Lucas Bowler - Academia.edu (original) (raw)

Papers by Lucas Bowler

Open research Europe, Jul 17, 2023

Background: The fungal component of the gut microbiome has been underrepresented in most gut micr... more Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While metabarcoding approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

Humana Press eBooks, Nov 14, 2003

Successful pathogens have evolved a variety of specific gene products that facilitate their survi... more Successful pathogens have evolved a variety of specific gene products that facilitate their survival and growth within the host, as well as mechanisms to regulate expression of these virulence-associated genes in response to their environment. In comparison with commensals, the pathogenic phenotype can thus be seen as a consequence of both differences in gene content and gene expression. Not surprisingly, identification of these differences is a frequent goal in modern biomedical research, and as a result, a variety of differential screening methods have been developed over the last few years (1,2).

Gene, May 1, 1997

The molecular diversity of protein D of nonencapsulated Haemophilus influenzae strains isolated f... more The molecular diversity of protein D of nonencapsulated Haemophilus influenzae strains isolated from persistently infected patients with chronic bronchitis was studied by sequencing the hpd gene of four independently obtained isolates. The nucleotide (nt) sequences of the hpd genes of two strains were identical. The other two hpd sequences showed nt substitutions which were mostly synonymous. As a consequence the deduced amino acid (aa) sequences differed from the consensus sequence only by a few aa. No changes in the hpd genes were observed among the four variants of the four strains persisting in chronic bronchitis patients for 9, 11, 8 and 3 months, respectively, although variation in their major outer membrane proteins P2 and P5 occurred. We conclude that the hpd gene is conserved during chronic infections of nonencapsulated H. influenzae.

Archives of Biochemistry and Biophysics, Oct 1, 2007

We previously described a caspase-like activity, which we termed KIPase that is implicated in the... more We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27 KIP1. KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27 KIP1 , and inhibitors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the b 1 subunit of the 20S proteasome. Further analyses revealed that the activity of the b 1 subunit is upregulated as cells enter the cell cycle without concomitant change in the levels of the proteasome b 1 , b 2 or b 5 subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity of the 20S proteasome.

Molecular Microbiology, Nov 1, 1987

SummaryThe coding region for the mature form of TEM β–lactamase was fused to random positions wit... more SummaryThe coding region for the mature form of TEM β–lactamase was fused to random positions within the coding region of the penicillin–binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in–frame fusions were determined. All fusion proteins that contained at least the NH2–terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the β–lactamase moiety had been translocated to the periplasm. Fusion proteins that contained ≤ 63 residues of PBP 1B possessed β–lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the 3–lactamase moiety of these fusion proteins remained in the cytoplasm. The β–lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic trans–membrane segment (residues 64–87), with a short NH2–terminal domain (residues 1–63), and the remainder of the polypeptide (residues 68–844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.

Journal of Bacteriology, 1994

base plus supplements in an atmosphere of 5% CO2 and 95% air.

Humana Press eBooks, Feb 17, 2004

... 4 Representational Difference Analysis of cDNA Lucas D. Bowler Abstract In this chapter I des... more ... 4 Representational Difference Analysis of cDNA Lucas D. Bowler Abstract In this chapter I describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). ... In addition, the exponential degree of enrich-Page 3. ...

Open Research Europe

Disorders associated with substance abuse are a major public health crisis with few treatment opt... more Disorders associated with substance abuse are a major public health crisis with few treatment options. According to World Health Organization (WHO) ethanol is the most widely used drug in the world, and it represents a risk factor for the advent of disease, disability, and eventually death. Foetal Alcoholic Spectrum Disorders (FASD) is a diagnostic term to describe the range of effects that can occur in an individual whose mother drank alcohol during pregnancy. These effects encompass both physical, mental, behavioural and further lifelong disabilities. Besides, ethanol can harm the gut microbiota. Gut microbiome is firstly acquired from the mother and it is crucial for intestinal homeostasis during hosts’ lifetime. It is responsible for producing metabolites that benefits and protects the host from harm microbial colonization. Knowledge about the interactions between human gut microbes and the developing nervous system is still scarce. Nevertheless, animal models have shown that gu...

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is ess... more The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as foetal alcohol spectrum disorders. Here we assessed the effectiveness of targeted and untargeted (metagenomic) nanopore sequencing approaches to profile the gut microbiota of infant mice exposed to ethanolin utero. DNA extracts from the gut content of 12 infant mice exposed to ethanol in utero were analysed using one untargeted and two targeted (full-length 16S rRNA gene and the 16S-ITS-23S region of the ribosomal RNA operon) nanopore sequencing approaches. The targeting of the full-length 16S rRNA gene provided the most comprehensive analysis of the mouse gut microbiota. The differences in diversity between approaches were accounted by the sequencing target (p-value < 0.001). Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial...

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is ess... more The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as Foetal Alcohol Spectrum Disorder. Foetal Alcohol Spectrum Disorder represents a cluster of abnormalities including growth deficiencies and neurological impairments, which are not easily diagnosed nor treated. Here the effect of ethanol exposurein uteroon the gut microbial profiles of 16 infant mice (nine exposedin uteroand seven non-exposed) was assessed by targeted nanopore sequencing and Illumina sequencing approaches. The nanopore sequencing was implemented using MinION system targeting PCR-amplified amplicons made from the full-length 16S rRNA gene. The Illumina sequencing was performed using Miseq system targeting the V3-V4 region of the 16S rRNA gene. Ethanol exposure did not affect the microbial profiles. Several low prevalent taxa, likeAkkermansia muciniphila, were detected but further studies must be performe...

Elsevier eBooks, 2002

Publisher Summary Representational difference analysis (RDA) is a subtractive hybridization techn... more Publisher Summary Representational difference analysis (RDA) is a subtractive hybridization technique that combines polymerase chain reaction (PCR) amplification with kinetic enrichment to isolate DNA sequences present in one population, but not in another. For comparisons between bacterial genomes (genomic RDA), populations of chromosomal DNA restriction fragments (called “representations”) are used as the starting material. Defined oligonucleotide adapters are then ligated on to the 5'-ends of the DNA molecules, comprising one population (known as the “tester”) but not the other (the “driver”). As with RDA, complementary DNA (cDNA) RDA can be divided into a number of phases: the generation of representations (the PCR amplicons, representing the RNAs isolated from given bacterial populations), the PCR-coupled subtractive hybridization of the different representations, and the cloning and screening of the resultant products that represent the differences between the two populations that were compared. Detailed protocols for both RDA and cDNA RDA are described in the chapter.

Frontiers in Microbiology

Umber of features being reduced and the Y-axis represents the average training error in percentag... more Umber of features being reduced and the Y-axis represents the average training error in percentage over 100 training times counted in percentage. The training error increases significantly when 23 less relevant features are removed, as indicated by the red arrow. It is then suggested that at most 22 features could be eliminated.Copyright information:Taken from "A machine learning approach to explore the spectra intensity pattern of peptides using tandem mass spectrometry data"http://www.biomedcentral.com/1471-2105/9/325BMC Bioinformatics 2008;9():325-325.Published online 30 Jul 2008PMCID:PMC2529326.

Erimental counterpart. Figure 7-A: The red line represents the sorted scores calculated with the ... more Erimental counterpart. Figure 7-A: The red line represents the sorted scores calculated with the predicted intensity information. The blue line represents the corresponding scores calculated without intensity information. The two score use the same variances predicted by the Bayesian neural network model. Figure 7-B: The red line represents the sorted scores calculated with the predicted intensity information. The blue line represents the corresponding scores calculated without intensity information. Variances for intensity-free scores are set to 1.Copyright information:Taken from "A machine learning approach to explore the spectra intensity pattern of peptides using tandem mass spectrometry data"http://www.biomedcentral.com/1471-2105/9/325BMC Bioinformatics 2008;9():325-325.Published online 30 Jul 2008PMCID:PMC2529326.

Luence on cleavage at its C-terminus is illustrated in the right panel (red dots). The most influ... more Luence on cleavage at its C-terminus is illustrated in the right panel (red dots). The most influential residues are marked with arrows. Down arrows indicate inhibition whereas up arrows indicate enhancement. Figure 3-A: Mobile status. Figure 3-B: Partial-mobile status. Figure 3-C: Non-mobile status.Copyright information:Taken from "A machine learning approach to explore the spectra intensity pattern of peptides using tandem mass spectrometry data"http://www.biomedcentral.com/1471-2105/9/325BMC Bioinformatics 2008;9():325-325.Published online 30 Jul 2008PMCID:PMC2529326.

E input layer representing 35 features. 40 nodes in binary are used to represent the presence of ... more E input layer representing 35 features. 40 nodes in binary are used to represent the presence of 20 different residues at N and C terminus to the target peptide bond. Every node in the input layer has an independent coefficient to reveal its "relevance" to the network output. The hidden layer has 40 nodes and the activation function of the hidden layer is sigmoidal.Copyright information:Taken from "A machine learning approach to explore the spectra intensity pattern of peptides using tandem mass spectrometry data"http://www.biomedcentral.com/1471-2105/9/325BMC Bioinformatics 2008;9():325-325.Published online 30 Jul 2008PMCID:PMC2529326.

Journal of the International Society of Antioxidants in Nutrition & Health, 2016

Glutathionylation, i.e. formation of mixed disulfides between protein cysteines and glutathione (... more Glutathionylation, i.e. formation of mixed disulfides between protein cysteines and glutathione (GSH), is a mechanism by which the redox state of the cell regulates protein functions. Most studies on identification of glutathionylated proteins focused on intracellular proteins. We used a redox proteomic technique based on preloading the cells with biotinylated GSH followed by affinity purification and mass spectrometry to identify glutathionylated proteins in the secretome of macrophages stimulated with lipopolysaccharide (LPS). We confirmed the LPS-induced secretion of a selected set of proteins: peroxiredoxin (PRDX)1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin1 (TXN1). We found that PRDXs are released as glutathionylated dimers; moreover, we were able to confirm that the released TXN1 is glutathionylated. The release of the proteins was inhibited by the anti-inflammatory drug, dexamethasone and by thiol antioxidants GSH derivative GSH-C4 and N-acetylcysteine. We found...

Currents in Pharmacy Teaching and Learning, 2017

Background. Antibiotic resistance has become a global public health concern. In this study we inv... more Background. Antibiotic resistance has become a global public health concern. In this study we investigated the knowledge and awareness of antibiotic use, resistance and stewardship, held by the pharmacy students currently studying at the University of Brighton. Study design. This was a cross-sectional, online survey, and email invitations to participate were sent to all students attending our Master of Pharmacy (MPharm) course (n = 583). Students' knowledge was assessed with 29 items; responses for these were totaled before comparison among students. Comparison of scores between groups of students was performed using the Kruskal-Wallis or the MannWhitney test, as appropriate. Results. The response rate was 32%. The overall median knowledge score was 7.9. There was a statistically significant difference in knowledge scores between years of study (p = 0.02), particularly between year of study 1 (7.6) and 4 (8.3). A statistically significant difference was found between the knowledge scores of male (8.4) and female (7.9) students (p = 0.03). Most students believed a strong knowledge of antibiotics, and microbiology and infection control is important for their pharmacy careers and more than 90% agreed that antibiotic resistance will be a greater clinical problem in the future. Conclusions. Although the MPharm students studied achieved good overall knowledge scores, a significant proportion showed a lack of understanding with regards to some important aspects of antibiotic resistance mechanisms, factors promoting the emergence and spread of antibiotic resistance, and antibiotic stewardship policies.

Open research Europe, Jul 17, 2023

Humana Press eBooks, Nov 14, 2003

Gene, May 1, 1997

Archives of Biochemistry and Biophysics, Oct 1, 2007

Molecular Microbiology, Nov 1, 1987

Journal of Bacteriology, 1994

base plus supplements in an atmosphere of 5% CO2 and 95% air.

Humana Press eBooks, Feb 17, 2004

Open Research Europe

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is ess... more The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as Foetal Alcohol Spectrum Disorder. Foetal Alcohol Spectrum Disorder represents a cluster of abnormalities including growth deficiencies and neurological impairments, which are not easily diagnosed nor treated. Here the effect of ethanol exposurein uteroon the gut microbial profiles of 16 infant mice (nine exposedin uteroand seven non-exposed) was assessed by targeted nanopore sequencing and Illumina sequencing approaches. The nanopore sequencing was implemented using MinION system targeting PCR-amplified amplicons made from the full-length 16S rRNA gene. The Illumina sequencing was performed using Miseq system targeting the V3-V4 region of the 16S rRNA gene. Ethanol exposure did not affect the microbial profiles. Several low prevalent taxa, likeAkkermansia muciniphila, were detected but further studies must be performe...

Elsevier eBooks, 2002

Frontiers in Microbiology

Journal of the International Society of Antioxidants in Nutrition & Health, 2016

Currents in Pharmacy Teaching and Learning, 2017