An automated microfluidic-based immunoassay cartridge for allergen screening and other multiplexed assays

A microfluidic cartridge and system for multiplexed immunoassays is described. The passive microfluidic cartridge was composed of three layers of injection molded plastic sealed together using a thermal staking technique. Using this platform technology, a specific immunoglobulin E (IgE) panel assay was constructed. Allergen extract targets, positive and negative controls, and IgE calibration standards were immobilized within the cartridge as a microarray. A computer-controlled solenoid array provided the necessary actuation force for pumping reagents within the cartridge to perform an automated, chemiluminescent indirect immunoassay. A 20-target allergen extract panel was demonstrated on the device with a total analysis time of 27 min. Allergen screening results showed 84% agreement for 3 house dust mites (N = 300) compared with a commercial test and 80% agreement overall (N = 978). Average coefficients of variation (N = 80) were measured as 20.5% for low/medium levels and 20.4% for medium/high levels. The average limit of detection (N = 160) was measured at 0.535 AU, and cutoff levels of 1.0 AU were estimated at less than 1 IU/ml (2.4 ng/ml). Such a system has potential applications in decentralized allergen screening as well as in other near-patient diagnostic immunoassays where multiplexed analysis, ease of use, and short analysis time are critical.     

Construction and characterization of isogenic O-antigen variants of Salmonella typhi

A 7.5 kb Kpnl-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype 09,12; Vi⁺ H-d), S. typhi Vi-negative strain H400 (i.e. serotype 09,12; Vi⁻; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi⁺; H-d and the serotype of H404 is O4,12; Vi⁻; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O-antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination     

Pedal Claw Curvature in Birds,Lizards and Mesozoic Dinosaurs – Complicated Categories and Compensating for Mass-Specific and Phylogenetic Control

Pedal claw geometry can be used to predict behaviour in extant tetrapods and has frequently been used as an indicator of lifestyle and ecology in Mesozoic birds and other fossil reptiles, sometimes without acknowledgement of the caveat that data from other aspects of morphology and proportions also need to be considered. Variation in styles of measurement (both inner and outer claw curvature angles) has made it difficult to compare results across studies, as have over-simplified ecological categories. We sought to increase sample size in a new analysis devised to test claw geometry against ecological niche. We found that taxa from different behavioural categories overlapped extensively in claw geometry. Whilst most taxa plotted as predicted, some fossil taxa were recovered in unexpected positions. Inner and outer claw curvatures were statistically correlated, and both correlated with relative claw robusticity (mid-point claw height). We corrected for mass and phylogeny, as both likely influence claw morphology. We conclude that there is no strong mass-specific effect on claw curvature; furthermore, correlations between claw geometry and behaviour are consistent across disparate clades. By using independent contrasts to correct for phylogeny, we found little significant relationship between claw geometry and behaviour. ‘Ground-dweller’ claws are less curved and relatively dorsoventrally deep relative to those of other behavioural categories; beyond this it is difficult to assign an explicit category to a claw based purely on geometry.     

The soft tissue of Jeholopterus (Pterosauria,Anurognathidae, Batrachognathinae) and the structure of the pterosaur wing membrane

The soft tissue preserved in the holotype (IVPP V12705) of Jeholopterus ningchengensis from the Daohugou Bed (Late Jurassic or Early Cretaceous) of China is described in detail. The plagiopatagium can be divided into the distal, comparatively more rigid actinopagatium and a proximal, more tensile tenopatagium. The actinopatagium extends from the wing finger to the articulation between the humerus and the forearm, and shows the presence of at least three layers containing actinofibrils. In each layer, the actinofibrils are parallel to subparallel, but this direction diverges from layer to layer. When distinct layers of actinofibrils are superimposed (owing to taphonomic compression), a reticular pattern is generated. The presence of layers with differently oriented actinofibrils is widespread in this pterosaur. A well-developed integumental covering formed by fibres (here named pycnofibres) that are thicker than the actinofibrils is present. Ungual sheaths that extend the length of the pedal and manual claws of this taxon are also observed. Although the understanding of the mechanical properties of the wing membrane is hampered by the lack of knowledge regarding the composition of the actinofibrils, the configuration observed in Jeholopterus might have allowed subtle changes in the membrane tension during flight, resulting in more control of flight movements and the organization of the wing membrane when the animal was at rest.     

A new specimen of the pterosaur Rhamphorhynchus

A previously undescribed and near-complete specimen of the non-pterodactyloid pterosaur Rhamphorhynchus is briefly described. Although purchased in 1878 and held in a public collection in Dublin, Ireland ever since, this specimen has never been described and was not even mentioned in the literature until 2010.     

Under what conditions do climate‐driven sex ratios enhance versus diminish population persistence?

For many species of reptile, crucial demographic parameters such as embryonic survival and individual sex (male or female) depend on ambient temperature during incubation. While much has been made of the role of climate on offspring sex ratios in species with temperature‐dependent sex determination (TSD), the impact of variable sex ratio on populations is likely to depend on how limiting male numbers are to female fecundity in female‐biased populations, and whether a climatic effect on embryonic survival overwhelms or interacts with sex ratio. To examine the sensitivity of populations to these interacting factors, we developed a generalized model to explore the effects of embryonic survival, hatchling sex ratio, and the interaction between these, on population size and persistence while varying the levels of male limitation. Populations with TSD reached a greater maximum number of females compared to populations with GSD, although this was often associated with a narrower range of persistence. When survival depended on temperature, TSD populations persisted over a greater range of temperatures than GSD populations. This benefit of TSD was greatly reduced by even modest male limitation, indicating very strong importance of this largely unmeasured biologic factor. Finally, when males were not limiting, a steep relationship between sex ratio and temperature favoured population persistence across a wider range of climates compared to the shallower relationships. The opposite was true when males were limiting – shallow relationships between sex ratio and temperature allowed greater persistence. The results highlight that, if we are to predict the response of populations with TSD to climate change, it is imperative to 1) accurately quantify the extent to which male abundance limits female fecundity, and 2) measure how sex ratios and peak survival coincide over climate.     

Three-dimensional geometric morphometric analysis of the skull of Protoceratops andrewsi supports a socio-sexual signalling role for the ceratopsian frill

Socio-sexual selection is predicted to be an important driver of evolution, influencing speciation, extinction and adaptation. The fossil record provides a means of testing these predictions, but detecting its signature from morphological data alone is difficult. There are, nonetheless, some specific patterns of growth and variation which are expected of traits under socio-sexual selection. The distinctive parietal-squamosal frill of ceratopsian dinosaurs has previously been suggested as a socio-sexual display trait, but evidence for this has been limited. Here, we perform a whole-skull shape analysis of an unprecedentedly large sample of specimens of Protoceratops andrewsi using a high-density landmark-based geometric morphometric approach to test four predictions regarding a potential socio-sexual signalling role for the frill. Three predictions—low integration with the rest of the skull, significantly higher rate of change in size and shape during ontogeny, and higher morphological variance than other skull regions—are supported. One prediction, sexual dimorphism in shape, is not supported, suggesting that sexual differences in P. andrewsi are likely to be small. Together, these findings are consistent with mutual mate choice or selection for signalling quality in more general social interactions, and support the hypothesis that the frill functioned as a socio-sexual signal in ceratopsian dinosaurs.     

The asymmetry of the carpal joint and the evolution of wing folding in maniraptoran theropod dinosaurs

In extant birds, the hand is permanently abducted towards the ulna, and the wrist joint can bend extensively in this direction to fold the wing when not in use. Anatomically, this asymmetric mobility of the wrist results from the wedge-like shape of one carpal bone, the radiale, and from the well-developed convexity of the trochlea at the proximal end of the carpometacarpus. Among the theropod precursors of birds, a strongly convex trochlea is characteristic of Coelurosauria, a clade including the highly derived Maniraptora in addition to tyrannosaurs and compsognathids. The shape of the radiale can be quantified using a ‘radiale angle’ between the proximal and distal articular surfaces. Measurement of the radiale angle and reconstruction of ancestral states using squared-change parsimony shows that the angle was small (15°) in primitive coelurosaurs but considerably larger (25°) in primitive maniraptorans, indicating that the radiale was more wedge-shaped and the carpal joint more asymmetric. The radiale angle progressively increased still further within Maniraptora, with concurrent elongation of the forelimb feathers and the forelimb itself. Carpal asymmetry would have permitted avian-like folding of the forelimb in order to protect the plumage, an early advantage of the flexible, asymmetric wrist inherited by birds.