Biology subjects

Lee, W.

Publications and source records attributed to Lee, W..

85 records · Page 5Linked to original sources

bioRxiv2019

Social status in mouse social hierarchies is associated with variation in oxytocin and vasopressin 1a receptor densities

The neuropeptides oxytocin and vasopressin and their receptors have established roles in the regulation of mammalian social behavior including parental care, sex, affiliation and pair-bonding, but less is known regarding their relationship to social dominance and subordination within social hierarchies. We have previously demonstrated that male mice can form stable linear dominance hierarchies with individuals occupying one of three classes of social status: alpha, subdominant, subordinate. Alpha males exhibit high levels of aggression and rarely receive aggression. Subdominant males exhibit aggression towards subordinate males but also receive aggression from more dominant individuals. Subordinate males rarely exhibit aggression and receive aggression from more dominant males. Here, we examined whether variation in social status was associated with levels of oxytocin (OTR) and vasopressin 1a (V1aR) receptor binding in socially relevant brain regions. We found that socially dominant males had significantly higher OTR binding in the nucleus accumbens core than subordinate animals. Alpha males also had higher OTR binding in the anterior olfactory nucleus, posterior part of the cortical amygdala and rostral lateral septum compared to more subordinate individuals. Conversely, alpha males had lower V1aR binding in the rostral lateral septum and lateral preoptic area compared to subordinates. These observed relationships have two potential explanations. Preexisting individual differences in the patterns of OTR and V1aR binding may underlie behavioral differences that promote or inhibit the acquisition of social status. More likely, the differential social environments experienced by dominant and subordinate animals may shift receptor expression, potentially facilitating the expression of adaptive social behaviors. HighlightsO_LIMice living in social hierarchies express different levels of oxytocin receptor (OTR) and vasopressin 1a receptor (V1aR) binding in various brain regions according to their social status. C_LIO_LIAlphas and subdominants have higher OTR binding in the nucleus accumbens compared to subordinates. C_LIO_LIAlphas have higher OTR binding in the anterior olfactory nucleus compared to subdominants and subordinates. C_LIO_LIAlphas have higher OTR and lower V1aR binding in the rostral lateral septum compared to subordinates. C_LIO_LIAlphas have lower V1aR binding in the lateral preoptic area compared to subordinates. C_LI

neuroscience↗

bioRxiv2019

A real-time PCR for the differentiation of typhoidal and non-typhoidal Salmonella

Rapid and accurate differentiation of Salmonella spp. causing enteric fever from non-typhoidal Salmonella is essential for clinical management of cases, laboratory risk management and implementation of public health measures. Current methods used for confirmation of identification including biochemistry and serotyping as well as whole genome sequencing analyses, takes several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies, for the rapid identification of typhoidal and non-typhoidal Salmonella. This novel two-hour assay identifies the genus Salmonella by detecting the ttr gene, encoding tetrathionate reductase, and defines typhoidal Salmonella by the detection of S. Typhi and Paratyphi-specific gene combinations. PCR assay performance was determined using 211 clinical cultures of Salmonella (114 non-typhoidal and 97 Typhoidal strains) and 7 clinical non-Salmonella cultures. In addition, the specificity of the assay was evaluated in silico using a diverse in-house collection of 1882 Salmonella whole genome sequences. The real-time PCR results for 218 isolates and the genomic analysis of the 1882 isolates produced 100% sensitivity and 100% specificity (based on a 7 gene profile) for identifying typhoidal Salmonella compared to the Salmonella whole genome sequening identification methods currently used at Public Health England. This paper describes a robust real-time PCR assay for the rapid, accurate identification of typhoidal and non-typhoidal Salmonella which will be invaluable for the urgent screening of isolates from symptomatic individuals, the safe processing of isolates in laboratories and for assisting the management of public health risks.

microbiology↗

bioRxiv2019

Protocol-dependent differences in IC50 values measured in hERG assays occur in a predictable way and can be used to quantify state preference of drug binding.

Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of Kv11.1 channels as a surrogate for risk. A shortcoming of this approach is that the measured IC50 of Kv11.1 block varies widely depending on the voltage protocol used in electrophysiological assays. In this study, we aimed to investigate the factors that that contribute to these differences and to identify whether it is possible to make predictions about protocol-dependent block that might facilitate comparison of potencies measured using different assays Our data demonstrate that state preferential binding, together with drug binding kinetics and trapping, is an important determinant of the protocol-dependence of Kv11.1 block. We show for the first time that differences in IC50 measured between protocols occurs in a predictable way, such that machine learning algorithms trained using a selection of simple voltage protocols can indeed predict protocol-dependent potency. Furthermore, we also show that a drugs preference for binding to the open versus the inactivated state of Kv11.1 can also be inferred from differences in IC50 measured between protocols. Our work therefore identifies how state preferential drug binding is a major determinant of the protocol dependence of IC50 measured in preclinical Kv11.1 assays. It also provides a novel method for quantifying the state dependence of Kv11.1 drug binding that will facilitate the development of more complete models of drug binding to Kv11.1 and improve our understanding of proarrhythmic risk associated with compounds that block Kv11.1.

pharmacology and toxicology↗

bioRxiv2019

Social hierarchy position in female mice is associated with plasma corticosterone levels and hypothalamic gene expression

Social hierarchies emerge when animals compete for access to resources such as food, mates or physical space. Wild and laboratory male mice have been shown to develop linear hierarchies, however, less is known regarding whether female mice have sufficient intrasexual competition to establish significant social dominance relationships. In this study, we examined whether groups of outbred CD-1 virgin female mice housed in a large vivaria formed social hierarchies. We show that females use fighting, chasing and mounting behaviors to rapidly establish highly directionally consistent social relationships. Notably, these female hierarchies are less linear, steep and despotic compared to male hierarchies. Female estrus state was not found to have a significant effect on aggressive behavior, though dominant females had elongated estrus cycles (due to increased time in estrus) compared to subordinate females. Plasma estradiol levels were equivalent between dominant and subordinate females. Subordinate females had significantly lower levels of basal corticosterone compared to dominant females. Analyses of gene expression in the ventromedial hypothalamus indicated that subordinate females have elevated ER, ER{beta} and OTR mRNA compared to dominant females. This study provides a methodological framework for the study of the neuroendocrine basis of female social aggression and dominance in laboratory mice.

animal behavior and cognition↗

bioRxiv2018

Levels of histidine-rich glycoprotein in human blood predict accelerated aging

1.Despite recognizing aging as risk factor of human diseases, little is still known about the molecular traits of biological age and mortality risk. To identify age-associated proteins circulating human blood, we screened 156 subjects aged 50-92 years using an exploratory and multiplexed affinity proteomics approach. We corroborated the top age-associated protein profile (adjusted P < 0.001) in eight additional study sets (N = 4,044 individuals), and confirmed a consistent age-associated increase (P = 6.61 x 10-6) by meta-analysis. Applying antibody validation determined circulating histidine-rich glycoprotein (HRG) as the target, and we observed that sequence variants influenced the antibodies ability to bind to the protein. Profiles of circulating HRG were associated to several clinical traits and predicted the risk of mortality during a follow-up period of 8.5 years (IQR = 7.7-9.3 years) after blood sampling (HR = 1.25 per SD; 95% CI = 1.12-1.39; P = 7.41 x 10-5). In conclusion, our affinity proteomics analysis found associations between the molecular traits of circulating HRG with age and all-cause mortality. This suggests that the profiles of multi-purpose protein HRG could serve as an accessible indicator of physiological processes related to aging.

epidemiology↗

bioRxiv2018

In silico Drug Repositioning of bortezomib to reverse metastatic effect of GALNT14 in lung cancer

Although many molecular targets for cancer therapy have been discovered, they often show poor druggability, which is a major obstacle to develop targeted drugs. As an alternative route to drug discovery, we adopted an in silico drug repositioning (in silico DR) approach based on large-scale gene expression signatures, with the goal of identifying inhibitors of lung cancer metastasis. Our analysis of clinicogenomic data identified GALNT14, an enzyme involved in O-linked N-acetyl galactosamine glycosylation, as a putative driver of lung cancer metastasis leading to poor survival. To overcome the poor druggability of GALNT14, we leveraged Connectivity Map approach, an in silico screening for drugs that are likely to revert the metastatic expression patterns. It leads to identification of bortezomib (BTZ) as a potent metastatic inhibitor, bypassing direct inhibition of poorly druggable target, GALNT14. The anti-metastatic effect of BTZ was verified in vitro and in vivo. Notably, both BTZ treatment and GALNT14 knockdown attenuated TGF{beta}-mediated gene expression and suppressed TGF{beta}-dependent metastatic genes, suggesting that BTZ acts by modulating TGF{beta} signalingTaken together, these results demonstrate that our in silico DR approach is a viable strategy to identify a candidate drug for undruggable targets, and to uncover its underlying mechanisms.

cancer biology↗

bioRxiv2018

Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods have provided new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we developed a genome editing strategy using a cytidine deaminase fused with inactive Cas9 (dCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns served as a genetic barcode, which was induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.

bioengineering↗

bioRxiv2018

Multiplexed single-cell RNA-seq via transient barcoding for drug screening

To simultaneously analyze multiple samples of various conditions with scRNA-seq, we developed a universal sample barcoding method through transient transfection of SBOs. A 48-plex drug treatment experiment of pooled samples analyzed by a single run of Drop-Seq revealed a unique transcriptome response for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for single-cell profiling of multiple experimental conditions.

genomics↗

bioRxiv2018

FtsW is a peptidoglycan polymerase that is activated by its cognate penicillin-binding protein

The peptidoglycan cell wall is essential for the survival and shape maintenance ofbacteria.1 For decades it was thought that only penicillin-binding proteins (PBPs) effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the Rod complex involved in side wall peptidoglycan synthesis, acts as a peptidoglycan polymerase.2-4 RodA is absent or dispensable in many bacteria that contain a cell wall; however, all of these bacteria have a RodA homologue, FtsW, which is a core member of the divisome complex that is essential for septal cell wall assembly.5,6 FtsW was previously proposed flip the peptidoglycan precursor Lipid II to the peripasm,7,8 but we report here that FtsW polymerizes Lipid II. We show that FtsW polymerase activity depends on the presence of the class B PBP (bPBP) that it recruits to the septum. We also demonstrate that the polymerase activity of FtsW is required for its function in vivo. Our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

microbiology↗

bioRxiv2018

Immediate early gene activation throughout the brain is associated with dynamic changes in social context

Social competence is dependent on successful processing of social context information. The social opportunity paradigm is a methodology in which dynamic shifts in social context are induced through removal of the alpha male in a dominance hierarchy, leading to rapid ascent in the hierarchy of the beta male and of other subordinate males in the social group. In the current study, we use the social opportunity paradigm to determine what brain regions respond to this dynamic change in social context, allowing an individual to recognize the absence of the alpha male and subsequently perform status-appropriate social behaviors. Replicating our previous work, we show that following removal of the alpha male, beta males rapidly ascend the social hierarchy and attain dominant status by increasing aggression towards more subordinate individuals. Analysis of patterns of Fos immunoreactivity throughout the brain indicates that in individuals undergoing social ascent, there is increased activity in regions of the social behavior network, as well as the infralimbic and prelimbic regions of the prefrontal cortex and areas of the hippocampus. Our findings demonstrate that male mice are able to respond to changes in social context and provide insight into the how the brain processes these complex behavioral changes.

animal behavior and cognition↗

bioRxiv2018

The Autoimmune Disease Risk Allele rs6897932 Modulates Monocyte IL7R Surface and Soluble Receptor Levels in a Context-Specific Manner

IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. IL7R mRNA is induced in stimulated monocytes in a genetically determined manner, yet a role for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level across multiple cell subsets and conditions in healthy individuals. We find monocyte surface and soluble IL7R (sIL7R) protein are markedly expressed in response to lipopolysaccharide (LPS). We further demonstrate alleles of rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, form the key determinant of both surface IL7R and sIL7R in the context of inflammation. No effect of this allele was observed in unstimulated monocytes or across lymphoid subsets. Production of sIL7R by monocytes greatly exceeded that of CD4+ T-cells, and was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. Stimulated monocytes were sensitive to exogenous IL-7, which elicits a defined transcriptional signature. Flow cytometry and single-cell sequencing of synovial fluid derived monocytes from patients with spondyloarthritis showed an enlarged subset of IL7R+ monocytes with a unique transcriptional profile that markedly overlaps that induced by IL-7 in-vitro and shows similarity to the previously described Mono4 subset. These data demonstrate disease-associated genetic variants at IL7R specifically impact monocyte surface IL7R and sIL7R following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.

immunology↗

bioRxiv2017

Analysis of archaic human haplotypes suggest 5-hmC to act as epigenetic guide for meiotic point recombination

Meiotic \"point recombination\" refers to homologue recombination events affecting only individual SNPs. Driven mostly by gene conversion, it is common process that allows for a gradual adaptation and maturation of haplotypes during genetic evolution. In contrast to crossover recombination it is not tied to predetermined recombination sites and therefore assumed to occur largely randomly. Our analysis of archaic human haplotypes however revealed striking differences in the local point recombination rate. A linkage-study of 1.9 million SNPs defined by the sequence of denisovan hominids revealed low rates in introns and quiescent intergenic regions but high rates in splice sites, exons, 5- and 3-UTRs, and CpG islands. Correlations with ChIP-Seq tracks from ENCODE and other public sources identified a number of epigenetic modifications, that associated directly with these recombination events. A particularly tight association was observed for 5-hydroxymethylcytosine marks (5hmC). The mark was enriched in virtually all of the functional regions associated with elevated point recombination rates, including CpG islands and poised bivalent regions. As intermediate of oxidative demethylation, 5hmC is also a marker of recently opened gene loci. The data, thus, supports a model of guided evolution, in which point recombination is directed by 5hmC marks towards the functionally relevant regions.

evolutionary biology↗

bioRxiv2017

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts and Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infection in mammals. While the uptake of cholesterol by Mtb is mediated by the Mce4 transporter, the route(s) of uptake of fatty acids remain unknown. Here, we demonstrate that an uncharacterized protein LucA, integrates the assimilation of both cholesterol and fatty acids in Mtb. LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters. We also demonstrate that Mce1 functions as an important fatty acid transporter in Mtb and we determine that the integration of cholesterol and fatty acid transport by LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key role for LucA in coordinating both transport activities.

microbiology↗