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Robinson, A.

Publications and source records attributed to Robinson, A..

At least 37 records · Page 2Linked to original sources
bioRxiv2023

Spatiotemporal dynamics of single-stranded DNA intermediates in Escherichia coli

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

biophysics↗
bioRxiv2022

Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test

Neutralization assays are important in understanding and quantifying neutralizing antibody responses towards SARS-CoV-2. The SARS-CoV-2 Lentivirus Surrogate Neutralization Assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable, alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and pre-screening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity produced acceptable results with 100% (95% CI: 94-100) specificity and 100% (95% CI: 94-100) sensitivity against ancestral Wuhan spike pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike pseudotyped lentivirus resulted in 88.3% (95% CI: 77.8 to 94.2) and 100% (95% CI: 94-100), respectively. Assay precision measuring intra-assay variability produced acceptable results for High (1:[≥] 640 PRNT50), Mid (1:160 PRNT50) and Low (1:40 PRNT50) antibody titer concentration ranges based on the PRNT50, with %CV of 14.21, 12.47, and 13.28 respectively. Intermediate precision indicated acceptable ranges for the High and Mid concentrations, with %CV of 15.52 and 16.09, respectively. However, the Low concentration did not meet the acceptance criteria with a %CV of 26.42. Acceptable ranges were found in the robustness evaluation for both intra-assay and inter-assay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT.

scientific communication and education↗
bioRxiv2022

Single neurons and networks in the claustrum integrate input from widespread cortical sources

The claustrum is thought to be one of the most highly interconnected forebrain structures but its organizing principles have yet to be fully explored at the level of single neurons. Here, we investigated the identity, connectivity, and activity of identified claustrum neurons to understand how the structures unique convergence of input and divergence of output support binding information streams. We found that neurons in the claustrum communicate with each other across efferent projection-defined modules which were differentially innervated by sensory and frontal cortical areas. Individual claustrum neurons were responsive to inputs from more than one cortical region in a cell-type and projection-specific manner, particularly between areas of frontal cortex. In vivo imaging of claustrum axons revealed responses to both unimodal and multimodal sensory stimuli. Finally, chronic claustrum silencing specifically reduced animals sensitivity to multimodal stimuli. These findings support the view that the claustrum is a fundamentally integrative structure, consolidating information from around the cortex and redistributing it following local computations.

neuroscience↗
bioRxiv2022

RET Signaling Persists in the Adult Intestine and Stimulates Motility by Limiting PYY Release from Enteroendocrine Cells

Background & AimsRET receptor tyrosine kinase is necessary for enteric nervous system (ENS) development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, HSCR patients often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. MethodsRetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET signaling in these cells regulates adult gut motility in vivo, genetic and pharmacologic approaches were used to disrupt RET in either all RET-expressing cells, a major subset of enteric neurons, or intestinal epithelial cells. ResultsDistinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the intestinal epithelium, rather than in enteric neurons, slowed GI motility selectively in adult male mice. This effect was phenocopied by RET kinase inhibition. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin (5-HT) or L-cells that release peptide YY (PYY), both of which can alter motility. RET kinase inhibition exaggerated PYY release in a nutrient-dependent manner without altering 5-HT secretion. PYY receptor blockade fully rescued dysmotility in mice lacking epithelial RET. ConclusionRET signaling normally limits nutrient-dependent PYY release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to post-operative dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/488514v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@4c8e83org.highwire.dtl.DTLVardef@204d13org.highwire.dtl.DTLVardef@a429d8org.highwire.dtl.DTLVardef@1b75265_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗
bioRxiv2022

Superfast Evolution of Multi-drug Antibiotic Resistance

The killing mechanism of many antibiotics involves the induction of DNA damage, either directly or indirectly, which activates the SOS response. RecA, the master regulator of the SOS response, has shown to play a central role in the evolution of resistance to fluoroquinolones, even after short-term exposure. While this paradigm is well established for DNA-damaging antibiotics, it remains unclear whether {beta}-lactams elicit similar resistance dynamics or depend on RecA and SOS-mediated mechanisms. In this study, we observed a rapid and stable evolution of {beta}-lactam resistance (20-fold MIC increase within 8 hours) in Escherichia coli lacking RecA after a single exposure to ampicillin. Contrary to expectation, this resistance emerged through an SOS-independent mechanism involving two distinct evolutionary forces: increased mutational supply and antibiotic-driven selection. Specifically, we found that RecA deletion impaired DNA repair and downregulated base excision repair pathways, while concurrently repressing the transcription of antioxidative defence genes. This dual impairment led to excessive accumulation of reactive oxygen species (ROS), which in turn promoted the emergence of resistance-conferring mutations. While ampicillin treatment did not alter survival, it selectively enriched for rare mutants arising in the RecA-deficient and ROS-elevated background. Collectively, our findings demonstrate that this oxidative environment, together with compromised DNA repair capacity, increases genetic instability and creates a selective landscape favouring the expansion of resistant clones. These results highlight the repair-redox axis as a key determinant of bacterial evolvability under antimicrobial stress.

microbiology↗
bioRxiv2022

Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli tomultiple bactericidal antibiotics

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus re-sensitise bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant re-sensitisation towards five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full re-sensitisation. For ciprofloxacin and nitrofurantoin, sensitisation manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the pro-mutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could re-sensitise antibiotic-resistant bacteria to multiple classes of existing antibiotics and may supress the development of de novo antibiotic-resistance mutations.

microbiology↗
bioRxiv2021

Systemic influences of mammary cancer on monocytes in mice

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and blood, particularly the myeloid compartment. Here we show using mouse models of breast cancer caused by the mammary epithelial expression of the Polyoma middle T antigen that there is an increase in the number of circulating and splenic monocytes. In the circulation, cancer does not affect ratios of classical to non-classical populations monocytes nor their halflives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. In the bone marrow cancer does not change monocytic progenitor number is unaffected but the proliferation rate of monocytes is higher thus providing an explanation for expansion in the circulating number. Deep RNA sequencing of these monocytic populations reveals cancer causes changes in the classical monocyte compartment with changes evident in bone marrow monocytes but more in the blood suggesting influences in both compartments. Down regulation of interferon type 1 signalling and antigen presentation were the most prominent. Consistent with this analysis down regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, transcription factors required for type 1 interferon signalling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, mouse models of cancer may be insufficient to study the systemic influences of human cancer.

cancer biology↗
bioRxiv2021

ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition

Metastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

cancer biology↗
bioRxiv2020

Androgen regulation of bowel function in mice and humans

Many digestive disorders have prominent sex differences in incidence, symptomatology, and treatment response that are not well understood. Irritable bowel syndrome (IBS), for example, affects approximately 10% of the population worldwide and tends to have different manifestations in males and females. Androgens are steroid hormones present at much higher levels in post-pubertal males than females and could be involved in these sex differences, but their normal functions in the bowel are largely unknown. Here, we show that gonadal androgens are required for normal gastrointestinal motility in vivo. In the healthy mouse gut, we detected androgen receptors in smooth muscle cells and a subset of enteric neurons. Surgical or genetic disruption of androgen signaling in adult mice selectively and reversibly altered colonic motility by affecting neurons rather than smooth muscle. To determine if androgens also influence human bowel function, we measured androgen levels in 208 adults with IBS. Free testosterone levels were lower in patients with IBS compared to healthy controls and inversely correlated with symptom severity. Taken together, these observations establish a role for androgens in the regulation of colonic motility and link altered androgen signaling with a common digestive disorder. These findings advance the fundamental understanding of gut motility, with implications for normal aging and disorders involving the gut-brain axis.

neuroscience↗
bioRxiv2020

The Pancreatic Tumor Microenvironment Buffers Redox Imbalance Imposed by Disrupted Mitochondrial Metabolism

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and, consequently, access to blood-derived nutrients and oxygen, which impacts tumor growth. Intracellular redox imbalance is another restraint on cellular proliferation, yet it is unknown if the TME contributes to the maintenance of redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle, disturbs redox homeostasis and halts proliferation of PDA cells in vitro. In contrast, GOT2 inhibition has no effect on in vivo tumor growth or tumorigenesis in an autochthonous model. We propose that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts. More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Genetic or pharmacologic inhibition of pyruvate uptake or metabolism abrogated pyruvate-mediated alleviation of reductive stress from NADH buildup. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.

cancer biology↗
bioRxiv2020

The complete genome sequence of the Staphylococcus bacteriophage Metroid

Phages infecting bacteria of the genus Staphylococcus play an important role in their hosts ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities - including a conservation of the lysis cassette - with other Staphylococcus cluster C1 bacteriophages, several of which were previously characterized for therapeutic applications.

genomics↗
bioRxiv2020

Decoding Predictions and Violations of Object Position and Category in Time-resolved EEG

Classic models of predictive coding propose that sensory systems use information retained from prior experience to predict current sensory input. Any mismatch between predicted and current input (prediction error) is then fed forward up the hierarchy leading to a revision of the prediction. We tested this hypothesis in the domain of object vision using a combination of multivariate pattern analysis and time-resolved electroencephalography. We presented participants with sequences of images that stepped around fixation in a predictable order. On the majority of presentations, the images conformed to a consistent pattern of position order and object category order, however, on a subset of presentations the last image in the sequence violated the established pattern by either violating the predicted category or position of the object. Contrary to classic predictive coding when decoding position and category we found no differences in decoding accuracy between predictable and violation conditions. However, consistent with recent extensions of predictive coding, exploratory analyses showed that a greater proportion of predictions was made to the forthcoming position in the sequence than to either the previous position or the position behind the previous position suggesting that the visual system actively anticipates future input as opposed to just inferring current input.

neuroscience↗
bioRxiv2019

Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces

BackgroundThe putative vector of trachoma, Musca sorbens, prefers to lay its eggs on human faeces on the ground. This study sought to determine whether M. sorbens females were attracted to volatile odours from human faeces in preference to odours from the faeces of other animals, and to determine whether specific volatile semiochemicals mediate selection of the faeces.\n\nMethodology/Principal findingsTraps baited with the faeces of humans and local domestic animals were used to catch flies at two trachoma-endemic locations in The Gambia and one in Ethiopia. At all locations, traps baited with faeces caught more female M. sorbens than control traps baited with soil, and human faeces was the most successful bait compared with soil (mean rate ratios 44.40, 61.40, 10.50 [P<0.001]; 8.17 for child faeces [P=0.004]). Odours from human faeces and some domestic animals were sampled by air entrainment. Extracts of the volatiles from human faeces were tested by coupled gas chromatography-electroantennography with laboratory-reared female M. sorbens. Twelve compounds were electrophysiologically active and tentatively identified by coupled mass spectrometry-gas chromatography, these included cresol, indole, 2-methylpropanoic acid, butanoic acid, pentanoic acid and hexanoic acid.\n\nConclusions/SignificanceIt is possible that some of these volatiles govern the strong attraction of M. sorbens flies to human faeces. If so, a synthetic blend of these chemicals, at the correct ratios, may prove to be a highly attractive lure. This could be used in odour-baited traps for monitoring or control of this species in trachoma-endemic regions.\n\nAuthor summaryMusca sorbens, also known as the Bazaar Fly, visits peoples faces to feed on ocular and nasal discharge. While feeding, M. sorbens can transmit Chlamydia trachomatis, the bacterium that causes the infectious eye disease trachoma. Around 1.9 million people worldwide are visually impaired or blind from this disease. Although it is believed that M. sorbens transmits trachoma, very few studies have looked at ways to control this fly. A large-scale trial has shown that control of fly populations with insecticide reduces active trachoma disease prevalence. Odour-baited traps for the suppression of disease vector populations are an attractive option as there is no widespread spraying of insecticide, however, highly attractive baits are critical to their success. Here we demonstrate that the preference of these flies for breeding in human faeces is probably mediated by odour cues, and we isolate chemicals in the odour of human faeces that cause a response in the antennae of M. sorbens. These compounds may play a role in the specific attractiveness of human faeces to these flies, perhaps by being present in greater amounts or at favourable ratios. These may be developed into a chemical lure for odour-baited trapping to suppress M. sorbens populations.

ecology↗
bioRxiv2019

Pathogenic and uncertain genetic variants have clinical cardiac correlates in diverse biobank participants

BackgroundGenome sequencing coupled with electronic heath record data can uncover medically important genetic variation. Interpretation of rare genetic variation and its role in mediating cardiovascular phenotypes is confounded by variants of uncertain significance.\n\nMethods and ResultsWe analyzed the whole genome sequence of 900 racially and ethnically diverse biobank participants selected from a single US center. Participants were equally divided among European, African, Hispanic, and mixed race/ethnicities. We evaluated the American College of Medical Genetics and Genomics medically actionable list of 59 genes focusing on the cardiac genes. Variation was interpreted using the most recent reports in ClinVar, a database of medically relevant human variation. We identified 19 individuals with pathogenic/likely pathogenic variants in cardiac actionable genes (2%) and found evidence for clinical correlates in the electronic health record. African ancestry participants had more variants of uncertain significance in the medically actionable genes including the 30 cardiac actionable genes, even when normalized to total variant count per person. Longitudinal measures of left ventricle size, corrected for body surface area, from approximately 400 biobank participants (1,723 patient years) correlated with genetic findings. The presence of one or more uncertain variants in the actionable cardiac genes and a cardiomyopathy diagnosis correlated with increased left ventricular internal diameter in diastole and in systole. In particular, MYBPC3 was identified as a gene with excess variants of uncertain significance.\n\nConclusionsThese data indicate a subset of uncertain variants may confer risk and should not be considered benign.

genomics↗
bioRxiv2019

UmuD and RecA* modulate the DNA-binding activity of DNA polymerase IV in Escherichia coli

DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells suffering high levels of DNA damage. In a recent single-molecule imaging study, we demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD, which accumulates in DNA-damaged cells, inhibits binding. In agreement with proposed roles for pol IV in homologous recombination, up to 40% of pol IV foci colocalise with a probe for RecA* nucleoprotein filaments in ciprofloxacin-treated cells. A hyperactive RecA mutant, recA(E38K), allows pol IV to bind the nucleoid even in the absence of exogenous DNA damage. In vitro, RecA(E38K) forms RecA*-like structures that can recruit pol IV, even on double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results indicate that UmuD and RecA modulate the binding of pol IV to its DNA substrates, which frequently coincide with RecA* structures.

microbiology↗
bioRxiv2019

Untangling featural and conceptual object representations

How are visual inputs transformed into conceptual representations by the human visual system? The contents of human perception, such as objects presented on a visual display, can reliably be decoded from voxel activation patterns in fMRI, and in evoked sensor activations in MEG and EEG. A prevailing question is the extent to which brain activation associated with object categories is due to statistical regularities of visual features within object categories. Here, we assessed the contribution of mid-level features to conceptual category decoding using EEG and a novel fast periodic decoding paradigm. Our study used a stimulus set consisting of intact objects from the animate (e.g., fish) and inanimate categories (e.g., chair) and scrambled versions of the same objects that were unrecognizable and preserved their visual features (Long, Yu, & Konkle, 2018). By presenting the images at different periodic rates, we biased processing to different levels of the visual hierarchy. We found that scrambled objects and their intact counterparts elicited similar patterns of activation, which could be used to decode the conceptual category (animate or inanimate), even for the unrecognizable scrambled objects. Animacy decoding for the scrambled objects, however, was only possible at the slowest periodic presentation rate. Animacy decoding for intact objects was faster, more robust, and could be achieved at faster presentation rates. Our results confirm that the mid-level visual features preserved in the scrambled objects contribute to animacy decoding, but also demonstrate that the dynamics vary markedly for intact versus scrambled objects. Our findings suggest a complex interplay between visual feature coding and categorical representations that is mediated by the visual systems capacity to use image features to resolve a recognisable object.

neuroscience↗
bioRxiv2019

Yellow strawberries and red bananas: The influence of object-colour knowledge on emerging object representations in the brain

The ability to rapidly and accurately recognise complex objects is a crucial function of the human visual system. To recognise an object, we need to bind incoming visual features such as colour and form together into cohesive neural representations and integrate these with our pre-existing knowledge about the world. For some objects, typical colour is a central feature for recognition; for example, a banana is typically yellow. Here, we applied multivariate pattern analysis on time-resolved neuroimaging (magnetoencephalography) data to examine how object-colour knowledge affects emerging object representations over time. Our results from 20 participants (11 female) show that the typicality of object-colour combinations influences object representations, although not at the initial stages of object and colour processing. We find evidence that colour decoding peaks later for atypical object-colour combinations in comparison to typical object-colour combinations, illustrating the interplay between processing incoming object features and stored object-knowledge. Taken together, these results provide new insights into the integration of incoming visual information with existing conceptual object knowledge. Significance StatementTo recognise objects, we have to be able to bind object features such as colour and shape into one coherent representation and compare it to stored object knowledge. The magnetoencephalography data presented here provide novel insights about the integration of incoming visual information with our knowledge about the world. Using colour as a model to understand the interaction between seeing and knowing, we show that there is a unique pattern of brain activity for congruently coloured objects (e.g., a yellow banana) relative to incongruently coloured objects (e.g., a red banana). This effect of object-colour knowledge only occurs after single object features are processed, demonstrating that conceptual knowledge is accessed relatively late in the visual processing hierarchy.

neuroscience↗