Biology subjects

White, J. J.

Publications and source records attributed to White, J. J..

6 recordsLinked to original sources

bioRxiv2025

Microfluidics based exploration for quorum quenching genes in Antarctic microbiomes

Quorum sensing (QS) is a form of microbial communication that enables each cell in a population to sense the total population density, so that gene expression can be modified accordingly. Quorum quenching (QQ) is the antagonistic disruption of this communication by competing organisms, with potential use in the ongoing human effort to control microbial populations. Previous studies have taken advantage of functional metagenomics to retrieve new QS/QQ genes, but the frequency of obtaining positive clones remained very low, suggesting the need for increased screening efficiency. Here, a new ultrahigh-throughput screening method was developed to search for genes encoding novel QQ genes based on functional metagenomics, microfluidics, and the development of an Escherichia coli reporter strain whose fluorescence is repressed in the presence of AHLs but restored by the expression of any gene that interferes with the QS system. This strain was transformed with a metagenomic short-insert library collected from Antarctic plant rhizospheres; an understudied extreme environment. The library was encapsulated in droplets containing single clones and sorted using fluorescence-activated cell sorting (FACS). After screening approximately 7,000,000 clones, around 200 were recovered and one positive hit was confirmed, showing a previously unreported mode of action that would have been difficult to detect using rational computational methods. These findings underline the potential of microfluidics to dramatically increase screening efficiency, while reducing costs and processing time, and they act as a proof of concept for the discovery of more genes involved in QS and other molecular mechanisms of interest in microbial ecology. IMPORTANCEQuorum sensing (QS) regulates essential microbial behaviours, including virulence, biofilm formation, and symbiotic interactions, making it a key target for ecological and applied microbiology. Disrupting QS through quorum quenching (QQ) has major potential in agriculture, medicine, and biotechnology as an alternative to antibiotics or chemical treatments. However, the discovery of QQ genes through brute-force approaches is limited by large screening efforts, resulting in high costs and long processing times. Our study introduces a microfluidics-based, ultrahigh-throughput functional screening platform that strongly improves screening efficiency. It allowed us to identify a novel QQ gene, predicted to be involved in a previously undescribed mechanism of QS disruption, from an under-explored extreme environment (Antarctic rhizosphere). These results demonstrate how microfluidics can optimise screening technologies, while unlocking the hidden functional diversity of microbial communities useful for biotechnological applications in microbial control.

molecular biology↗

bioRxiv2024

Purkinje cell intrinsic activity shapes cerebellar development and function

The emergence of functional cerebellar circuits is heavily influenced by activity-dependent processes. However, the role of intrinsic activity in Purkinje neurons, independent of external input, in driving cerebellar development remains less understood. Here, we demonstrate that before synaptic networks mature, Purkinje cell intrinsic activity is essential for regulating dendrite growth, establishing connections with cerebellar nuclei, and ensuring proper cerebellar function. Disrupting this activity during the postnatal period impairs motor function, with earlier disruptions causing more severe effects. Importantly, only disruptions during early development lead to pronounced defects in cellular morphology, highlighting key temporal windows for dendritic growth and maturation. Transcriptomic analysis revealed that early intrinsic activity drives the expression of activity-dependent genes, such as Prkcg and Car8, which are essential for dendritic growth. Our findings emphasize the importance of temporally-specific intrinsic activity in Purkinje cells for guiding cerebellar circuit development, providing a potential common mechanism underlying cerebellum-related disorders.

neuroscience↗

bioRxiv2023

Detecting ataxia using automated analysis of motor coordination and balance of mice on the balance beam

BackgroundThe balance beam assay is a well-known paradigm to assess motor coordination in mouse models of neurodegenerative diseases. Classically, these experiments are quantified using manual annotation, which is time-consuming and prone to inter-experimenter variability. MethodsWe present an open-source analysis pipeline that allows for the automated quantification of motor function in mice crossing the balance beam. ResultsUsing an established Pcp2-Ppp3r1 ataxia mouse model, we validated the analysis pipeline by comparing the motor performance of Pcp2-Ppp3r1 animals to their wildtype littermates. Pcp2-Ppp3r1 animals showed a significant increase in the number of missteps and increased time to traverse the beam. Moreover, we compared the results of the automated classification of missteps and stops to that of 3 independent observers, which showed no significant differences between the classifier and the cumulative observer score for missteps. ConclusionWe show that our pipeline can reliably report crossing time and missteps, offering a high-throughput, automated option for the analysis of balance beam data. Method summaryOur method consists of an easy-to-follow and low-cost manual for building a balance beam setup and an analysis pipeline of mouse movement while crossing the beam. We present a MATLAB script combining FFMPEG and ImageJ (using the MIJ package) to pre-process the videos and extract the time for the mouse to cross the beam. Mice movements on the beam were then tracked using motr and stops and missteps were extracted using a trained JAABA classifier. The trained classifier, the code and all accompanying files were deposited in an open source repository

animal behavior and cognition↗

bioRxiv2019

Production of Insoluble Starch-Like Granules in Escherichia coli by Modification of the Glycogen Synthesis Pathway

While investigating the conversion of cellulosic biomass to starch-like materials for industrial use, it was observed that the overexpression of native ADP-glucose pyrophosphorylase GlgC in Escherichia coli led to the formation of insoluble polysaccharide granules within the cytoplasm, occupying a large fraction of the cell volume, as well as causing an overall increase in cellular polysaccharide content. TEM microscopy revealed that the granules did not have the lamellar structure of starch, but rather an irregular, clustered structure. On starvation, cells overexpressing GlgC appeared unable to fully degrade their polysaccharide material and granules were still clearly visible in cultures after 8 days of starvation. Interestingly, the additional overexpression of the branching enzyme GlgB eliminated the production of granules and led to a further increase in cellular polysaccharides. GlgC is generally thought to be responsible for the rate-limiting step of glycogen synthesis. Our interpretation of these results is that excess GlgC activity may cause the elongation of glycogen chains to outpace the addition of side branches, allowing the chains of adjacent glycogen molecules to reach lengths at which they spontaneously intertwine, forming dense clusters that are largely inaccessible to the host. However, upon additional upregulation of the GlgB branching enzyme, the branching of the polysaccharide is able to keep speed with the synthesis of linear chains, eliminating the granule phenotype. This study suggests potential avenues for increasing bacterial polysaccharide production and recovery. ImportanceIn this work, the polysaccharide stores of Escherichia coli were altered through the addition of extra copies of the bacterias own polysaccharide synthesis genes. In this way, bacteria were created that produced over twice the level of storage polysaccharide as a control strain, in the form of a granule that could potentially facilitate easy harvest. Another form of mutant Escherichia coli was created that produced over seven times the normal level of storage polysaccharide, and also grew to higher cell densities in liquid culture. In addition to increasing our understanding of glycogen synthesis, it is proposed that similarly modified bacteria, grown on inexpensive waste materials, may be a useful source of starch-like polysaccharides for industrial or agricultural use. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field.

microbiology↗

bioRxiv2019

Cerebellar Purkinje cell microcircuits are essential for tremor

Tremor is currently ranked as the most common movement disorder. The brain regions and neural signals that initiate the debilitating shakiness of different body parts remain unclear. Here, we found that genetically silencing cerebellar Purkinje cell activity blocked tremor in mice that were given the tremorgenic drug harmaline. We show in awake behaving mice that the onset of tremor is coincident with rhythmic Purkinje cell firing, which alters the output of their target cerebellar nuclei cells. We mimic the tremorgenic action of the drug with optogenetics and present evidence that highly patterned Purkinje cell activity drives a powerful tremor in otherwise normal mice. Modulating the altered activity with deep brain stimulation directed to the Purkinje cell output in the cerebellar nuclei reduced tremor in freely moving mice. Together, the data implicate Purkinje cell connectivity as a neural substrate for tremor and a gateway for signals that mediate the disease.

neuroscience↗

bioRxiv2018

Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells

One-sentence summaryCerebellar stellate cells and basket cells shape distinct Purkinje cell firing properties\n\nAbstractPurkinje cells receive synaptic input from several classes of interneurons. Here, we address the roles of inhibitory molecular layer interneurons in establishing Purkinje cell function in vivo. Using conditional genetics approaches in mice, we compare how the lack of stellate cell versus basket cell GABAergic neurotransmission sculpts the firing properties of Purkinje cells. We take advantage of an inducible Ascl1CreER allele to spatially and temporally target the deletion of the vesicular GABA transporter, Vgat, in developing neurons. Selective depletion of basket cell GABAergic neurotransmission increases the frequency of Purkinje cell simple spike firing and decreases the frequency of complex spike firing in adult behaving mice. In contrast, lack of stellate cell communication increases the regularity of Purkinje cell simple spike firing while increasing the frequency of complex spike firing. Our data uncover complementary roles for molecular layer interneurons in shaping the rate and pattern of Purkinje cell activity in vivo.

neuroscience↗