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Biology subjects

Christopher, J.

Publications and source records attributed to Christopher, J..

17 recordsLinked to original sources

Control of Guanine Exchange Factor Activity Using De Novo Designed Protein Inhibitors

Guanine Exchange Factors (GEF) of the Dbl family are the main activators of RhoA GTPases. GEF and GTPase activity is tightly regulated at the subcellular level with fast kinetics. Therefore, to fully understand the function of Dbl GEFs requires their study in living cells. Towards developing molecular tools that reversibly and rapidly modulate the activity of endogenous GEFs in living cells, here we developed a general platform for engineering inhibitors against members of the Dbl family of GEFs using generative protein design. Engineered proteins showed high affinity and remarkable specificity for the target GEFs and modulated GEF activity both in vitro and in cells. In a proof-of-principle example, a GEF inhibitor was coupled to a light-activated module, enabling the optogenetic control of its activity in cells. These findings show that generative protein design can create modulators of intracellular signaling and broaden the range of tools available for biological research.

cell biology↗

A rapid, low-cost approach to solid immersion lens fabrication for enhanced resolution in optical microscopy

Solid immersion lenses (SILs) enhance the spatial resolution of an optical microscope by increasing the effective numerical aperture (NA) without physical modification of the objective lens. However, SIL application remains limited by cost, fragility, and accessibility. We present a rapid, single-step fabrication process to create optical quality hemispherical SILs using consumer-grade UV-curable transparent resin which reduces material costs by over five orders of magnitude relative to commercial glass counterparts. Our method produced resin SILs within seconds which can be easily implemented into conventional microscopy setups for increasing the effective NA. Quantitative imaging of USAF resolution targets and histology muscle preparations demonstrated a resolution enhancement approaching theoretical limits and comparable performance to N-BK7 glass SILs. This enabled visualisation of features usually below the diffraction limit of low NA dry objectives at a fraction of the cost of otherwise required high-powered objective lenses. To demonstrate accessibility and translational potential, our workflow was taught in a practical tutorial of an international microscopy course, where non-expert participants successfully fabricated, characterised, and applied SILs within a single session, reporting high confidence in independent implementation. We established ultra-low-cost resin SILs as a practical, scalable option to enhance the spatial resolution of routine optical microscopes and as an accessible and cost-effective platform for optics education.

biophysics↗

A Mammalian Surface Display Platform to Optimize the Antigenicity of Viral Proteins for Vaccine Design

Vaccine development often involves modifying native viral proteins to enhance their stability and antigenicity, as seen in approved Covid-19 vaccines and multiple HIV vaccine candidates currently under investigation. High throughput screening on the surface of mammalian cells enables the rapid evaluation of oligomeric, glycosylated viral proteins and the identification of mutations that improve their properties for vaccine design. Here, we developed an experimental platform that uses the PiggyBac transposon system to display libraries of viral protein variants on the surface of mammalian cells for efficient screening. This approach addresses common challenges in existing mammalian display systems, including low transfection efficiency and the development of stable cell lines for iterative selection rounds. The new platform was validated by expressing and characterizing antigenically diverse viral proteins from influenza, SARS-CoV-2, and HIV. In a further application, library screening of influenza haemagglutinin libraries identified mutations that increased binding of broadly cross-reactive antibodies to a conserved, but partially occluded, epitope of interest for the development of a universal influenza vaccine. These results demonstrate the potential of this mammalian display platform to rapidly engineer immunogens with desired antigenic properties for vaccine design.

immunology↗

Iterative immunogen optimization to focus elicited responses on a conserved, subdominant influenza hemagglutinin epitope

Designing effective vaccination strategies against genetically diverse viruses, such as HIV or influenza, is hindered by the ability of these pathogens to mutate and readily evade immune responses. While these viruses contain conserved regions that cannot easily mutate without affecting viral fitness, these sites are subdominant, meaning that they are not the primary target of antibodies elicited by natural infection or current vaccines. Here, we integrated recent advances in machine learning methods for protein structure prediction and design to engineer protein immunogens that focus immune responses on a conserved, subdominant HA epitope targeted by broadly cross-reactive influenza antibodies. Iterating between computation-guided optimization and in vivo analyses generated immunogens that faithfully displayed the target epitope and redirected humoral responses toward it upon vaccination. These results provide a blueprint for applying recent protein engineering approaches to immunogen design and may inform the development of broadly protective influenza vaccines.

immunology↗

Low-Cost 3D Printed Optics for Super-Resolution Multifocal Structured Illumination Microscopy

We present a low-cost 3D printing method of fabricating optical quality lenslet arrays for integration in a multifocal structured illumination microscope (mSIM), achieving fluorescence imaging below the optical diffraction limit. We detail the design and manufacturing processes to produce high-quality 3D printed optics, showing their comparable surface roughness of 30 {+/-} 2.5 nm for the 3D printed elements compared to 37 {+/-} 1.4 nm for commercial glass optics. A 3D printed lenslet array with a honeycomb geometry and 1.2 mm lenslet diameter was compared to a high-end glass commercial lenslet array with 250 {micro}m lenslet diameter and a lower cost commercial lenslet array with a 1.2 mm by 1.6 mm lenslet footprint. The imaging performance of the different optics was benchmarked using a custom mSIM setup by quantifying the beam profile homogeneity and the experimental lateral resolution. The mSIM setup incorporating the different microlens arrays was tested using a commercial bovine pulmonary artery endothelial cell specimen, highlighting an achievable resolution enhancement from 229 nm {+/-} 11 nm with widefield illumination to 137 {+/-} 11 nm using the high-end commercial microlens array and 134 nm {+/-} 9 nm using the 3D printed honeycomb lenslet array. Advantages of improved background rejection through the custom lenslet geometry are discussed, highlighting the super-resolution microscope performance achievable through custom low-cost 3D printed optics.

biophysics↗

A large 3D-printed integrated lens-biprism element enhances contrast in transmission stereomicroscopy

Stereomicroscopes are routinely used across disciplines for material and surface characterisation due to their simplicity of use and minimal specimen preparation requirements. However, the stereomicroscope transillumination design is suboptimal, as a single incident beam at the specimen plane is shared and transmitted via two laterally offset detection axes. This flaw limits life science applications due to the transparent nature of samples which results in poor contrast images. We use a single additional element in the illumination path to correct the illumination uniformity across the field of view and, by doing so, enhance image contrast and facilitate detection of refractive structures in transparent biological specimens. We designed and fabricated an integrated lens-biprism element using low-cost, consumer-grade 3D printing methods and consumables. This 3D printed lens-biprism distributed diverging rays from a single incandescent light source into two parallel beams that converged at the specimen plane and transmitted through the respective left and right detection axes of a stereomicroscope. This improved transillumination setup increased the image contrast by up to 67.62% compared to the conventional stereomicroscope setup. We demonstrated the benefit of the lens-biprism element by visualising dynamic cellular events in live tissue and discerning refractive structures more easily in transparent specimens.

biophysics↗

Envirotyping Facilitates Understanding of Genotype x Environment Interactions and Highlights the Potential of Stay-green Traits in Wheat

Better understanding genotype by environment interaction (GxE) can help breeding for better adapted varieties. Envirotyping for environmental water status was applied to assist interpretation of GxE interactions for wheat yield in multi-environment trials conducted in drought-prone Australian environments. Genotypes from a multi-reference parent nested association mapping (MR-NAM) population were tested in 10 trials across the Australian wheatbelt. Genotype yield and phenology were measured in all trials, while traits associated with the stay-green phenotype were assessed for a subset of 5 trials. Envirotyping was conducted by characterizing water stress experienced by genotypes at each trial using crop modelling. Envirotyping facilitated the understanding of GxE interactions by explaining 75, 67, and 66% of the genotypic variance for yield in severe water-limited (ET3), mild terminal water-stress (ET2), and water-sufficient (ET1) environments, respectively. Yield and stay-green were negatively correlated with flowering time in most trials. However, when focusing on genotypes flowering at similar times within a trial, no significant correlation was found between yield and flowering. Importantly stay-green traits remained significantly correlated with yield. Stay-green traits such as delayed onset of senescence and slower senescence rate benefited yield by 0.2 to 1.1 t ha-1 across environments, highlighting the breeding potential for stay-green traits in both water-sufficient and water-limited environments. Hence, sustaining green leaf area during grain filling helped to enhance yield. Envirotyping to better understand GxE interactions for yield, coupled with screening for traits exhibiting superior adaptive mechanisms, are powerful assets in assisting plant breeders to select more effectively drought adapted genotypes. HighlightsGenotype x environment interaction for yield could be reduced with envirotyping. Envirotyping enabled a 75% gain in genotypic variance in severe drought conditions. Envirotyping clarified breeding potential of stay-green traits across environments. Stay-green has potential to enhance wheat yield across diverse environments.

physiology↗

Design of SARS-CoV-2 RBD Immunogens to Focus Immune Responses Towards Conserved Coronavirus Epitopes

SARS-CoV-2 continues to evolve, with new variants emerging that evade pre-existing immunity and limit the efficacy of existing vaccines. One approach towards developing superior, variant-proof vaccines is to engineer immunogens that preferentially elicit antibodies with broad cross-reactivity against SARS-CoV-2 and its variants by targeting conserved epitopes on spike. The inner and outer faces of the Receptor Binding Domain (RBD) are two such conserved regions targeted by antibodies that recognize diverse human and animal coronaviruses. To promote the elicitation of such antibodies by vaccination, we engineered "resurfaced" RBD immunogens that contained mutations at exposed RBD residues outside the target epitopes. In the context of pre-existing immunity, these vaccine candidates aim to disfavor the elicitation of strain-specific antibodies against the immunodominant Receptor Binding Motif (RBM) while boosting the induction of inner and outer face antibodies. The engineered resurfaced RBD immunogens were stable, lacked binding to monoclonal antibodies with limited breadth, and maintained strong interactions with target broadly neutralizing antibodies. When used as vaccines, they limited humoral responses against the RBM as intended. Multimerization on nanoparticles further increased the immunogenicity of the resurfaced RBDs immunogens, thus supporting resurfacing as a promising immunogen design approach to rationally shift natural immune responses to develop more protective vaccines.

immunology↗

A fully 3D-printed optical microscope for low-cost histological imaging

We present the manufacture and characterisation of a fully 3D printed, low-cost optical microscope using both a 3D printed chassis and 3D printed illumination and imaging optics. The required commercial components, consisting of a basic camera for image acquisition and light emitting diode controlled by a Raspberry Pi for illumination, are integrated into the 3D printed microscope with the full design shown for ease of replication. Our 3D printed microscope uses a single 3D printed objective lens with a 2.9x magnification and a numerical aperture of 0.07. To benchmark the imaging performance of the system, we used standard test targets and histological specimens, namely, a Giemsa-stained blood smear sample and a thin section of mouse kidney stained with Haemotoxylin and Eosin. We demonstrated that sub-cellular resolution was obtained, and we corroborated this by imaging individual red blood cells and intricate anatomical details of the stained mouse kidney section. All of this was achieved using entirely 3D printed hardware and optics, at a fraction of the cost of a commercial brightfield microscope, while presenting remarkable potential for customisation and increased accessibility for diagnostic imaging applications.

biophysics↗

Printing, Characterising, and Assessing Transparent 3D Printed Lenses for Optical Imaging

High-quality lens production has involved subtractive manufacturing methods for centuries. These methods demand specialist equipment and expertise that often render custom high-grade glass optics inaccessible. We aimed to develop a low-cost, accessible, and reproducible method to manufacture high-quality three-dimensional (3D) printed lenses using consumer-grade technology. Various planoconvex lenses were produced using a consumer-grade 3D printer and low-cost spin coating setup, and printed lenses were compared to commercial glass counterparts. A range of mechanical and optical methods are introduced to determine the surface quality and curvature of 3D printed lenses. Amongst others, high-resolution interference reflection microscopy methods were used to reconstruct the convex surface of printed lenses and quantify their radius of curvature. The optical throughput and performance of 3D printed lenses were assessed using optical transmissivity measurements and classical beam characterisation methods. We determined that 3D printed lenses had comparable curvature and performance to commercial glass lenses. Finally, we demonstrated the application of 3D printed lenses for brightfield transmission microscopy, resolving sub-cellular structures over a 2.3 mm field-of-view. The high reproducibility and comparable performance of 3D printed lenses present great opportunities for additive manufacturing of bespoke optics for low-cost rapid prototyping and improved accessibility to high-quality optics in low-resource settings.

biophysics↗

Low-cost 3D printed lenses for brightfield and fluorescence microscopy

We present the fabrication and implementation of low-cost optical quality 3D printed lenses, and their application as microscope objectives with different prescriptions. The imaging performance of the 3D printed lenses was benchmarked against commercially available optics including a 20 mm focal length 12.7 mm diameter NBK-7 plano-convex lens used as a low magnification objective, and a separate high magnification objective featuring three 6 mm diameter NBK-7 lenses with different positive and negative focal lengths. We describe the design and manufacturing processes to produce high-quality 3D printed lenses. We tested their surface quality using a stylus profilometer, showing that they conform to that of commercial glass counterpart lenses. The 3D printed lenses were used as microscope objectives in both brightfield and epi-fluorescence imaging of specimens including onion, cyanobacteria, and variegated Hosta leaves, demonstrating a sub-cellular resolution performance obtained with low-cost 3D printed optical elements within brightfield and fluorescence microscopy.

biophysics↗

Pre- and post-flowering impacts of natural heat events on yield components in wheat

O_LICONTEXT Wheat crops are highly sensitive to elevated temperatures and experience significant yield losses when short periods of heat occur at sensitive developmental phases. C_LIO_LIOBJECTIVE This research aimed at quantifying wheat responses of grain yield and yield components to heat indicators in fluctuating field conditions. C_LIO_LIMETHODS The impacts of high temperature on yield and its components were assessed for 20-35 wheat lines in irrigated multi-environment trials over three years. Genotypes were cultivated using a novel photoperiod extension method adjacent to some conventional yield plots with different sowing dates. In the photoperiod-extension method, either single stems or plant quadrates were tagged at specific growth stages and hand-harvested at maturity, while conventional plots were mechanically harvested at maturity. The impact of heat was estimated for events occurring at different developmental stages and different temperature thresholds (26 to 35{circ}C). C_LIO_LIRESULTS The closest correlation between heat events and grain number was observed for a threshold temperature of 28{circ}C pre-flowering. For individual grain weight, the correlation was statiscally the closest for threshold temperatures above 32{degrees}C post-flowering. In the tested environments, grain number was most sensitive to heat between 300 and 200{circ}Cd before flowering. For each hot hour (T > 28{circ}C) during this period, wheat genotypes lost an extra 0.25 grains spike-1 or 90 grains m-2 on average. Post-flowering heat reduced individual grain weight by 0.26 mg (at spike harvest) and grain yield by 2.44 g m-2 (at plot harvest) for every hot hour (T > 32{circ}C) between 0 and 500{circ}Cd after flowering. Heat impacts were the clearest for plants with synchronised phenology. In addition, results suggest that heat impacts can be quantified more reliably using finer time units (i.e. hot hours rather than days). C_LIO_LIONCLUSIONS Natural heat events strongly impacted grain number and individual grain weight of wheat for temperatures above 28-30{degrees}C in well-watered conditions. Reduction in grain number and individual grain weight were strongly associated with accumulated hot hours that occurred during 200-300{degrees}Cd before and 0-500{degrees}Cd after flowering, respectively. C_LIO_LIIMPLICATIONS The findings from this study will assist improvement for crop modelling in response to heat, development of relevant phenotyping methods and selection of cultivars with better adaptation to warmer environments. C_LI

plant biology↗

Miniaturised structured illumination microscopy using two 3-axis MEMS micromirrors

We present the development and performance characterisation of a novel structured illumination microscope (SIM) in which the grating pattern is generated using two optical beams controlled via 2 micro-electro-mechanical system (MEMS) three-axis scanning micromirrors. The implementation of MEMS micromirrors to accurately and repeatably control angular, radial and phase positioning delivers flexible control of the fluorescence excitation illumination, with achromatic beam delivery through the same optical path, reduced spatial footprint and cost-efficient integration being further benefits. Our SIM architecture enables the direct implementation of multi-colour imaging in a compact and adaptable package. The two-dimensional SIM system approach is enabled by a pair of 2 mm aperture electrostatically actuated three-axis micromirrors having static angular tilt motion along the x- and y- axes and static piston motion along the z-axis. This allows precise angular, radial and phase positioning of two optical beams, generating a fully controllable spatial interference pattern at the focal plane by adjusting the positions of the beam in the back-aperture of a microscope objective. This MEMS-SIM system was applied to fluorescent bead samples and cell specimens, and was able to obtain a variable lateral resolution improvement between 1.3 and 1.8 times the diffraction limited resolution.

cell biology↗

Hippocampal Concentrations Drive Seizures in a Rat Model for Cefepime-induced Neurotoxicity

BackgroundIn high dose, cefepime causes neurotoxicity in patients with kidney injury; however, the relationship between exposure and observed neurotoxicity is not clear, and no animal model presently recapitulates the human condition. ObjectivesThis study sought to describe plasma and tissue pharmacokinetics and pharmacodynamics (PK/PD) of cefepime in rats experiencing neurotoxicity. MethodsMale Sprague-Dawley rats (n=21) received escalating cefepime total daily doses ranging from 531-1593 mg/kg body weight/day administered as a short infusion (0.5 mL/min) every 24h for 5 days. Cefepime was quantified in plasma, cerebral cortex and hippocampus via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple PK/PD models of cefepime transit between plasma and brain compartments (i.e. cerebral cortex and hippocampus) and neurotoxic response were explored using Monolix 2021R1 (LixoftPK). ResultsExposure estimation of cerebral cortex demonstrated a median (IQR) AUC0 -24 and Cmax 0 -24 of 181.8 (85.2-661.3) mg {middle dot} 24 h/liter and 13.9 (1.0-30.1) mg/L, respectively. The median cerebral cortex/blood percentage of penetration was 1.7%. Exposure estimation of hippocampus demonstrated a median (IQR) AUC0 -24 and Cmax 0 -24 of 291.4 (126.6-1091.6) mg {middle dot} 24 h/liter and 8.8 (3.4-33.4) mg/L, respectively. The median hippocampus/blood percentage of penetration was 4.5%. Rats that reached a cefepime Cmax of {square}17 mg/L in the hippocampus exhibited signs of neurotoxicity. A hippocampal cefepime concentration of 4.1 {micro}g/100 mg brain tissue best described seizure stages >1 for cefepime-induced neurotoxicty. ConclusionsA cefepime plasma AUC0 -24 of 28,000 mg*24h/L and hippocampal concentrations of 4.1 {micro}g/100 mg brain tissue may be a threshold for cefepime-induced neurotoxicity. This model provides a methodology for future interrogation of the relationship between plasma concentrations, brain tissue concentrations, and neurotoxicity.

pharmacology and toxicology↗

Physiological and genetic drivers underpinning canopy development are associated with durum wheat yield in rainfed environments

New durum wheat (Triticum turgidum L. ssp. Durum) cultivars with improved adaptation to variable rainfall environments are required to sustain productivity in the face of climate change. Physiological traits related to canopy development underpin the production of biomass and yield, as they interact with solar radiation and affect the timing of water use throughout the growing season. This study explored the temporal canopy dynamics of durum wheat using a nested-association mapping population evaluated for longitudinal normalized difference vegetation index (NDVI) measurements. Association mapping was performed to identify quantitative trait loci (QTL) for time-point NDVI and spline-smoothed NDVI trajectory traits. Yield effects associated with QTL for canopy development were investigated using data from four rainfed field trials. Four QTL associated with slower canopy closure, improved yield in specific environments, and notably, were not associated with a yield penalty in any environment. This was likely due to optimised timing of water-use and pleiotropic effects on yield component traits, including spike number and spike length. Overall, this study suggests that slower canopy closure is beneficial for durum wheat production in rainfed environments. Selection for traits or loci associated with canopy development may improve yield stability of durum wheat in water limited environments.

genetics↗

A robust field-based method to screen heat tolerance in wheat

Wheat is highly sensitive to elevated temperatures, particularly during pollen meiosis and early-to-mid grain filling. The impact of heat stress greatly depends on the plant developmental stage. Thus, germplasm ranking for heat tolerance in field trials may be confounded by variations in developmental phase between genotypes at the time of heat events. A photoperiod-extension method (PEM) was developed allowing screening of 35 diverse genotypes at matched developmental phase despite phenological variations. Paired trials were conducted to compare the new PEM against conventional field screening in plots. In the PEM, plants were sown in single rows or small plots. Artificial lighting was installed at one end of each row or plot to extend day length, inducing a gradient of flowering times with distance from the lights. Individual stems or plot quadrats of each genotype were tagged at flowering. Late-sown plants received more heat shocks during early to mid grain filling than earlier sowings, suffering reductions in both individual grain weight (IGW) and yield. IGW was reduced by 1.5 mg for each additional post-flowering day with temperature > 30{degrees}C. Significant genotypic differences in heat tolerance ranking were observed between PEM versus conventional plot screening. Strong correlations between trials experiencing similar degree of heat were found both for IGW and for total grain weight with the PEM either with individual-stem tagging (e.g. average r of 0.59 and 0.54, respectively for environments with moderate postflowering heat) or quadrat tagging (r of 0.53 and 0.47). However, correlations for IGW and yield in these environments were either poor or negative for conventional trials (e.g. average r of 0.11 and 0.12, respectively for environments with moderate postflowering heat). Accordingly, a PCA grouped genotypes consistently for heir performance across environments with similar heat stress in PEM trials but not in conventional trials. In this study, most consistent genotype ranking for heat tolerance was achieved with the PEM with tagging and harvesting individual spikes at matched developmental phase. The PEM with quadrat sampling provided slightly less consistent rankings but appears overall more suitable for high-throughput phenotyping. The method promises to improve the efficiency of heat tolerance field screening, particularly when comparing genotypes of different maturity types.

plant biology↗

Extensive phylogenies of human development reveal variable embryonic patterns

Starting from the zygote, all cells in the developing and adult human body continuously acquire mutations. A mutation shared between two different cells implies a shared progenitor cell and can thus be used as a naturally occurring marker for lineage tracing. Here, we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissected samples from multiple organs. Early embryonic progenitor cells inferred from the phylogenies often contribute in different proportions to the adult body and the extent of this asymmetry is variable between individuals, with ratios between the first two reconstructed cells ranging from 56:44 to 92:8. Asymmetries also pervade subsequent cell generations and can differ between tissues in the same individual. The phylogenies also resolve the spatial embryonic origins and patterning of tissues, revealing a spatial effect in the development of the human brain. Supplemented by data on eleven men, we timed the split between soma and germline, with the earliest observed segregation occurring at the first cell divisions. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals.

genomics↗