Biology subjects

Houston, D. W.

Publications and source records attributed to Houston, D. W..

4 recordsLinked to original sources

bioRxiv2025

Interdomain disulfide bonds of rabbit kappa light chain allotypes influence mouse-rabbit chimeric antibody performance

Chimeric monoclonal antibodies have emerged as powerful tools to extend the capabilities of traditional monoclonal antibodies. These antibodies are made by replacing the variable region of a monoclonal antibody with the variable region of another antibody, typically from a different species, enabling use in a wider range of applications. Although theoretically compatible, interspecies differences in antibody structure can complicate chimeric antibody design and performance. In this study, we evaluated the impact of rabbit light chain allotype on the expression and function of chimeric antibodies containing mouse light chain variable regions. We found that constructs using the rabbit kappa 1 b4 (K1-b4) allotype frequently exhibited poor recombinant expression and, in some cases, lost antigen recognition. Structural analysis implicated disruption of an intrachain, interdomain disulfide bond as a contributing factor. Restoration of key residues predicted to re-establish this bond partially rescued both expression and activity. Additionally, chimeric antibodies incorporating the rabbit kappa 1 b9 (K1-b9) allotype, which contains a disulfide bond not disrupted by mouse variable region sequences, consistently maintained robust production and antigen-binding activity across multiple applications, including immunoblotting, immunoprecipitation, and immunostaining, for several test antibodies. Our findings underscore the importance of light chain scaffold selection in recombinant antibody engineering and provide practical guidance for optimizing chimeric antibody design to preserve both expression and function.

molecular biology↗

bioRxiv2025

Novel monoclonal antibodies against the C-terminal HEAT domain of Huntingtin

BACKGROUNDReliable detection of huntingtin (HTT) is essential for understanding Huntingtons disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVEWe sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODSWe immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTSThree novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONSThese C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.

neuroscience↗

bioRxiv2025

Alternative approaches to single-cell trajectory inference using a commute time matrix

Single-cell technology has enhanced the high-resolution analysis of dynamic developmental cell fate decisions. A number of elegant mathematical and computational approaches have been developed for using single-cell genomics data to identify gene regulatory events and cell state changes during embryonic development and related processes. These approaches are typically used in combination to model dynamic cell differentiation trajectories but have different underlying mathematical foundations. The extent to which commonly used algorithms for trajectory modeling, such as data imputation, pseudotemporal ordering, and cell fate probability modeling, might be derived from the same underlying approach has not been widely explored. This work describes the use of a matrix based on the commute time of a graph as a single consistent kernel for cell fate trajectory modeling. The commute time kernel is derived from significant eigenvectors of the pseudo-inverse of the graph Laplacian in a manner that preserves commute time. This kernel matrix is used directly in trajectory inference methods and recapitulates the results obtained using different algorithms using three benchmark datasets. Additionally, a comparison of commute time kernels between spliced and unspliced counts was effective for identifying populations of circadian-cycling progenitor cells in differentiating pancreatic endocrine cells. Overall, this work identifies the commute time kernel as a potential parsimonious measure for multiple aspects of trajectory inference.

bioinformatics↗

bioRxiv2022

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions, namely the dorsal morula, yolk syncytial layer, and distal hypoblast/anterior visceral endoderm (in amphibians, teleosts and mammals, respectively), require the localised stabilisation of nuclear Beta-catenin (Ctnnb1), implying that localised Wnt/Beta-catenin signalling activity is critical in their establishment. However, it is becoming increasingly apparent that the stabilisation of Beta-catenin in this context may be initiated independently of secreted Wnt growth factor activity. In Xenopus, dorsal Beta-catenin stabilisation is initiated by a requisite microtubule-mediated symmetry-breaking event in the fertilised egg: "cortical rotation". Vegetally-localised wnt11b mRNA has been implicated upstream of Beta-catenin in this context, as has the dorsal enrichment of Wnt ligand-independent activators of Beta-catenin, but the extent that each of these processes contribute to axis formation in this paradigm remains unclear. Here we describe a maternal effect mutation in Xenopus laevis wnt11b.L, generated by CRISPR mutagenesis. We demonstrate a maternal requirement for timely and complete gastrulation morphogenesis and a zygotic requirement for proper left-right asymmetry. We also show that a subset of maternal wnt11b mutants have axis and dorsal gene expression defects, but that Wnt11b likely does not act through the Wnt coreceptor Lrp6 or through Dishevelled, which we additionally show (using exogenous constructs) do not exhibit patterns of activity consistent with roles in early Beta-catenin stabilisation. Instead, we find that microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs, leading to less organised and directed vegetal microtubule arrays. In conclusion, we propose that Wnt11b signals to the cytoskeleton in the egg or early zygote to enable robust cortical rotation, and thus acts in the distribution of putative dorsal determinants rather than as a component or effector of the determinants themselves.

developmental biology↗