Biology subjects

Jones, D.

Publications and source records attributed to Jones, D..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Junctophilin-2 promotes cardiomyocyte survival by blocking MURF1-mediated Junctin ubiquitination and proteasome-dependent degradation

AimsJunctophilin-2 is required for the development, maturation and integrity of the t-tubule system and the gating stability of RyR2 in cardiomyocytes. This study investigated whether and how junctophilin-2 maintained junctin, a scaffold protein stabilizing RyR2, to prevent cardiomyocyte death under stress. MethodsCardiomyocytes were exposed to conditions of stress including palmitate, doxorubicin, or hypoxia/re-oxygenation. Adenoviral vectors were employed to manipulate expression of junctophilin-2 and junctin in cardiomyocytes. Molecular/cellular/biochemical analyses were conducted. ResultsDifferent conditions of stress decreased junctophilin-2 expression through aberrant autophagy and concomitantly induced a reduction of junctin protein in cardiomyocytes. Over-expression of junctophilin-2 preserved the protein levels of junctin and attenuated cytosolic Ca2+ and apoptosis in cardiomyocytes under stress. Knockdown of junctophilin-2 reproduced the detrimental phenotypes of stress in cardiomyocytes. Notably, over-expression of junctin prevented cardiomyocyte death under stress whereas knockdown of junctin offset the protective effects conferred by junctophilin-2 over-expression. Mechanistically, junctophilin-2 blocked MURF1-junctin interaction thereby preventing junctin ubiquitination and proteasome-dependent degradation. Mass spectrometry analysis identified multiple ubiquitination sites on the junctin protein and the non-ubiquitinated junctin mutant (K8A/K102A/K107A/K140A) was resistant to degradation. ConclusionsThis study uncovers an unrecognized role of junctophilin-2 in preventing junctin ubiquitination and degradation in maintaining cytosolic Ca2+ homeostasis. Both junctophilin-2 and junctin represent two new survival factors of cardiomyocytes and thus, may be new therapeutic targets for cardiac protection.

molecular biology↗

bioRxiv2022

Distinct Neutralizing Antibody Escape of SARS-CoV-2 Omicron Subvariants BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2

Continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants, including BQ.1, BQ. 1.1, BA.4.6, BF.7 and BA.2.75.2. Here we examine the neutralization resistance of these subvariants, as well as their ancestral BA.4/5, BA.2.75 and D614G variants, against sera from 3-dose vaccinated health care workers, hospitalized BA.1-wave patients, and BA.5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially the BQ.1 and BQ.1.1 subvariants driven by a key N460K mutation, and to a lesser extent, R346T and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its F486S mutation. The BQ.1 and BQ.1.1 subvariants also exhibited enhanced fusogenicity and S processing dictated by the N460K mutation. Interestingly, the BA.2.75.2 subvariant saw an enhancement by the F486S mutation and a reduction by the D1199N mutation to its fusogenicity and S processing, resulting in minimal overall change. Molecular modelling revealed the mechanisms of receptor-binding and non-receptor binding monoclonal antibody-mediated immune evasion by R346T, K444T, F486S and D1199N mutations. Altogether, these findings shed light on the concerning evolution of newly emerging SARS-CoV-2 Omicron subvariants.

microbiology↗

bioRxiv2022

Cannabidiol targets a modulatory system for excitatory-inhibitory synaptic coordination, contributing to its anti-seizure action

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsy, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3[->]CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABAAR{gamma}2 and gephyrin puncta. Effects of LPI at both excitatory and inhibitory synapses were eliminated by CBD pretreatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated levels of GPR55 and LPI, and chronic lithium pilocarpine-induced epileptogenesis potentiated the pro-excitatory effects of LPI. We propose that CBD exerts potential therapeutic effect both by blocking synaptic effects of LPI and dampening hyperexcitability.

neuroscience↗

bioRxiv2022

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here we show glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its activity and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRPs thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its non-glycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data shows that glycosylation does not just have a passive effect on HRP stability but can exert long range effects that mediate the native enzymes activity and stability through changes in inherent dynamics.

biochemistry↗

bioRxiv2022

Disruption of lysosomal proteolysis in astrocytes facilitates midbrain proteostasis failure in an early-onset PD model.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompany cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early on-set Parkinsons Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, -synuclein (-syn) phosphorylation to increase, and proteins to aggregate. These processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in PD.

neuroscience↗

bioRxiv2022

Optimisation and Pre-clinical Demonstration of Temporal Diffusion Ratio for Imaging Restricted Diffusion

Temporal Diffusion Ratio (TDR) is a recently proposed dMRI technique (DellAcqua, 2019) which provides contrast between areas with restricted diffusion and areas either without restricted diffusion or with length scales too small for characterisation. Hence, it has a potential for mapping pore sizes, in particular large axon diameters or other cellular structures. TDR employs the signal from two dMRI acquisitions obtained with the same, large, b-value but with different diffusion times and gradient settings. TDR is advantageous as it employs standard acquisition sequences, does not make any assumptions on the underlying tissue structure and does not require any model fitting, avoiding issues related to model degeneracy. This work for the first time optimises the TDR diffusion sequences in simulation for a range of different tissues and scanner constraints. We extend the original work (which considers substrates containing cylinders) by additionally considering the TDR signal obtained from spherical structures, representing cell soma in tissue. Our results show that contrasting an acquisition with short gradient duration and short diffusion time with an acquisition with long gradient duration and long diffusion time improves the TDR contrast for a wide range of pore configurations. Additionally, in the presence of Rician noise, computing TDR from a subset (50% or fewer) of the acquired diffusion gradients rather than the entire shell as proposed originally further improves the contrast. In the last part of the work the results are demonstrated experimentally on rat spinal cord. In line with simulations, the experimental data shows that optimised TDR improves the contrast compared to non-optimised TDR. Furthermore, we find a strong correlation between TDR and histology measurements of axon diameter. In conclusion, we find that TDR has great potential and is a very promising alternative (or potentially complement) to model-based approaches for mapping pore sizes and restricted diffusion in general. HighlightsO_LITemporal Diffusion Ratio (TDR) 2-seq approach maps areas with restricted diffusion C_LIO_LIOptimised gradient waveform pair is: long {delta} + low G and short {delta} + high G C_LIO_LIIf data is noisy calculating TDR using HARDI acquisition subsets increases accuracy C_LIO_LIWe demonstrate TDR for the first time pre-clinically in rat spinal cord C_LIO_LIPre-clinical TDR values are strongly correlated with axon diameter C_LI

bioengineering↗

bioRxiv2022

Genome-wide analysis of dendritic cell differentiation

Dendritic cells (DCs) are immune sentinel cells that comprise antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs). Cytokine Flt3 ligand (Flt3L) supports the proliferation of hematopoietic progenitors, and is also necessary and sufficient for DC differentiation. Here we characterized the spontaneous differentiation of a Flt3L-dependent murine progenitor cell line into pDCs and "myeloid" cDCs (cDC2s), and interrogated it using a genome-wide CRISPR/Cas9 dropout screen. The screen revealed multiple regulators of DC differentiation including the glycosylphosphatidylinositol transamidase complex, the Nieman-Pick type C cholesterol transporter and arginine methyltransferase Carm1; the role of Carm1 in pDC and cDC2 differentiation was confirmed by conditional targeting in vivo. We also found that negative regulators of mTOR signaling, including the subunits of TSC and GATOR1 complexes, restricted progenitor growth but enabled DC differentiation. The results provide a comprehensive forward genetic analysis of DC differentiation, and help explain how the opposing processes of proliferation and differentiation could be driven by the same cytokine.

immunology↗

bioRxiv2022

The arthropod associates of 155 North American cynipid oak galls

The identities of most arthropod associates of cynipid-induced oak galls in the western Palearctic are generally known. However, a comprehensive accounting of associates has been performed for only a small number of the galls induced by the estimated 700 species of cynipid gall wasp in the Nearctic. This gap in knowledge stymies many potential studies of diversity, coevolution, and community ecology, for which oak gall systems are otherwise ideal models. We report rearing records of insects and other arthropods from more than 527,306 individual galls representing 201 different oak gall types collected from 32 oak tree species in North America. Of the 201 gall types collected, 155 produced one or more animals. A total of 151,075 animals were found in association with these 155 gall types, and of these 61,044 (40.4%) were gall wasps while 90,031 (59.6%) were other arthropods. We identified all animals to superfamily, family, or, where possible, to genus. We provide raw numbers and summaries of collections, alongside notes on natural history, ecology, and previously published associations for each taxon. For eight common gall-associated genera (Synergus, Ceroptres, Euceroptres, Ormyrus, Torymus, Eurytoma, Sycophila, and Euderus), we also connect rearing records to gall wasp phylogeny, geography, and ecology - including host tree and gall location (host organ), and their co-occurrence with other insect genera. Though the diversity of gall wasps and the large size of these communities is such that many Nearctic oak gall-associated insects still remain undescribed, this large collection and identification effort should facilitate the testing of new and varied ecological and evolutionary hypotheses in Nearctic oak galls.

zoology↗

bioRxiv2021

Multiplexed Single-Molecule Epigenetic Analysis of Plasma-Isolated Nucleosomes for Cancer Diagnostics

The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine, providing information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed EPINUC, a single-molecule multi-parametric assay to comprehensively profile the Epigenetics of Plasma Isolated Nucleosomes, DNA methylation and cancer-specific protein biomarkers. Our system allows high-resolution detection of six active and repressive histone modifications, their ratios and combinatorial patterns, on millions of individual nucleosomes by single-molecule imaging. In addition, it provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins such as mutant p53. Applying this analysis to a cohort of plasma samples detected colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue-of-origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multi-layered clinical-relevant information from limited liquid biopsy material, establishing a novel approach for cancer diagnostics.

cancer biology↗

bioRxiv2021

The mechanobiology of craniosynostosis in Crouzon syndrome

Children with syndromic forms of craniosynostosis undergo a plethora of surgical interventions to resolve the clinical features caused by the premature fusion of cranial sutures. While surgical correction is reliable, the need for repeated rounds of invasive treatment puts a heavy burden on the child and their family. This study explores a non-surgical alternative using mechanical loading of the cranial joints to prevent or delay craniofacial phenotypes associated with Crouzon syndrome. We treated Crouzon syndrome mice before the onset of craniosynostosis by cyclical mechanical loading of cranial joints using a custom designed set-up. Cranial loading applied to the frontal bone partially restores normal skull morphology, significantly reducing the typical brachycephalic appearance. This is underpinned by the delayed closure of the coronal suture and of the intersphenoidal synchondrosis. This study provides a novel treatment alternative for syndromic craniosynostosis which has the potential to be an important step towards replacing, reducing or refining the surgical treatment of all craniosynostosis patients.

biophysics↗

bioRxiv2021

Somatic mutation rates scale with lifespan across mammals

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including [~]30-fold variation in lifespan and [~]40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of [~]3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

genomics↗

bioRxiv2021

H3-K27M-Mutant Nucleosomes Interact with MLL1 to Shape the Glioma Epigenetic Landscape

Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate Polycomb Repressive Complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2, but also directly interact with MLL1, thus leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of both repressive and active chromatin marks leads to unbalanced bivalent chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.

cancer biology↗

bioRxiv2021

The heterogeneity of the DNA damage response landscape determines patient outcomes in ovarian cancer

Defective DNA damage response (DDR) pathways allow cancer cells to accrue genomic aberrations and evade normal cellular growth checkpoints. Defective DDR also determines response to chemotherapy. However, the interaction and overlap between the two double strand repair pathways and the three single strand repair pathways is complex, and has remained poorly understood. Here we show that, in ovarian cancer, a disease hallmarked by chromosomal instability, explant cultures show a range of DDR abrogation patterns. Defective homologous recombination (HR) and non-homologous end joining (NHEJ) are near mutually exclusive with HR deficient (HRD) cells showing increased abrogation of the single strand repair pathways compared to NHEJ defective cells. When combined with global markers of DNA damage, including mitochondrial membrane functionality and reactive oxygen species burden, the pattern of DDR abrogation allows the construction of DDR signatures which are predictive of both ex vivo cytotoxicity, and more importantly, patient outcome. SignificanceHolistic assessment of the DDR is possible, shows improved ability to predict response to chemotherapy over single pathway assessment, and is applicable to a variety of ovarian cancer types. Such an assessment has clinical utility in settings of therapeutic dilemma such as retreatment for relapse.

cell biology↗

bioRxiv2021

Structure and dynamics of a cold-active esterase reveals water entropy and active site accessibility as the likely drivers for cold-adaptation

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical /{beta} hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests the EstN7 cap region is proportionally less flexible. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7s cold adaptation rather than changes in dynamics.

biochemistry↗

bioRxiv2021

Structure guided engineering of a cold active esterase expands substrate range though a stabilisation mutation that allows access to a buried water chamber

Cold active esterases represent an important class of enzymes capable of undertaking useful chemical transformations at low temperatures. EstN7 from Bacillus cohnii represents a true psychrophilic esterase with a temperature optimum below 20{degrees}C. We have recently determined the structure of EstN7 and have used this knowledge to understand substrate specificity and expands its substrate range through protein engineering. Substrate range is determined by a plug at the end of acyl binding pocket that blocks access to a buried water filled cavity, so limiting EstN7 to turnover of C2 and C4 substrates. Data mining revealed a potentially important commercial reaction, conversion of triacetin to only the 1,2-glyceryl diacetate isomer, which the EstN7 could achieve. Residues M187, N211 and W206 were identified as plug residues. M187 was identified as the key plug residue but mutation to alanine destabilised the structure as whole. Another plug mutation, N211A had a stabilising effect on EstN7 and suppressed the destabilising M187A mutation. The M187A-N211A variant had the broadest substrate range, capable of hydrolysing a C8 substrate. Thus, the structure of EstN7 together with focused engineering has provided new insights into the structural stability and substrate specificity that allowed expansion of substrate range.

biochemistry↗

bioRxiv2021

Extending the Near Infrared Emission Range of Indium Phosphide Quantum Dots for Multiplexed In Vivo Imaging

This report of the reddest emitting indium phosphide quantum dots (InP QDs) to date demonstrates tunable, near infrared (NIR) photoluminescence and fluorescence multiplexing in the first optical tissue window with a material that avoids toxic constituents. This synthesis overcomes the InP synthesis "growth bottleneck" and extends the emission peak of InP QDs deeper into the first optical tissue window using an inverted QD heterostructure. The ZnSe/InP/ZnS core/shell/shell structure is designed to produce emission from excitons with heavy holes confined in InP shells wrapped around larger-bandgap ZnSe cores and protected by a second shell of ZnS. The InP QDs exhibit InP shell thickness-dependent tunable emission with peaks ranging from 515 - 845 nm. The high absorptivity of InP leads to effective absorbance and photoexcitation of the QDs with UV, visible, and NIR wavelengths in particles with diameters of eight nanometers or less. These nanoparticles extend the range of tunable direct-bandgap emission from InP-based nanostructures, effectively overcoming a synthetic barrier that has prevented InP-based QDs from reaching their full potential as NIR imaging agents. Multiplexed lymph node imaging in a mouse model shows the potential of the NIR-emitting InP particles for in vivo imaging.

bioengineering↗

bioRxiv2021

Trophic position derived from amino-acid nitrogen isotopes reflect physiological status of both predator and prey over four decades

Nitrogen isotope analyses of amino-acids ({delta}15N-AA) are increasingly used to decipher food webs. Interpretation of {delta}15N-AA in consumers relies on the assumption that physiological status has a negligible influence on the trophic enrichment factor (TEF). Recent experiments have shown that this is not always the case and there is a need to validate derived trophic position (TP) estimates using ecological data. We analyzed {delta}15N-AA in cod and herring (1980-2019) from the Baltic Sea, a species-poor system where dramatic reduction in condition status of cod has occurred. We expected that TEFcod-herring in trophic AAs would increase during periods of poor cod growth, resulting in inflated TP estimates. We found that TEF and TP estimates were negatively linked to individual condition status, prey fat content and the hypoxic state of the ecosystem. Statistically adjusting for these variables resulted in lower cod TP, highlighting the importance of including ecological knowledge when interpreting TP. Scientific Significance StatementNitrogen stable isotope analyses in amino acids are increasingly used in ecology to understand how environmental change impacts food-webs. Specifically, it is used to more accurately calculate trophic position (TP) of consumers. Controlled experiments have shown that physiological status can alter amino acid isotope composition and TP interpretation, but field studies are lacking. We use 40 years of archived material to demonstrate that TP estimates in Baltic Sea cod and its prey herring are directly related to physiological status. This has important implications for interpreting the real trophic ecology of consumers under environmental stress. By simultaneously measuring condition status in both predator and prey it is possible to adjust for them as confounding variables and decipher actual consumer TP.

ecology↗

bioRxiv2021

Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio

Following the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).

genomics↗