Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range. Read the paper here.

More and more evidence suggest that circadian clock disruption or misalignment is a feature of many diverse chronic diseases including metabolic syndrome, depression but also a number of cancers. For the latter, recent mechanistic studies in cancer models have established an understanding of how the circadian clock influences onset, progression and therapeutic outcomes. Moreover, it has been proposed that tumours might have disrupted circadian oscillators. In patients, however, this is more difficult to establish as usually only single samples, e.g., tumour biopsies, are available. Therefore, novel tools to measure the functional state of the molecular circadian clock are needed.

Here, we introduce TimeTeller, a machine learning tool that analyses the clock as a system and aims to estimate circadian clock function from a single sample’s transcriptome by modelling the multi-dimensional state of the clock. We demonstrate TimeTeller’s utility for analysing experimental in vitro and in vivo, as well as healthy human and patient samples from various platforms (microarray, RNA-Seq and NanoString) and highlight TimeTeller’s potential relevance for advancing circadian medicine. The project is an inter-disciplinary collaboration including significant work by Warwick’s MRCDTP students Laura Usselmann and Vadim Vasilyev and is setting the stage for further applications of TimeTeller in experimental models and human breast tumours.
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This work is based on the discovery of diurnal variations impacting cancer therapy. Especially, use of chronomodulated treatment with 5-fluorouracil (5-FU) has gained significance. Studies indicate high inter-individual variability in diurnal variations in dihydropyrimidine dehydrogenase (DPD) activity – a key enzyme for 5-FU metabolism. However, the influence of individual chronotypes on chronomodulated therapy was unclear but is needed to optimize precision dosing of chronomodulated 5‑FU. Lead by the Thorsten Lehr's PKPD group at the University of Saarland, this collaborative paper is taking a treasure trove of patient 5-FU PK data amalgamated with DPD enzyme activity data from health people to establish a novel PKPD model of 5-FU that captures the extent of diurnal variations in DPD activity and can help investigate individualized chronomodulated 5-FU therapy through testing alternative personalized dosing strategies. Read the paper here.

Work by recent WMS PhD graduate Agnieszka Nagorska and PDRA Andreas Zaucker shows that translational control of an enzyme, FurinA, is important for normal positioning of the heart, and for cardiac valve function. FurinA cleaves the growth factor signal and morphogen, Nodal. Mutant zebrafish embryos with mis-regulated furina show premature and increased levels of FurinA, ectopic Nodal signalling, and defects in heart positioning and valve development. This is similar to human patients with mitral valve regurgitation. The findings pave the way for potential diagnostic tests for patients with heart valve dysfunctions. The work was supported by grants from the Leverhulme Trust, UKRI-BBSRC, MLSRF, and doctoral studentships from Warwick Medical School, MRC DTP, and Warwick-ARAP.
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Gram negative bacterial pathogens use so called Type VI secretion systems (T6SSs) to deliver virulence effectors into target cells (either animal cells or other bacteria). Besides structural and effector proteins, many other proteins, such as adaptors, co-effectors and accessory proteins, are involved in this process. MIX domains can assist in the delivery of T6SS effectors when encoded as a stand-alone gene or fused at the N-terminal of the effector. However, whether there are other conserved domains exhibiting similar encoding forms to MIX in T6SS remains obscure.

In this work, we scanned publicly available bacterial genomes and established a database which include 130,825 T6SS vgrG loci from 45,041 bacterial genomes. Based on this, we revealed six domain families encoded within vgrG loci, which are either fused at the C-terminus of VgrG/N-terminus of T6SS toxin or encoded by an independent gene. Among them, DUF2345 was further validated and shown to be indispensable for the T6SS effector delivery and LysM was confirmed to assist the interaction between VgrG and the corresponding effector. Together, our results implied that these widely distributed domain families with similar genetic configurations may be required for the T6SS effector recruitment process.

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The dramatic global impact of the coronavirus pandemic has increased consideration on epidemiological progressions of pandemics. Measures implemented to reduce viral transmission have been largely historical, comparable in nature with the 1918 and 2009 influenza pandemics, demonstrating the importance of clinicians’ awareness on historical pandemics.

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Liver hepatocytes are the front-line cells for screening new compounds for toxicity. However, hepatocytes are stored frozen in vials, not in monolayers meaning they need substantial processing to be ‘usable’ especially for high throughput screening. To solve this the Gibson and Dallman groups have collaborated, so show that immortalised and primary hepatocytes can be cryopreserved whilst attached to 96 well microplates. These can then be taken from the freezer, and simple thawed and are ready to use in under 24 hours. Post-thaw the cells show equal performance to fresh. This was achieved by careful consideration of the molecular mechanism damage during freezing, with the team using patent-pending controlled ice nucleation technology, rather than a traditional re-formulation of cryoprotectants approach. This work was in collaboration with Cryologyx, a University of Warwick Spin out, which has commercialised aspects of this technology.  
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Trafficking of proteins, lipids, and other molecules between cellular compartments is carried out by vesicular carriers. Material destined for transfer is packaged into a small trafficking vesicle at the donor compartment; the vesicle must then travel to its destination, before fusing with the target compartment to deliver the material.

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Dr John James' lab have used a cellular reconstitution of preTCR function to investigate the trafficking dynamics of this developmentally important immune receptor, work which has just been published in the Journal of Cell Biology.

Expression of the pre-T cell receptor (preTCR) is an important checkpoint during the development of T cells, an essential cell type of our adaptive immune system. The preTCR complex is only transiently expressed and rapidly internalized in developing T cells and is thought to signal in a ligand-independent manner. However, identifying a mechanistic basis for these unique features of the preTCR compared with the final TCR complex has been confounded by the concomitant signaling that is normally present. Thus, we have reconstituted preTCR expression in non-immune cells to uncouple receptor trafficking dynamics from its associated signaling. We find that all the defining features of the preTCR are intrinsic properties of the receptor itself, driven by exposure of an extracellular hydrophobic region, and are not the consequence of receptor activation. Finally, we show that transitory preTCR cell surface expression can sustain tonic signaling in the absence of ligand binding, suggesting how the preTCR can nonetheless drive αβTCR lineage commitment.

Read the paper here.

Advanced cell-based therapies are often delivered to the patient frozen. Hence, any technology which increases the number of recovered, viable, cells post-thaw may improve clinical outcomes or allow more treatments per donation. The GibsonGroup have previously shown strategies to protect cells during freezing. In this latest work, in collaboration with Cytiva, the team show that incubating a model t-cell line with proline before cryopreservation leads to increased post-thaw cell yields. All proline is removed before cryopreservation so the actual freezing and thawing processes are unchanged. It was shown that proline limits cell proliferation, which might be contributing to its mode of action similar to ‘metabolic pre-conditioning’ which has been shown before.

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Glycans (sugars) dictate cell-cell communication, are sites for pathogen invasion and are a key part of our immune systems. Current synthetic platforms to display glycans to investigate their biology almost always are imperfect, with heterogeneity in terms of number of glycans and the synthesis is not reproducible batch to batch. The Gibson and Ward (chemistry) groups have collaborated on a Leverhulme-Trust funded project to create ‘programmable’ glyco-clusters - before entering the lab, this method enables a research to know exactly how many glycans and their 3-D location are present on a material, and gives zero heterogeneity. This is achieved using metal co-ordination cages - 3D structures formed by spontaneous self-assembly. The team used these with model glycan-binding proteins to identify key interactions which would not be possible with traditional materials. The team are now using this to interrogate a range of targets including toxins, for diagnostics.

Read the paper here.

Spheroids (and organoids) can reproduce key aspects of human biological responses, and since the FDA simplification act it is possible (in some cases) to bypass animal testing in the development of new drugs where quality tissue models exist. However, these are not accessible ‘off the shelf’ so are not widely used, with monolayer culture then animal studies common. The GibsonGroup working with the WhaleGroup have recently shown how controlled nucleation (making ice form) can actually improve cryopreservation outcomes by reducing intracellular ice formation. In this latest work they combine nucleation with proline-pre conditioning which ‘prepares’ cells for cryopreservation. This shows how joined-up thinking of cryopreservation as a biochemical and biophysical problem can make a major impact on cell-storage platform technologies.

Read the paper here.

Bacteriophage (phage) are present wherever their bacteria hosts are. Phage have huge biotechnological potential, but lytic phages can also cause complete loss of bacterial cultures. For example in the food industry, or in every research laboratory, where rigorous sterile handing is the primary containment strategy. For industrial biotechnology using microorganisms to enable sustainable of chemicals, materials and drugs, phage infection must be addressed. In our latest (patent pending) work, in collaboration with the SagonaLab at Warwick, and Cytiva, we discovered that a simple polymer can prevent phage infection of bacteria when applied to the growth media. This process is simple, requires no change to working practises and prevents phage infections. We are still investigating the mechanistic aspects, but this is virustatic (inhibitory) rather than virucidal.

Read the press release here.
Read the paper here.

The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules.

Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Read the paper here.

Dr Timothy Saunders and team have been awarded almost £300,000 by the British Heart foundation to study Fruit Fly Hearts!

Read more here:
Fruit fly helps Warwick scientists understand human heart development - BHF

The GibsonGroup, in collaboration RCSI (Dublin), have demonstrated that polyproline is a structurally simple mimic of antifreeze glycoproteins. The GibsonGroup have a large interest in developing materials which can control ice growth/formation, and their application in biotechnology. This is inspired by antifreeze proteins, which can be challenging to obtain and are not suitable for scale up. The antifreeze glycoproteins are known to adopt a PPII helix in solution, and in this latest work the team show that polyproline itself is sufficient for ice binding and inhibiting ice growth, when it has sufficiently high molecular weight. This is significantly simpler than using a glycoprotein and supports growing evidence that the ‘hydrophobic’ face of AFGPs binds the ice, rather than the glycans, and that hydrogen bonding to the ice is not always essential for activity. Finally, this also shows that bio-renewable resources can be used to obtain ice growth inhibitors which themselves could be biodegradable.

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Liquid biopsies of circulating tumor cells (CTCs) have the potential to transform cancer management through non-invasive, real-time feedback on patient conditions. However, immunoaffinity-based liquid biopsies typically suffer from low throughput, relative complexity, and postprocessing limitations. Here, we addressed these issues simultaneously by decoupling and independently optimizing the nano-, micro-, and macro-scales of an enrichment device that is simple to fabricate and operate. Unlike other affinity-based devices, our scalable mesh approach enables optimum capture conditions at any flow rate. The device detected CTCs under experimental conditions and in the blood of cancer patients where it also allowed for postprocessing and, thus, identification of clinically relevant biomarkers such as HER2, but also has the potential to predict patient response to therapies such as immune checkpoint inhibition therapy in the future. This suggests that our approach can overcome major limitations associated with affinity-based liquid biopsies and help improve cancer management.

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Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other in Bacillus anthracis: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all B. anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and haemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25 °C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37 °C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25 °C compared to 37 °C. Furthermore, results suggest that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37 °C, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to ‘switch’ between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA.

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Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. . Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37˚C in comparison to 25˚C. At 37˚C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The figure below shows the first image of the anthrax G9241 cross-over strain linear chromosome bacteriophage (unusual in bacteriophage itself).

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We reveal that the developing Drosophila nerve cord has a distinctive architectural structure, which is driven by JNK signalling.
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A collaborative study by the Thompson group with Dr Robin Thompson's (Mathematics Institute, University of Warwick) and Dr Uri Obolski's (Tel Aviv University) groups. The study examines the impact of prior immunity conferred by SARS-CoV-2 or seasonal coronavirus infection on the emergence of new variants using mathematical modelling . We find that, if cross-reactive immunity is complete (i.e. someone infected by the previously circulating virus is not susceptible to the novel variant), the novel variant must be more transmissible than the previous virus to invade the population. However, in a more realistic scenario in which cross-reactive immunity is partial, we show that it is possible for novel variants to invade, even if they are less transmissible than previously circulating viruses. Finally, we find that if previous infection with the antigenically related virus assists the establishment of infection with the novel variant, as has been proposed following some experimental studies, then even variants with very limited transmissibility are able to invade the host population.

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The GibsonGroup have a programme of research to investigate how glycans (sugars) can be used in biosensing or diagnosis of disease. In the latest publication from the team, they show how otherwise identifical proteins with different glycosylation patterns can be identified and discriminated between. This is achieved by using antibodies immobilised on biolayer interferometry sensors which can first target all glycoforms (and hence are are not specific). In a second step, gold nanoparticles labelled with lectins (carbohydrate binding proteins) are used to identify which glycoform is present, and due to the large mass of the gold particles leads to signal enhancement. This is demonstrated for prostate specific antigen - a key biomarker for prostate diseases including cancer. It is known that the glycosylation pattern, not just protein concentration, is a hallmark of disease state but current techniques do not distinguish glycoforms. The method shown her can be automated and takes < 90 minutes to complete in this proof of concept study.
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3-D tissue models (such as spheroids and organoids) better predict physiological responses than 2D monolayers and may play a role in reducing animal usage, particularly in toxicology. Spheroids are more challenging, however, to work with than cell monolayers and hence there is a barrier to their use. Spheroids can also not (always) be easily cryopreserved and hence buying them ‘off the shelf’ and ‘ready to use’ is not common or is expensive. The GibsonGroup, working with Cryologyx, have show in this latest work that their macromolecular cryoprotectants can protect live cell spheroids during cryopreservation allowing the recovery of viable spheroids direct from the freezer.
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