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Biology subjects

Harris, A.

Publications and source records attributed to Harris, A..

34 records · Page 2Linked to original sources

Fracture in Living Cell Monolayers

The ability of tissues to sustain mechanical stress and avoid rupture is a fundamental pillar of their function. Rupture in response to physiological levels of stress can be undesired, for example resulting from disease or genetic mutations, or be an integral part of developmental processes, such as during blastocoel formation in mouse or leg eversion in flies. Despite its importance, we know very little about rupture in cellularised tissues because it is a multi-scale phenomenon that necessitates comprehension of the interplay between mechanical forces and processes at the molecular and cellular scales. Using a combination of mechanical measurements, live imaging and computational modelling, we characterise rupture in epithelial monolayers. We show that, despite consisting of only a single layer of cells, monolayers can withstand surprisingly large deformations, often accommodating several-fold increases in their length before rupture. At large deformation, epithelia increase their stiffness multiple-fold in a process controlled by a supracellular network of keratin filaments. Perturbing keratin organisation fragilised monolayers and prevented strain stiffening. Using computational approaches, we show that, although the kinetics of adhesive bond rupture ultimately control tissue strength, tissue rheology and the history of deformation prior to failure set the strain and stress that the tissue reaches at the onset of fracture. Our data paint a picture of epithelia as versatile materials that combine resistance to shocks with deformability when subjected to low strain rates.

biophysics↗

Quantum analysis of P3a and P3b from auditory single trial ERPs differentiates borderline personality disorder from schizophrenia

Traditional approaches to EEG modelling use the methods of classical physics to reconstruct scalp potentials in terms of explicit physical models of cortical neuron ensembles. The principal difficulty is that the multiplicity of cellular processes with an intricate array of deterministic and random factors prevents creation of consistent biophysical parameter sets. An original, empirically-testable solution has been recently achieved in our previous studies by a radical departure from the deterministic equations of classical physics to the probabilistic reasoning of quantum mechanics. This crucial step relocates elementary bioelectric sources of EEG signals from the cellular to the molecular level where positively and negatively ions are considered as elementary sources of electricity. The rationale is that despite dramatic differences in cellular machineries, statistical factors governed by the rules of central limit theorem produce EEG waveforms as a statistical aggregate of the synchronized activity of multiple closely-located microscale sources. Using the formalism of nonhomogeneous birth-and-death processes (BDP) the quantum models of microscale events are deduced and linked to the dynamics of macroscale EEG waveforms. This study expands these methods with new features for comprehensive analysis of event related potentials directly from single trials, i.e. the EEG segments which are closely related in timing to cognitive events. We derive a universal model of the components of single trial ERPs both in frequency and time domains. This, for the first time, enables us to quantify all significant cognitive components in single trial ERPs, providing an alternative to the traditional method of averaging. Given P300 as an important objective marker of psychiatric disorders, a methodology which reliably discloses the component compositions of this potential, may have specific diagnostic importance. In this study, reliable identification of the P3a and P3b components from an auditory oddball paradigm provided a means of differentiating borderline personality disorder from schizophrenia.

neuroscience↗

A genomic timescale for placental mammal evolution

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signal using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic timetrees reveal an accumulation of cladogenic events before and immediately following the KPg boundary implying important roles for Cretaceous continental vicariance and the KPg extinction in the placental radiation. One-Sentence SummaryA comprehensive whole genome phylogeny of extant placental mammals reveals timing and patterns of ordinal diversification.

evolutionary biology↗

RNA-seq sample preparation kits strongly affect transcriptome profiles of a gas-fermenting bacterium

Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. This rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation such as ribosomal RNA (rRNA) depletion and first-strand cDNA synthesis are tailored to a specific group of organisms (e.g. eukaryotes vs. prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products by NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand-specificity, and, most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies. ImportanceRNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. Our work is important as we show that RNA-seq library preparation kits can strongly affect the outcome of the RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investment infeasible to most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting of sample processing workflows for any given organism.

systems biology↗

Eight week dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology, increases oxidative stress, alters metabolism and causes gut dybiosis in APCmin mice

Colorectal cancer (CRC) is the second most prevelant malignancy in Europe and diet is an important modifiable risk factor. Processed meat consumption, including meats with preservative salts such as sodium nitrite, have been implicated in CRC pathogenesis. This study investigated how the CRC pathology and metabolic status of adenomatous polyposis coli (APC) multiple intestinal neoplasia (min) mice was perturbed following 8 weeks of pork meat consumption. Dietary inclusions (15%) of either nitrite-free pork, nitrite-free sausage or nitrite-containing sausage (frankfurter) were compared against a parallel control group (100% chow). Comprehensive studies investigated: gastrointestinal tract histology (tumours, aberrant crypt foci (ACF) and mucin deplin foci (MDF), lipid peroxidation (urine and serum), faecal microbiota and serum metabolomics (599 metabolites). After 8 weeks mice consuming the frankfurter diet had 53% more (P=0.014) gastrointestinal tumours than control, although ACF and MDF did not differ. Urine and serum lipid peroxidation markers were 59% (P=0.001) and 108% (P=0.001) higher, respectively in the frankfurter group. Gut dysbiosis was evident in these mice with comparably fewer Bacteriodes and more Firmicutes. Fasting serum levels of trimethylamine N-oxide (TMAO) and numerous triglycerides were elevated. Various serum phosphotidylcholine species were decreased. These results demonstrate that nitrite-containing sausages may exaccerbate the development of CRC pathology in APCMin mice to a greater extent than nitrite-free sausages, and this is associated with greater lipid peroxidation, wide-ranging metabolic alternation and gut dysbiosis.

biochemistry↗

Faster growth enhances low carbon fuel and chemical production through gas fermentation

Gas fermentation offers both fossil carbon-free sustainable production of fuels and chemicals and recycling of gaseous and solid waste using gas-fermenting microbes. Bioprocess development, systems-level analysis of biocatalyst metabolism, and engineering of cell factories are advancing the widespread deployment of the commercialised technology. Acetogens are particularly attractive biocatalysts but effects of the key physiological parameter - specific growth rate () - on acetogen metabolism and the gas fermentation bioprocess have not been established yet. Here, we investigate the -dependent bioprocess performance of the model-acetogen Clostridium autoethanogenum in CO and syngas (CO+CO2+H2) grown chemostat cultures and assess systems-level metabolic responses using gas analysis, metabolomics, transcriptomics, and metabolic modelling. We were able to obtain steady-states up to ~2.8 day-1 (~0.12 h-1) and show that faster growth supports both higher yields and productivities for reduced by-products ethanol and 2,3-butanediol. Transcriptomics data revealed differential expression of 1,337 genes with increasing and suggest that C. autoethanogenum uses transcriptional regulation to a large extent for facilitating faster growth. Metabolic modelling showed significantly increased fluxes for faster growing cells that were, however, not accompanied by gene expression changes in key catabolic pathways for CO and H2 metabolism. Cells thus seem to maintain sufficient "baseline" gene expression to rapidly respond to CO and H2 availability without delays to kick-start metabolism. Our work advances understanding of transcriptional regulation in acetogens and shows that faster growth of the biocatalyst improves the gas fermentation bioprocess.

systems biology↗

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

microbiology↗

Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage

The connexin family is a diverse group of highly regulated non-{beta}-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3,5-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 s simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 s of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner. Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-{beta}-barrel wide-pore channels.

biophysics↗

Tumor irradiation combined with vascular-targeted photodynamic therapy enhances anti-tumor effects in preclinical prostate cancer.

RationaleThere is an important clinical need to improve the treatment of high risk localized and locally advanced prostate cancer (PCa), and to reduce the side effects of these treatments. We hypothesized that multi-modality therapy combining radiotherapy and vascular-targeted photodynamic therapy (VTP) could PCa tumour control compared against monotherapy with each of these treatments alone. This could provide proof-of-concept to take to the clinic. VTP is a focal therapy for localized PCa, which rapidly destroys targeted tumors through vascular disruption. Tumor vasculature is characterized by vessel immaturity, increased permeability, aberrant branching and inefficient flow. Fractionated radiotherapy (FRT) alters the tumor microenvironment and promotes transient vascular normalization. ObjectiveWe investigated whether sequential delivery of FRT followed by VTP 7 days later improves PCa tumor control compared to monotherapy with FRT or VTP alone. FindingsFRT induced vascular normalization changes in PCa flank tumor allografts, improving vascular function as demonstrated using dynamic contrast enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumor growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP alone, and improved overall survival. ConclusionTaken together, these results suggest that combining FRT and VTP could become a promising multimodal clinical strategy in PCa therapy. This provides proof-of-concept for this multi-modality therapy approach to take forward to early phase clinical trials.

cancer biology↗

Hippocampal place cells encode global location but not changes in environmental connectivity in a 4-room navigation task

Flexible navigation relies on a cognitive map of space, thought to be implemented by hippocampal place cells: neurons that exhibit location-specific firing. In connected environments, optimal navigation requires keeping track of ones location and of the available connections between subspaces. We examined whether the dorsal CA1 place cells of rats encode environmental connectivity in four geometrically-identical boxes arranged in a square. Rats moved between boxes by pushing saloon-type doors that could be locked in one or both directions. While rats demonstrated knowledge of environmental connectivity, their place cells did not respond to connectivity changes, nor did they represent doorways differently from other locations. Importantly, place cells coded the space in a global frame, expressing minimal repetitive fields despite the repetitive geometry (global coding). These results suggest that CA1 place cells provide a spatial map that does not explicitly include connectivity.

neuroscience↗

GABA AND GLUTAMATE CHANGES IN PEDIATRIC MIGRAINE

Despite migraine being one of the top five most prevalent childhood diseases, a lack of knowledge about pediatric migraine limits effective treatment strategies; standard adult pharmaceutical therapies are less effective in children and can carry undesirable side-effects. Non-pharmacological therapies have shown some success in adults; however, to appropriately apply these in children we need to understand pediatric migraines underlying biology. One theory is that migraine results from an imbalance in cortical excitability. Magnetic resonance spectroscopy (MRS) studies show changes in GABA and glutamate levels (the primary inhibitory and excitatory neurotransmitters in the brain, respectively) in multiple brain regions. Although there is indirect evidence of abnormal excitability in pediatric migraine, GABA and glutamate levels have yet to be assessed. The purpose of this study was to measure levels of GABA and glutamate in the thalamus, sensorimotor cortex and visual cortex of children with migraine using MRS. We found that children with migraine and aura had significantly lower glutamate levels in the visual cortex as compared to control children, opposite to results seen in adults. Additionally, we found significant correlations between metabolite levels and migraine characteristics; higher GABA levels were associated with a higher migraine burden. We also found that higher glutamate in the thalamus and higher GABA/Glx ratios in the sensorimotor cortex were associated with duration since diagnosis, i.e., having migraines longer. Lower GABA levels in the sensorimotor cortex were associated with being closer to their next migraine attack. Together this indicates that GABA and glutamate disturbances occur early in migraine pathophysiology and emphasizes that evidence from adults with migraine cannot be immediately translated to paediatric sufferers. This highlights the need for further mechanistic studies of migraine in children, to aid in the development of more effective treatments.

neuroscience↗

Biofluid modeling of the coupled eye-brain system and insights into simulated microgravity conditions

This work aims at investigating the interactions between the flow of fluids in the eyes and the brain and their potential implications in the development of visual impairment in astronauts, a condition also known as spaceflight associated neuro-ocular syndrome (SANS). To this end, we propose a reduced (0-dimensional) mathematical model of fluid flow in the eyes and brain, which is embedded into a simplified whole-body circulation model. In particular, the model accounts for: (i) the flows of blood and aqueous humor in the eyes; (ii) the flows of blood, cerebrospinal fluid and interstitial fluid in the brain; and (iii) their interactions. The model is used to simulate variations in intraocular pressure, intracranial pressure and blood flow due to microgravity conditions, which are thought to be critical factors in SANS. Specifically, the model predicts that both intracranial and intraocular pressures increase in microgravity, even though their respective trends may be different. In such conditions, ocular blood flow is predicted to decrease in the choroid and ciliary body circulations, whereas retinal circulation is found to be less susceptible to microgravity-induced alterations, owing to a purely mechanical component in perfusion control associated with the venous segments. These findings indicate that the particular anatomical architecture of venous drainage in the retina may be one of the reasons why most of the SANS alterations are not observed in the retina but, rather, in other vascular beds, particularly the choroid. Thus, clinical assessment of ocular venous function may be considered as a determinant SANS factor, for which astronauts could be screened on earth and in-flight.

physiology↗

BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors

Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, BRCA2 inactivation in tumors is associated with uncontrolled cell proliferation. We set out to investigate this conundrum by exploring modalities of cell adaptation to loss of BRCA2 and focused on genome-wide transcriptome alterations. Human cells in which BRCA2 expression was inhibited using a doxycycline (DOX)-inducible shRNA for 4 or 28 days were subjected to RNA-seq analyses. Gene sets differentially expressed in BRCA2-deficient versus -proficient cells revealed a biphasic response to BRCA2 abrogation. The early, acute response consisted of downregulation of genes involved in cell cycle progression, DNA replication and repair and was associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consisted predominantly of upregulation of innate immune response genes controlled by interferon. Activation of the cGAS-STING pathway detected in these cells further substantiated the concept that long-term BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we found that treatment with PARP inhibitors stimulated the interferon response in cells and tumors lacking BRCA2. We propose that PARP inhibitors may suppress growth of BRCA2-deficient cells and tumors, in part, by activating interferon signaling.

cancer biology↗

Onecut factors and Pou2f2 regulate diversification and migration of V2 interneurons in the mouse developing spinal cord

Acquisition of proper neuronal identity and position is critical for the formation of neural circuits. In the embryonic spinal cord, cardinal populations of interneurons diversify into specialized subsets and migrate to defined locations within the spinal parenchyma. However, the factors that control interneuron diversification and migration remain poorly characterized. Here, we show that the Onecut transcription factors are necessary for proper diversification and distribution of the V2 interneurons in the developing spinal cord. Furthermore, we uncover that these proteins restrict and moderate the expression of spinal isoforms of Pou2f2, a transcription factor known to regulate B-cell differentiation. By gain- or loss-of-function experiments, we show that Pou2f2 contribute to regulate the position of V2 populations in the developing spinal cord. Thus, we uncovered a genetic pathway that regulates the diversification and the distribution of V2 interneurons during embryonic development.\n\nSignificance statementIn this study, we identify the Onecut and Pou2f2 transcription factors as regulators of spinal interneuron diversification and migration, two events that are critical for proper CNS development.

developmental biology↗

Accounting for taste: A multi-attribute neurocomputational model explains the neural dynamics of choices for self and others

How do we make choices for others with different preferences from our own? Although neuroimaging studies implicate similar circuits in representing preferences for oneself and others, some models propose that additional corrective mechanisms come online when choices for others diverge from ones own preferences. Here we used event-related potentials (ERP) in humans, in combination with computational modeling, to examine how social information is integrated in the time leading up to choices for oneself and others. Hungry male and female participants with unrestricted diets selected foods for themselves, a similar unrestricted eater, and a dissimilar, self-identified healthy eater. Across choices for both oneself and others, ERP value signals emerged within the same time window but differentially reflected taste and health attributes based on the recipients preferences. Choices for the dissimilar recipient were associated with earlier activity localized to brain regions implicated in social cognition including temporoparietal junction (TPJ). Finally, response-locked analysis revealed a late ERP component specific to choices for the similar recipient, localized to the parietal lobe, that appeared to reflect differences in the response threshold based on uncertainty. A multi-attribute computational model supported the link between specific ERP components and distinct model parameters, and was not significantly improved by adding time-dependent dual processes. Model simulations suggested that longer response times (RTs) previously associated with effortful correction may alternatively arise from higher choice uncertainty. Together these results provide a parsimonious neurocomputational mechanism for social decision-making, additionally explaining divergent patterns of choice and RT data in decisions for oneself and others.\n\nSignificance StatementHow do we choose for others, particularly when they have different preferences? Whereas some studies suggest that similar neural circuits underlie decision-making for oneself and others, others argue for additional, slower perspective-taking mechanisms. Combining event-related potentials with computational modeling, we found that integration of others preferences occurs over the same timescale as for oneself, while differentially tracking recipient-relevant attributes. Although choosing for others took longer and produced differences in late-emerging neural responses, computational modeling attributed these patterns to greater response caution rather than egocentric bias correction. Computational simulations also correctly predicted when and why choosing differently for others takes longer, suggesting that a model incorporating value integration and evidence accumulation can parsimoniously account for complex patterns in social decision-making.

neuroscience↗

Stress relaxation in epithelial monolayers is controlled by actomyosin

Epithelial monolayers are one-cell thick tissue sheets that separate internal and external environments. As part of their function, they withstand extrinsic mechanical stresses applied at high strain rate. However, little is known about how monolayers respond to mechanical deformations. In stress relaxation tests, monolayers respond in a biphasic manner and stress dissipation is accompanied by an increase in monolayer resting length, pointing to active remodelling of cell architecture during relaxation. Consistent with this, actomyosin remodels at a rate commensurate with mechanical relaxation and governs the rate of monolayer stress relaxation - as in single cells. By contrast, junctional complexes and intermediate filaments form stable connections between cells, enabling monolayers to behave rheologically as single cells. Together, these data show actomyosin cytoskeletal dynamics govern the rheological properties of monolayers by enabling active, ATP-dependent changes in the resting length. These findings have far-reaching consequences for our understanding of developmental morphogenesis and tissue response to mechanical stress.

biophysics↗