Successful transmission and transcriptional deployment of a human chromosome via mouse male meiosis

Most human aneuploidies originate maternally, due in part to the presence of highly stringent checkpoints during male meiosis. Indeed, male sterility is common among aneuploid mice used to study chromosomal abnormalities, and male germline transmission of exogenous DNA has been rarely reported. Here we show that, despite aberrant testis architecture, males of the aneuploid Tc1 mouse strain produce viable sperm and transmit human chromosome 21 to create aneuploid offspring. In these offspring, we mapped transcription, transcriptional initiation, enhancer activity, non-methylated DNA, and transcription factor binding in adult tissues. Remarkably, when compared with mice derived from female passage of human chromosome 21, the chromatin condensation during spermatogenesis and the extensive epigenetic reprogramming specific to male germline transmission resulted in almost indistinguishable patterns of transcriptional deployment. Our results reveal an unexpected tolerance of aneuploidy during mammalian spermatogenesis, and the surprisingly robust ability of mouse developmental machinery to accurately deploy an exogenous chromosome, regardless of germline transmission.This research was supported by Cancer Research UK (CE, CK, FC, TFR, ML, DTO), the European Molecular Biology Laboratory (NE), the Wellcome Trust (106563/Z/14/A: SJA and 098024/Z/11/Z: RJK) and the European Research Council (DTO)

Longitudinal analysis of the microbiome and metabolome in the 5xfAD mouse model of Alzheimer's disease

Here we evaluate longitudinal microbiomes and metabolomes from a popular transgenic mouse model for familial Alzheimer's disease (5xfAD). This data is comprised of metagenomics of both cecal and fecal samples, metabolomics of fecal samples, and metabolomics of blood plasma. Samples were collected from 5xfAD mice and their WT B6J littermates from 4 – 18 months of age. Differences in microbiome and metabolite composition with relation to genotype, sex, age, and housing were explored. This study was conducted as part of the MODEL-AD consortium (model-ad.org).No special programs of software are required to open the data files. The R scripts that were used to analyze the data can be found on GitHub (https://github.com/sjbd1/5xfAD_mBio2022). PRJNA902000 is the bioaccession number for the raw sequence reads on the SRA: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA902000/Funding provided by: National Institutes of HealthCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000002Award Number: U54AG054349Funding provided by: National Institute on AgingCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000049Award Number: T32 AG00096-38This dataset consists of four text files, each of which has undergone extensive pre-processing in R. The relevant metadata and data are combined in each file. The files include: PlasmaMX_data.txt: plasma metabolomics data FecalMX_data.txt: fecal metabolomics data Taxonomy_data.txt: fecal and cecal microbiome data Turicibacter_data.txt: Bowtie2 data for three species of Turicibacter in fecal and cecal samples Please see the manuscipt methods for more information (https://doi.org/10.1128/mbio.01794-22)

Secondary ion mass spectrometry imaging of surface-bound microbial communities

As we move into the heart of the 21st century we are finally beginning to understand the extent of microbial influence over everyday life. Truth be told, we humans are but guests on a planet ruled by a largely unseen, and often undiscovered, microbial population. In order to understand how the microbial dimension influences our own we must first understand the microbes themselves. Far from existing as a collection of single cells floating about aimlessly, bacteria largely reside in highly cooperative multicellular communities where they work in concert to colonize and mold their surrounding environments, harvest nutrients, and wage warfare. How can these seemingly simple life forms facilitate such complicated behaviors? The answer largely resides in chemistry, for the backbone of microbial influence is built with the chemicals that they produce, secrete, sense, and consume. With clever implementation of the proper analytical tools this chemical information is ripe for our discovery and exploitation. This dissertation primarily focuses on the adaptation and application of an existing chemical imaging technique, secondary ion mass spectrometry (SIMS) imaging, to study the chemistry underlying surface-bound microbial communities. The first chapter provides a general overview of the dissertation. The second chapter provides an introduction to mass spectrometry imaging – which encompasses a broad family of techniques including SIMS imaging – with a focus on its application to microbiology. The remaining six chapters, detailed below, describe both method development for SIMS imaging and application of the technique to explore several questions in microbiology. SIMS is applied in conjunction with a complimentary analytical technique, confocal Raman microscopy (CRM), to study early stage biofilms formed by the gram-negative bacterium Pseudomonas aeruginosa, which is an opportunistic pathogen for both plants and humans. In addition to methodological advancements, this work revealed that P. aeruginosa accumulates highly concentrated clusters of alkyl-quinolines – including 2-heptyl-4-quinoline-N-oxide (HQNO) and 2-nonyl-4-quinoline-N-oxide (NQNO) – during the early stages of biofilm formation. HQNO and NQNO are known to disrupt the formation of healthy communities of gram-positive bacteria, and their high abundance during biofilm development suggests that P. aeruginosa utilizes these molecules for a competitive advantage for establishing new colonies. In a purely methodological study, we examined the effects of applying a thin (~2 nm) layer of gold to the biofilm surface prior to SIMS imaging. This investigation revealed a signal enhancement for cluster-SIMS that, remarkably, only applied to analytes contained within biological samples. Examination of gold coated standards deposited on hard silicon wafers did not yield a signal enhancement, suggesting that the SIMS community needs to look beyond simple standard formulations when developing or adapting sample treatment strategies. Separately, a simple nitrogen desiccation procedure was developed for imaging microbial communities on semi-solid agar, which can be a challenging substrate due to the high water content. Traditionally, most SIMS studies are carried out using hard, conductive surfaces, however microbiology assays commonly require growth on semi-solid agar. Both analyte-to-analyte differences in ionization efficiency and interfering signal from compounds with the same or similar mass-to-charge ratio (m/z) have largely prevented biological SIMS imaging from becoming a quantitative technique; the distribution of an analyte can be determined however the absolute quantity usually cannot. We therefore developed a quantitative SIMS imaging strategy where (1) SIMS product ion imaging is used to increase analyte specificity, (2) analyte-analyte differences in ionization efficiency are accommodated through calibration to an external quadratic calibration curve, and (3) competing ion signal is algebraically removed from each image pixel. This strategy is demonstrated by imaging the surface density of two different alkyl quinolone/quinoline isomeric pairs across several agar-based P. aeruginosa bacterial biofilms. Another major challenge for SIMS imaging is the regiospecific differences in ionization efficiency, which impede direct comparison of ion intensity to analyte abundance. Heterogeneous ionization efficiency arises from a myriad of factors, including changes in the chemical microenvironment, local morphology, and conductivity of the sample. A microspot array methodology for evaluating regiospecific differences in ionization efficiency is presented, and its utility is demonstrated by evaluating several different strains of P. aeruginosa cultivated on semi-solid agar. In a highly collaborative study that required the expertise of three separate research groups, the spatiochemical response of P. aeruginosa to antibiotic exposure was examined with SIMS imaging, CRM, and a number of traditional microbiology techniques. This study showed that P. aeruginosa swarms migrate away from the antibiotic source and increase their production of several alkyl quinolones in a dose-dependent manner. Interestingly, the quorum sensing molecule known as Pseudomonas quinolone signal (PQS) was found to be more abundant in regions closest to the antibiotic. These results suggest that PQS is regulated independent of the other alkyl quinolones, and acts as a transient, short-range signal. In an effort with relevance to microbial induced corrosion, a method is presented for cultivating, preparing, and examining drip-flow biofilms on metallic surfaces. Two species of bacteria, Pseudomonas putida and Shewanella oneidensis, were cultivated on both stainless and low carbon steel and examined with SIMS imaging, matrix-assisted laser desorption/ionization mass spectrometry imaging, scanning electron microscopy, and energy-dispersive x-ray spectroscopy. This study partially identifies and maps the distribution of 25 lipids and polysaccharides on P. putida drip-flow biofilms, examines the spatiochemical interactions between P. putida and S. oneidensis grown adjacent to one another, and examines the chemical and morphological environment of the two bacteria on corroding low-carbon steel. Taken together, the research described in this dissertation enhances our fundamental knowledge of the chemistry underlying microbial communities. The developed analytical methodologies can be applied by other researchers to further advance our collective understanding of the microbial world.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

What Lies Beneath? Taking the Plunge into the Murky Waters of Phage Biology

The sequence revolution revealed that bacteria-infecting viruses, known as phages, are Earth's most abundant biological entities. Phages have far-reaching impacts on the form and function of microbial communities and play a fundamental role in ecological processes. However, even well into the sequencing revolution, we have only just begun to explore the murky waters around the phage biology iceberg. Many viral reads cannot be assigned to a culturable isolate, and reference databases are biased toward more easily collectible samples, which likely distorts our conclusions. This minireview points out alternatives to mapping reads to reference databases and highlights innovative bioinformatic and experimental approaches that can help us overcome some of the challenges in phage research and better decipher the impact of phages on microbial communities. Moving beyond the identification of novel phages, we highlight phage metabolomics as an important influencer of bacterial host cell physiology and hope to inspire the reader to consider the effects of phages on host metabolism and ecosystems at large. We encourage researchers to report unassigned/unknown sequencing reads and contigs and to continue developing alternative methods to investigate phages within sequence data

Vacuum-assisted sorbent extraction: An analytical methodology for the determination of ultraviolet filters in environmental samples

Vacuum-assisted sorbent extraction (VASE) has been applied for the first time in the determination of UV filters in water samples in combination with gas chromatography-mass spectrometry. VASE is a variant of headspace extraction which was developed in conjunction with the sorbent pen (SP) technology. This technique combines the advantages of both stir-bar assisted extraction and headspace solid-phase microextraction. The SP traps allowed both reduced pressure in-vial extraction and direct thermal desorption via a unique gas chromatographic injection port. The main parameters that affect the performance of VASE, including both extraction and desorption conditions, were extensively optimized. Under optimum conditions, extraction required 10 mL of sample within 40 mL vials, pH 3.5, ∼30 s of air-evacuation, 14 h incubation at 70 °C, stirring at 200 rpm, and a final water management step conducted at ∼ −17 °C for 15 min. Optimal thermal desorption required preheating at 260 °C for 2 min followed by desorption at 300 °C for 2 min. The beneficial effect of reduced-pressure extraction was demonstrated by comparing the UV filter extraction time profiles collected using VASE to an analogous atmospheric pressure procedure, resulting in up to a 3-fold improvement under optimized conditions. The VASE methodology enabled simultaneous extractions using different SPs without compromising the method reproducibility, which increases the overall sample throughput. The method was characterized by low limits of detection, from 0.5 to 80 ng L−1, and adequate reproducibility, with inter-SP and inter-day relative standard deviation lower than 14%. Tap and lake water was successfully analyzed with the proposed methodology, resulting in relative recoveries of spiked samples ranging between 70.0 and 120%.This is a manuscript of an article published as Trujillo-Rodríguez, María J., Jared L. Anderson, Sage JB Dunham, Victoria L. Noad, and Daniel B. Cardin. "Vacuum-assisted sorbent extraction: An analytical methodology for the determination of ultraviolet filters in environmental samples." 208 Talanta (2019): 120390. DOI: 10.1016/j.talanta.2019.120390. Posted with permission.</p

Mass Spectrometry Imaging of Complex Microbial Communities

ConspectusIn the two decades since mass spectrometry imaging (MSI) was first applied to visualize the distribution of peptides across biological tissues and cells, the technique has become increasingly effective and reliable. MSI excels at providing complementary information to existing methods for molecular analysissuch as genomics, transcriptomics, and metabolomicsand stands apart from other chemical imaging modalities through its capability to generate information that is simultaneously multiplexed and chemically specific. Today a diverse family of MSI approaches are applied throughout the scientific community to study the distribution of proteins, peptides, and small-molecule metabolites across many biological models.The inherent strengths of MSI make the technique valuable for studying microbial systems. Many microbes reside in surface-attached multicellular and multispecies communities, such as biofilms and motile colonies, where they work together to harness surrounding nutrients, fend off hostile organisms, and shield one another from adverse environmental conditions. These processes, as well as many others essential for microbial survival, are mediated through the production and utilization of a diverse assortment of chemicals. Although bacterial cells are generally only a few microns in diameter, the ecologies they influence can encompass entire ecosystems, and the chemical changes that they bring about can occur over time scales ranging from milliseconds to decades. Because of their incredible complexity, our understanding of and influence over microbial systems requires detailed scientific evaluations that yield both chemical and spatial information. MSI is well-positioned to fulfill these requirements. With small adaptations to existing methods, the technique can be applied to study a wide variety of chemical interactions, including those that occur inside single-species microbial communities, between cohabitating microbes, and between microbes and their hosts.In recognition of this potential for scientific advancement, researchers have adapted MSI methodologies for the specific needs of the microbiology research community. As a result, workflows exist for imaging microbial systems with many of the common MSI ionization methods. Despite this progress, there is substantial room for improvements in instrumentation, sample preparation, and data interpretation. This Account provides a brief overview of the state of technology in microbial MSI, illuminates selected applications that demonstrate the potential of the technique, and highlights a series of development challenges that are needed to move the field forward. In the coming years, as microbial MSI becomes easier to use and more universally applicable, the technique will evolve into a fundamental tool widely applied throughout many divisions of science, medicine, and industry

Longitudinal Analysis of the Microbiome and Metabolome in the 5xfAD Mouse Model of Alzheimer's Disease.

Recent reports implicate gut microbiome dysbiosis in the onset and progression of Alzheimer's disease (AD), yet studies involving model animals overwhelmingly omit the microbial perspective. Here, we evaluate longitudinal microbiomes and metabolomes from a popular transgenic mouse model for familial AD (5xfAD). Cecal and fecal samples from 5xfAD and wild-type B6J (WT) mice from 4 to 18 months of age were subjected to shotgun Illumina sequencing. Metabolomics was performed on plasma and feces from a subset of the same animals. Significant genotype, sex, age, and cage-specific differences were observed in the microbiome, with the variance explained by genotype at 4 and 18 months of age rising from 0.9 to 9% and 0.3 to 8% for the cecal and fecal samples, respectively. Bacteria at significantly higher abundances in AD mice include multiple Alistipes spp., two Ligilactobacillus spp., and Lactobacillus sp. P38, while multiple species of Turicibacter, Lactobacillus johnsonii, and Romboutsia ilealis were less abundant. Turicibacter is similarly depleted in people with AD, and members of this genus both consume and induce the production of gut-derived serotonin. Contradicting previous findings in humans, serotonin is significantly more concentrated in the blood of older 5xfAD animals compared to their WT littermates. 5xfAD animals exhibited significantly lower plasma concentrations of carnosine and the lysophospholipid lysoPC a C18:1. Correlations between the microbiome and metabolome were also explored. Taken together, these findings strengthen the link between Turicibacter abundance and AD, provide a basis for further microbiome studies of murine models for AD, and suggest that greater control over animal model microbiomes is needed in AD research. IMPORTANCE Microorganisms residing within the gastrointestinal tract are implicated in the onset and progression of Alzheimer's disease (AD) through the mediation of inflammation, exchange of small-molecules across the blood-brain barrier, and stimulation of the vagus nerve. Unfortunately, most animal models for AD are housed under conditions that do not reflect real-world human microbial exposure and do not sufficiently account for (or meaningfully consider) variations in the microbiome. An improved understanding of AD model animal microbiomes will increase model efficacy and the translatability of research findings into humans. Here, we present the characterization of the microbiome and metabolome of the 5xfAD mouse model, which is one of the most common animal models for familial AD. The manuscript highlights the importance of considering the microbiome in study design and aims to lay the groundwork for future studies involving mouse models for AD

Phage Cocktails Constrain the Growth of Enterococcus.

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance