The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Longer development provides first-feeding fish time to escape hydrodynamic constraints

What is the functional effect of prolonged development? By controlling for size, we quantify first-feeding performance and hydrodynamics of zebrafish and guppy offspring (5 ± 0.5 mm in length), which differ fivefold in developmental time and twofold in ontogenetic state. By manipulating water viscosity, we control the hydrodynamic regime, measured as Reynolds number. We predicted that if feeding performance were strictly the result of hydrodynamics, and not development, feeding performance would scale with Reynolds number. We find that guppy offspring successfully feed at much greater distances to prey (1.0 vs. 0.2 mm) and with higher capture success (90 vs. 20%) compared with zebrafish larvae, and that feeding performance was not a result of Reynolds number alone. Flow visualization shows that zebrafish larvae produce a bow wave ~0.2 mm in length, and that the flow field produced during suction does not extend beyond this bow wave. Due to well-developed oral jaw protrusion, the similar-sized suction field generated by guppy offspring extends beyond the horizon of their bow wave, leading to successful prey capture from greater distances. These findings suggest that prolonged development and increased ontogenetic state provides first-feeding fish time to escape the pervasive hydrodynamic constraints (bow wave) of being small.     

pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.     

Functional morphology of the pectoral fins in bamboo sharks, Chiloscyllium plagiosum: benthic vs. pelagic station-holding

Bamboo sharks (Chiloscyllium plagiosum) are primarily benthic and use their relatively flexible pectoral and pelvic fins to rest on and move about the substrate. We examined the morphology of the pectoral fins and investigated their locomotory function to determine if pectoral fin function during both benthic station-holding and pelagic swimming differs from fin function described previously in leopard sharks, Triakis semifasciata. We used three-dimensional kinematics and digital particle image velocimetry (DPIV) to quantify pectoral fin function in five white-spotted bamboo sharks, C. plagiosum, during four behaviors: holding station on the substrate, steady horizontal swimming, and rising and sinking during swimming. During benthic station-holding in current flow, bamboo sharks decrease body angle and adjust pectoral fin angle to shed a clockwise fluid vortex. This vortex generates negative lift more than eight times that produced during open water vertical maneuvering and also results in an upstream flow that pushes against the posterior surface of the pectoral fin to oppose drag. In contrast, there is no evidence of significant lift force in the wake of the pectoral fin during steady horizontal swimming. The pectoral fin is held concave downward and at a negative dihedral angle during steady horizontal swimming, promoting maneuverability rather than stability, although this negative dihedral angle is much less than that observed previously in sturgeon and leopard sharks. During sinking, the pectoral fins are held concave upward and shed a clockwise vortex with a negative lift force, while in rising the pectoral fin is held concave downward and sheds a counterclockwise vortex with a positive lift force. Bamboo sharks appear to sacrifice maneuverability for stability when locomoting in the water column and use their relatively flexible fins to generate strong negative lift forces when holding position on the substrate and to enhance stability when swimming in the water column.     

Differential Diagnosis of Some Conditions of the Canine and Feline Thorax : (First Installment)

Routine clinical examination of the chest is described. The basic principles of viewing X-ray plates are mentioned. The conditions of the canine and feline thorax dealt with are mostly those which can be confirmed radiologically during life. The major points of differential diagnosis of each condition are dealt with within groups determined by some cardinal clinical sign. For convenience each condition is dealt with only under one groupheading, although it may have additional signs of some of the other groups. The groups and conditions are as follows:Gross DyspnoeaHydrothoraxPyothoraxPneumothoraxDiaphragmatic herniaPneumoniaCoughDistemperTuberculosisSecondary lung tumourFilaroides osleri infestation of the tracheaKennel coughFeline influenzaTiring on exerciseCardiac valvular diseaseCongenital cardiac diseaseDifficulty in swallowingOesophageal foreign bodyFeline oesophageal annular carcinomaOesophageal diverticulumMega-oesophagus (Achalasia)Oesophageal dilatation associated withvascular anomalies of the right aortic arch.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Keratan sulfates from bovine tracheal cartilage structural studies of intact polymer chains using H and 13C NMR spectroscopy.

Intact keratan sulfate chains derived from bovine tracheal cartilage have been examined using both one-dimensional methods and the two-dimensional experiments COSY-45 and TOCSY for homonuclear shift correlations and a modified COLOC (correlated spectroscopy for long-range couplings) approach for 13C-1H shift correlations. Partial 1H and 13C NMR signal assignments for residues within the intact polymer chain are reported; data derived from the repeat region signals and from chain cap residues are assigned by comparison with published data derived from oligosaccharides obtained through cleavage of keratan sulfate polymer chains using keratanase and keratanase II and are discussed in detail. The one-dimensional spectra for both 1H and 13C nuclei contain highly crowded signal clusters for which data analysis is not directly possible. COSY-45 analysis allow the correlation and assignment of many proton resonances located within the 3.4-4.8 p.p.m. chemical shift region while from the C/H correlation spectrum data are assignable for some signals within the complex set of carbon resonances which fall in the region between 68 and 86 p.p.m., This work using material from tracheal cartilage has permitted the first detailed combined 1H and 13C NMR examination of the primary keratan sulfate polymer structure; this sequence forms the basis for the more complex members of the keratan sulfate family present in other tissues such as articular cartilage and cornea where further residues such as (alpha1-3)-linked fucose and (alpha2-6)-linked N-acetylneuraminic acid are also present. This nondestructive method of analysis complements the currently available degradative methods for structure determination which may then subsequently be utilized.