth-dependent mechanical properties, which have been shown to localize strain and energy dissipation. This paradigm proposes that these properties arise from the depth-dependent collagen fiber orientation. Nevertheless, this structure-function relationship has not yet been quantified. Here, we use confocal elastography, quantitative polarized light microscopy, and Fourier-transform infrared imaging to make same-sample measurements of the depth-dependent shear modulus, collagen fiber organization, and extracellular matrix concentration in neonatal bovine articular cartilage. We find weak correlations between the shear modulus |G^∗| and both the collagen fiber orientation and polarization. We find a much stronger correlation between |G^∗| and the concentration of collagen fibers. Interestingly, very small changes in collagen volume fraction v_c lead to orders-of-magnitude changes in the modulus with |G^∗| scaling as (v_c – v₀)^ξ. Such dependencies are observed in the rheology of other biopolymer networks whose structure exhibits rigidity percolation phase transitions. Along these lines, we propose that the collagen network in articular cartilage is near a percolation threshold that gives rise to these large mechanical variations and localization of strain at the tissue’s surface.     

Nanomechanical phenotype of chondroadherin-null murine articular cartilage

Chondroadherin (CHAD), a class IV small leucine rich proteoglycan/protein (SLRP), was hypothesized to play important roles in regulating chondrocyte signaling and cartilage homeostasis. However, its roles in cartilage development and function are not well understood, and no major osteoarthritis-like phenotype was found in the murine model with CHAD genetically deleted (CHAD^−/−). In this study, we used atomic force microscopy (AFM)-based nanoindentation to quantify the effects of CHAD deletion on changes in the biomechanical function of murine cartilage. In comparison to wild-type (WT) mice, CHAD-deletion resulted in a significant ≈ 70–80% reduction in the indentation modulus, E_ind, of the superficial zone knee cartilage of 11 weeks, 4 months and 1 year old animals. This mechanical phenotype correlates well with observed increases in the heterogeneity collagen fibril diameters in the surface zone. The results suggest that CHAD mainly plays a major role in regulating the formation of the collagen fibrillar network during the early skeletal development. In contrast, CHAD-deletion had no appreciable effects on the indentation mechanics of middle/deep zone cartilage, likely due to the dominating role of aggrecan in the middle/deep zone. The presence of significant rate dependence of the indentation stiffness in both WT and CHAD^−/− knee cartilage suggested the importance of both fluid flow induced poroelasticity and intrinsic viscoelasticity in murine cartilage biomechanical properties. Furthermore, the marked differences in the nanomechanical behavior of WT versus CHAD^−/− cartilage contrasted sharply with the relative absence of overt differences in histological appearance. These observations highlight the sensitivity of nanomechanical tools in evaluating structural and mechanical phenotypes in transgenic mice.     

On the use of stable oxygen isotope (δ18O) measurements for tracking avian movements in North America

Tracking migratory animals has benefitted using measurements of naturally occurring stable isotopes of hydrogen (δ²H) in keratinous tissues such as hair and feathers to link animal origins to continental patterns or isoscapes of δ²H in precipitation. However, for most taxa, much less information exists on the use of stable oxygen isotope ratios (δ¹⁸O) despite the fact that δ²H and δ¹⁸O are strongly linked in environmental waters through the meteoric relationship and the possibility of using both isotopes to infer greater information on origins and climatic conditions where tissues are grown. A fundamental requirement of using stable isotopes to assign individuals and populations to origins is the development of a rescaling function linking environmental food web signals to the tissue of interest and for birds, this has not been carried out. Here, we derived the relationship between H and O isotopes in known source feathers of 104 individuals representing 11 species of insectivorous passerines sampled across the strong precipitation isoscape of North America. We determined again a strong expected relationship between feather δ²H (δ²H_f) and long‐term amount‐weighted precipitation δ²H (δ²H_p; r² = 0.77), but the corresponding relationship between δ¹⁸O_f and δ¹⁸O_p was poor (r² = 0.32) for the same samples. This suggests that δ²H measurements are currently more useful for assignment of insectivorous songbirds to precipitation isoscapes but does not preclude other uses of the δ¹⁸O_f data. Currently, mechanisms responsible for the decoupling of H and O isotopes in food webs is poorly known, and we advocate a much broader sampling of both isotopes in the same keratinous tissues across precipitation isotope gradients and across taxa to resolve this issue and to increase the power of using water isotopes to track migratory animals.     

Relation between mechanical foam-breaking difficulty and the foaming characteristics of solutions

Using a small foaming apparatus, liquid foaming characteristics such as liquid holdup in foamate flow (φ_t), rate of liquid drainage from foam (ν), foam velocity (u_f) and foam size (d_f) were examined for various liquids. A parameter φ_f, incorporating φ_t, ν and u_f, was obtained. The relationship among φ_f, d_f and the liquid holdup in foam, φ_b, in the actual gas-bubbling systems under foam control was expressed in the form of φ_b ∞ φ_fd_f^1.83, regardless of the type of foaming liquid or its concentration. This relationship was useful for the prediction of φ_b as related to mechanical foam-breaking difficulty.     

Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, φ_m, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K⁺ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the φ_m-sensitive fluorescent probe 3,3′-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3′-dihexyloxacarbocyanine iodine increased with |φ_m| up to 118 mV, and its dependence on |φ_m| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of φ_m on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, V_entry, increased with an increase in |φ_m|. The rim intensity due to CF-TP10 increased with an increase in |φ_m|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |φ_m|. These results indicate that the φ_m-induced elevation in V_entry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |φ_m|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.     

Structure-Function Relations and Rigidity Percolation in the Shear Properties of Articular Cartilage

Among mammalian soft tissues, articular cartilage is particularly interesting because it can endure a lifetime of daily mechanical loading despite having minimal regenerative capacity. This remarkable resilience may be due to the depth-dependent mechanical properties, which have been shown to localize strain and energy dissipation. This paradigm proposes that these properties arise from the depth-dependent collagen fiber orientation. Nevertheless, this structure-function relationship has not yet been quantified. Here, we use confocal elastography, quantitative polarized light microscopy, and Fourier-transform infrared imaging to make same-sample measurements of the depth-dependent shear modulus, collagen fiber organization, and extracellular matrix concentration in neonatal bovine articular cartilage. We find weak correlations between the shear modulus |G^∗| and both the collagen fiber orientation and polarization. We find a much stronger correlation between |G^∗| and the concentration of collagen fibers. Interestingly, very small changes in collagen volume fraction v_c lead to orders-of-magnitude changes in the modulus with |G^∗| scaling as (v_c – v₀)^ξ. Such dependencies are observed in the rheology of other biopolymer networks whose structure exhibits rigidity percolation phase transitions. Along these lines, we propose that the collagen network in articular cartilage is near a percolation threshold that gives rise to these large mechanical variations and localization of strain at the tissue’s surface.     

On the mechanical stabilization of filopodia

We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15–30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width <v_1/2>, ∼0.02 μm s⁻¹) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10⁻²³ J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca²⁺ by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.     

Prediction of peak occurrence of Dendrolimus punctatus larvae based on Bayes discriminant method

To improve the accuracy of forecasting the peak occurrence of Dendrolimus punctatus Walker, we here used the Bayes discriminant analysis to predict this peak occurrence for the first and second generation of Dendrolimus punctatus larvae based on these data from 1983 to 2016 in Qianshan County, Anhui Province. Our present results showed that this discriminant equation for the first generation was as follows: f⁽¹⁾ = ?3.2588‐6.2700x₁ + 1.2870x₂ + 0.7920x₃ + 0.4152x₄; f⁽²⁾ = ?14.5215‐8.5710x₁ + 2.9790x₂ + 2.0280x₃ + 0.5031x₄; f⁽³⁾ = ?3.5264; f⁽⁴⁾ = ?66.8312‐12.5216x₁ + 5.1740x₂ + 4.7162x₃ + 0.6033x₄. And that the prediction accuracy for the first generation was 97.22%. Whilst this discriminant equation for the second generation was as follows: f⁽¹⁾ = ?3.536‐1.192x₅ + 1.338x₆ + 0.638x_7?0.025x₈; f⁽²⁾ = ?7.317‐1.337x₅ + 4.240x₆ + 1.010x_7?0.295x₈; f⁽³⁾ = ?16.488‐3.192x₅ + 4.955x₆ + 1.900x₇–0.411x₈; f⁽⁴⁾ = ?34.502‐4.184x₅ + 7.484x₆ + 2.583x₇–0.443x₈. The prediction accuracy for the second generation was 85.71%. Overall, our findings revealed that the Bayes discriminant analysis could screen out key factors to significantly improve the prediction accuracy of peak occurrence of Dendrolimus punctatus larvae.     

Geographical origin of dabbling ducks wintering in Iberia: sex differences and implications for pair formation

Natural and anthropogenic Iberian wetlands in southern Europe are well known for supporting large numbers of migratory Palaearctic waterbirds each winter. However, information on the geographical origin of dabbling ducks overwintering in these wetlands is scarce and mostly limited to data from ringing recoveries. Here, we used intrinsic isotopic markers to determine the geographical origin of male and female Northern Pintails Anas acuta and Eurasian Teal Anas crecca in Extremadura, inland Iberia, a key site for overwintering dabbling ducks. Additionally, we fitted six Northern Pintails with GPS‐GSM tags to complement the data derived from stable isotope analysis. Most (> 70%) first calendar‐year Northern Pintails were assigned to regions above 55°N, flying 2600–5600 km from their main natal regions to Extremadura. Mean values of δ²H_f varied significantly between male and female Northern Pintails, suggesting that the sexes had different geographical origins. Data from tagged adult Northern Pintails supported the isotopic data, one male flying more than 5000 km to the coast of the Pechora Sea (Russia). Most (> 70%) first calendar‐year Eurasian Teal were assigned to the region between 48° and 60°N, travelling 1500–4500 km to arrive in Extremadura. Male and female Eurasian Teal showed marginal differences in mean values of δ²H_f. In migratory dabbling ducks, pairing typically occurs on the wintering grounds, and ducks in their first winter can breed the following spring. For Northern Pintails, pair formation in Extremadura could occur between individuals with different geographical origins, which could contribute to the genetic variability of their offspring.     

Optimization of the Bowtie Gap Geometry for a Maximum Electric Field Enhancement

Optimization of the geometry of a metallic bowtie gap at radio frequency is presented. We investigate the geometry of the bowtie gap including gap size, tip width, metal thickness and tip angle at macroscale to find the maximum electric field enhancement across the gap. The results indicate that 90^° bowtie with 0.06 λ gap size has the most |E _t |² enhancement. Effects of changing the permittivity and conductivity of the material across the gap are also investigated. NEC-2 simulations show that the numerical calculations agree with the experimental results. Since the design and fabrication of a plasmonic device (nanogap) at nanoscale is challenging, the results of this study can be used to estimate the best design parameters for nanogap structure. Different amounts of enhancement at different frequency ranges are explained by mode volume. The product of the mode volume and |E _t |² enhancement is constant for different gap structures and different frequencies.     

Trophic discrimination factor of nitrogen isotopes within amino acids in the dobsonfly Protohermes grandis (Megaloptera: Corydalidae) larvae in a controlled feeding experiment

The trophic discrimination factor (TDF) of nitrogen isotopes (¹⁵N/¹⁴N) within amino acids, between a stream‐dwelling dobsonfly larva (Protohermes grandis: Megaloptera; Corydalidae) and its diet (chironomid larvae), was determined in controlled feeding experiments. Last‐instar larvae of P. grandis were collected from the Yozawa‐gawa River, central Japan, and reared in the laboratory. After fed to satiation for 1 month, one group of larvae was each fed one living chironomid larva per day for 4 weeks, while a second group was starved for 8 weeks. The larvae were harvested at intervals and the nitrogen isotopic composition of glutamic acid (δ¹⁵N_Glu) and phenylalanine (δ¹⁵N_Phe) were determined to calculate TDF. The mean TDF of satiated and starved larvae were 7.1‰ ± 0.5‰ (n = 3) and 7.3‰ ± 0.5‰ (n = 5), respectively. Thus, the TDF for P. grandis larvae in this study was similar to that reported for other arthropods (approximately 7‰) and was independent of satiation or starvation. A previous study of wild P. grandis larvae, based on the δ¹⁵N_Glu and δ¹⁵N_Phe values, estimated its trophic position (TP) as approximately 2.0 ± 0.1 (n = 5), a low value close to that of algivores, although they are generally characterized as carnivores (usually accepted as TP ≥ 3). The TDF for P. grandis larvae suggests that their low TPs in nature were caused by incorporation of vascular plant‐derived amino acids (with a different δ¹⁵N profile from that of algae) and not by an unusually low TDF or by the effects of the satiation/starvation on amino acid metabolism.     

Natal origins and timing of migration of two passerine species through the southern Alps: inferences from multiple stable isotopes (δ2H, δ13C, δ15N, δ34S) and ringing data

Understanding spatial linkages between areas used by migratory animals during the annual cycle is fundamental to their conservation. Stable isotope measurements of animal tissues can be a valuable tool in understanding spatial connectivity and migration phenology of migratory wildlife. We inferred natal origins of two migratory passerines, European Pied Flycatcher Ficedula hypoleuca and European Robin Erithacus rubecula, captured during autumn migration in the Italian Alps, by combining feather δ²H (δ²H_f) and ring recovery data. We used a spatially explicit likelihood-based method to assign individuals to a precipitation δ²H surface calibrated to represent feather δ²H, together with the directional probability of origin from ring recoveries. The highest probabilities of origin for most individuals of both species were in central and north-eastern Europe. Seasonal trends in δ²H_f, which described the species’ migratory phenology through the Italian Alps, were correlated with feather δ¹³C, δ¹⁵N and δ³⁴S values, indicating strong spatial discrimination related to continental patterns for these isotopes. We demonstrate how this combined information can define catchment areas and migratory connectivity of birds intercepted in the Alps. We highlight the importance of ringing data in defining directional priors to define Bayesian-based probability surfaces using continental δ²H_f isoscapes, and how such information can be used to inform estimates of migratory connectivity.     

An Annular Plasmonic Lens Under Illumination of Circularly Polarized Light

In this paper, we consider a circular central aperture surrounded with annular depth-tuned grooves and investigate the beaming effect of the structure under illumination of a circularly polarized (CP) plane wave. As a CP plane wave is equivalent to the superposition of two linearly polarized plane waves (TM and TE) with a phase difference of π/2, the superposition of the electric field intensity, ( | E_x |² + | E_y |² ) \left( {{{\left| {E_x} \right|}^2} + {{\left| {E_y} \right|}^2}} \right) , is observed in the transmission field. In addition, two plasmonic modes are found at the resonant wavelengths λ ₁ and λ ₂ with each consisting of multiple wavelengths. At the wavelength λ ₁ = 420 nm, the significant near-field collimation is formed along the direction z, having a long propagation distance up to 1.75 μm (≈4λ) away from the exit plane of the new plasmonic lens.     

Worsening of neck and shoulder complaints in humans are correlated with frequency parameters of electromyogram recorded 1-year earlier

The aim was to investigate whether output and electromyogram (EMG) variables obtained from an isokinetic endurance test of the shoulder flexor muscles of 23 women with neck and shoulder problems in a car and truck industry correlated with improvement or worsening of complaints 1 year later. Each subject performed 100 maximal isokinetic shoulder forward flexions at 60° · s⁻¹. Surface EMG of the trapezius, deltoid, biceps brachii and infraspinatus muscles and mechanical output (peak torque) were determined for each contraction. The EMG was used to determine mean frequency f _mean and the ratio between the signal amplitudes of the EMG of the passive relaxation and active flexion parts of each contraction cycle (SAR). The subjects also rated the degree of fatigue they experienced throughout the test. The magnitude of the shift in f _mean was correlated with whether improvement or worsening occurred for complaints in the neck and or shoulders; a significant relationship (r ² = 0.44; P = 0.001) existed between the total frequency shift of the four muscles and the variables measuring improvement in complaints. In the multivariate predictions other f _mean variables and perception of fatigue were also of significance. The present study would indicate that a high degree of f _mean shift correlates with improvement in neck and shoulder complaints 1 year later. One possible reason could be that f _mean reflects the muscle morphology and/or a pathological situation for the type-1 muscle fibres. Accepted: 27 May 1998     

Dentine oxygen isotopes (δ18O) as a proxy for odontocete distributions and movements

Spatial variation in marine oxygen isotope ratios (δ¹⁸O) resulting from differential evaporation rates and precipitation inputs is potentially useful for characterizing marine mammal distributions and tracking movements across δ¹⁸O gradients. Dentine hydroxyapatite contains carbonate and phosphate that precipitate in oxygen isotopic equilibrium with body water, which in odontocetes closely tracks the isotopic composition of ambient water. To test whether dentine oxygen isotope composition reliably records that of ambient water and can therefore serve as a proxy for odontocete distribution and movement patterns, we measured δ¹⁸O values of dentine structural carbonate (δ¹⁸O_SC) and phosphate (δ¹⁸O_P) of seven odontocete species (n = 55 individuals) from regional marine water bodies spanning a surface water δ¹⁸O range of several per mil. Mean dentine δ¹⁸O_SC (range +21.2 to +25.5‰ VSMOW) and δ¹⁸O_P (+16.7 to +20.3‰) values were strongly correlated with marine surface water δ¹⁸O values, with lower dentine δ¹⁸O_SC and δ¹⁸O_P values in high‐latitude regions (Arctic and Eastern North Pacific) and higher values in the Gulf of California, Gulf of Mexico, and Mediterranean Sea. Correlations between dentine δ¹⁸O_SC and δ¹⁸O_P values with marine surface water δ¹⁸O values indicate that sequential δ¹⁸O measurements along dentine, which grows incrementally and archives intra‐ and interannual isotopic composition over the lifetime of the animal, would be useful for characterizing residency within and movements among water bodies with strong δ¹⁸O gradients, particularly between polar and lower latitudes, or between oceans and marginal basins.     

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors

In one series of experiments, heart frequency (f _H), blood pressure (P _a), gill ventilation frequency (f _R ), ventilation amplitude (V _AMP) and total gill ventilation (V _TOT) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO₂ ~140, normoxia to 17 mmHg, severe hypoxia), they increased f _R , V _AMP, V _TOT and P _a and decreased f _H. In a second series of experiments, air-breathing frequency (f _RA), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 μg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 μg NaCN in 1.0 mL water) on f _R , V _AMP, V _TOT, P _a and f _H we conclude that the O₂ receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O₂ levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.