Biology subjects

Settles, M. L.

Publications and source records attributed to Settles, M. L..

6 recordsLinked to original sources

bioRxiv2023

NeuroMabSeq: high volume acquisition, processing, and curation of hybridoma sequences and their use in generating recombinant monoclonal antibodies and scFvs for neuroscience research

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

neuroscience↗

bioRxiv2022

Biofilms preserve transmissibility of a multi-drug resistance plasmid

Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations, and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizonal transfer of plasmids. In contrast, in biofilms most plasmids retained their transfer genes, but on average plasmid persistence improved less over time. Our results showed that biofilms can act as refugia of MDR plasmids and favor the horizontal mode of plasmid transfer, which has important implications for the spread of MDR.

microbiology↗

bioRxiv2021

Protective heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens

T cells are critical for control of viral infection and effective vaccination. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diphtheria-pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. Using co-cultures of antigen presenting cells (APC) loaded with antigens and autologous T cells, we found a high correlation between responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap vaccine proteins in both SARS-CoV-2 infected individuals and individuals immunized with mRNA-based SARS-CoV-2 vaccines. The overlapping T cell population contained effector memory T cells (TEMRA) previously implicated in anti-viral immunity and their activation required APC-derived IL-15. TCR- and scRNA-sequencing detected cross-reactive clones with TEMRA features among the cells recognizing SARS-CoV-2, MMR and Tdap epitopes. A propensity-weighted analysis of 73,582 COVID-19 patients revealed that severe disease outcomes (hospitalization and transfer to intensive care unit or death) were reduced in MMR or Tdap vaccinated individuals by 38-32% and 23-20% respectively. In summary, SARS-CoV-2 re-activates memory T cells generated by Tdap and MMR vaccines, which may reduce disease severity.

immunology↗

bioRxiv2021

Wilson disease: intersecting DNA methylation and histone acetylation regulation of gene expression in a mouse model of hepatic copper accumulation

The pathogenesis of Wilson disease (WD) is multi-factorial, involving hepatic and brain copper accumulation due to pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior DNA methylation investigations in human WD liver and blood and in a WD mouse model revealed an epigenetic signature of WD, including alterations in the histone deacetylase HDAC5. To test the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD, we investigated class IIa histone deacetylases (HDAC4 and HDAC5) and H3K9/H3K27 histone acetylation in the Jackson Laboratory toxic milk (tx-j) mouse model of WD compared to C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet (HFD), D-penicillamine (PCA, copper chelator), and choline (methyl group donor). HDAC5 levels significantly increased in 9-week tx-j livers after 8 days of HFD compared to chow. In 24-week tx-j livers, HDAC4/5 levels were reduced 5- to 10-fold compared to C3H likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were also affected by disease progression and accompanied by increased acetylation. PCA and choline partially restored HDAC4, HDAC5, H3K9ac, and H3K27ac levels to that of CH3 liver. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development were, in turn, regulated by histone acetylation in tx-j mice compared to C3H, with Ppar and Ppar{gamma} among the most relevant targets. These results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation, can regulate gene expression in key metabolic pathways in the pathogenesis of WD. Significance StatementWilson disease is considered a monogenic disease caused by pathogenic variants in the ATP7B copper transporter, resulting in hepatic and brain copper accumulation. Given the lack of genotype-phenotype correlation, evidence of epigenetic and metabolic mechanisms regulating phenotype in patients and in animal models could explain the high phenotype variability observed in WD. In this study, we identify class IIa histone deacetylases as players involved in the epigenetic regulation of key metabolic pathways that can affect WD severity as well as targets sensitive to dietary modulations, which is an important characteristic for designing effective and feasible therapies. Understanding the epigenetic mechanisms in WD pathogenesis contributes to a better understanding of the phenotypic variability in WD and other common liver conditions.

molecular biology↗

bioRxiv2020

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen. Author SummaryEntamoeba histolytica is a globally important pathogen that is dramatically understudied. One of the major limitations of this organism is its challenging genome. RNAi is the state-of-the-art tool for genetic manipulation in E. histolytica, though the RNAi pathway has several noncanonical features. Here, we harnessed the RNAi pathway to enable RNAi-based forward genetics for the first time in this organism. We validated the RNAi library by performing the first E. histolytica RNAi screen and identified slow growth mutants. We showed that independently-generated mutants also exhibited slow growth phenotypes, and we characterized protein localization and domains for some of the identified slow growth genes. The RNAi library that we constructed enables modern, quantitative Illumina deep sequencing analysis to identify mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. Our approach differs from previous approaches for analysis of RNAi screens, and it better represents the actual DNA fragments and their quantities. This study dramatically improves the tractability of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms. Author ContributionsA.B. designed and performed the experiments, except fragmentation of gDNA, immunofluorescence, and growth curves with expression of full-length or truncated proteins. R.L.S. performed SG1 and SG2 immunofluorescence experiments. M.C.R. performed growth curves with expression of full-length or truncated proteins. W.H. generated transfectants expressing full-length SG2. C.G.B. fragmented gDNA and processed the fragments to enable subsequent plasmid library construction. T.S.Y.T. and H.W.M. performed Gal/GalNAc lectin immunofluorescence experiments. S.S.H. designed the analysis strategy for Illumina sequencing data, and analyzed all Illumina sequencing data. M.L.S. oversaw the Illumina analysis approach. K.S.R. performed growth curves, conceived of the overall approach and oversaw the design and analysis of the experiments. A.B., S.S.H., and K.S.R. wrote the manuscript.

microbiology↗

bioRxiv2018

Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

Amplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.\n\nImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

microbiology↗