Biology subjects

Needham, D.

Publications and source records attributed to Needham, D..

6 recordsLinked to original sources

bioRxiv2024

Arctic Ocean virus communities: seasonality, bipolarity, and prokaryotic interactions

Viruses play important roles in ocean environments as agents of mortality and genetic transfer, influencing ecology, evolution and biogeochemical processes. However, we know little about the diversity, seasonality, and host interactions of viruses in polar waters. To address this, we studied dsDNA viruses in the Arctic Fram Strait across four years via 47 long-read metagenomes of the cellular size-fraction. Among 5,662 vOTUs, 69%, 30% and 1.4% were bacteriophages (Myoviridae, Podoviridae, and Siphoviridae), Unassigned, and Phycodnaviridae, respectively. Viral coverage was, on average, 5-fold higher than prokaryotic coverage, and 8-fold higher in summer. Viral community composition showed annual peaks in similarity and was strongly correlated with prokaryotic community composition. Using a Convergent Cross Mapping network, we identified putative virus-host interactions and six ecological modules, each associated with distinct environmental conditions. The network also revealed putative novel cyanophages with time-lagged correlations to their hosts (late summer) as well as diverse viruses correlated with Nitrososphaerales (winter). By comparison with global metagenomes, we found that 42% of Fram Strait vOTUs peaked in abundance in high latitude regions of both hemispheres (average 61{degrees}N and 51{degrees}S), and encoded proteins with biochemical signatures of cold adaptation. Our study reveals a rich diversity of polar viruses with pronounced seasonality, providing a foundation for understanding how they regulate and impact ecosystem functionality in changing polar oceans.

microbiology↗

bioRxiv2022

GBP2 aggregates LPS and activates the caspase-4 inflammasome independent of the bacterial encapsulation factor GBP1

Sensing and killing of intracellular bacterial pathogens are important features of cell-autonomous immunity. The cytokine gamma-interferon (IFN{gamma}) enhances cell-autonomous immunity through upregulation of interferon stimulated genes such as guanylate binding proteins (GBPs). GBPs promote defense against Gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate caspase-4 sensing of the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). There are seven human GBP paralogs and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS and recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore Gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates free LPS through protein polymerization. This provides a novel mechanistic framework for non-canonical inflammasome activation where GBP1 or GBP2 assemble cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to Gram-negative bacterial infections. Significance StatementSensing Gram-negative bacterial lipopolysaccharide by human caspase-4 is critical for host defense to intracellular Gram-negative bacterial pathogens. Human guanylate binding proteins (GBPs) facilitate caspase-4 activation in response to Gram-negative infections by a poorly understood mechanism. The prevailing model suggests GBP1 binding to bacteria and consequential recruitment of caspase-4 to the bacterial surface are essential for triggering this host response. Here, we show GBP1 binding to bacteria is dispensable for caspase-4 activation and identify GBP2 as an additional lipopolysaccharide-binding protein that can functionally replace GBP1. We demonstrate that GBP1 and GBP2 share the ability to form lipopolysaccharide-protein complexes, which, we propose, allow caspase-4 activation. Our study provides a new mechanistic framework for cytosolic LPS sensing.

immunology↗

bioRxiv2021

Investigation of the futalosine pathway for menaquinone biosynthesis as a novel target in the inhibition of Chlamydia trachomatis infection

Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow spectrum targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through LC-MS/MS. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.

biochemistry↗

bioRxiv2021

The pH Dependence of Niclosamide Solubility, Dissolution and Morphology Motivates a Potentially More Bioavailable Mucin-Penetrating Nasal and Throat Spray for COVID19, it's Contagious Variants, and Other Respiratory Viral Infections

MotivationWith the coronavirus pandemic still raging, prophylactic nasal and early treatment throat sprays could help prevent infection and reduce viral load. Niclosamide has the potential to treat a broad range of viral infections if local bioavailability is optimized as mucin-penetrating solutions as opposed to more traditional microparticle-based sprays that cannot penetrate the mucin. ExperimentalpH-dependence of supernatant concentrations and dissolution rates of niclosamide were measured in buffered solutions by Nanodrop-UV/Vis-spectroscopy for niclosamide from different suppliers, as precipitated material, and as cosolvates. Data was compared to predictions from Henderson Hasselbalch and precipitation pH models. Optimal microscopy was used to observe the morphologies of precipitated and converted niclosamide. ResultsSupernatant-concentrations of niclosamide increased with increasing pH, from 1.77uM at pH 3.66 to 30uM at pH 8, and more rapidly from 90uM at pH8.5 to 300uM at pH 9.1, reaching 641uM at pH 9.5. Logarithmic rates for dissolution increased by [~]3x for pHs 8.62 to 9.44. However, when precipitated from supersaturated solution, niclosamide equilibrated to much lower final supernatant concentrations, reflective of more stable polymorphs at each pH that were also apparent for niclosamide from other suppliers and cosolvates. ConclusionsGiven niclosamides activity against COVID19, its more contagious variants, and other respiratory viral infections, these niclosamide solutions, that put the virus in lockdown, could represent universal prophylactic nasal and early treatment throat sprays. As solutions they would be the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, with no cold chain. They now just need testing.

pharmacology and toxicology↗

bioRxiv2020

Designing Topographically Textured Microparticles For Induction and Modulation of Osteogenesis in Mesenchymal Stem Cell Engineering

Mesenchymal stem cells have been the focus of intense research in bone development and regeneration. We demonstrate the potential of microparticles as modulating moieties of osteogenic response by utilizing their architectural features. Topographically textured microparticles of varying microscale features were produced by exploiting phase-separation of a readily-soluble sacrificial component from polylactic acid. The influence of varying topographical features on primary human mesenchymal stem cell attachment, proliferation and markers of osteogenesis was investigated. In the absence of osteoinductive supplements, cells cultured on textured microparticles exhibited notably increased expression of osteogenic markers relative to conventional smooth microparticles. They also exhibited varying morphological, attachment and proliferation responses. Significantly altered gene expression and metabolic profiles were observed, with varying histological characteristics in vivo. This study highlights how tailoring topographical design offers cell-instructive 3D microenvironments which allow manipulation of stem cell fate by eliciting the desired downstream response without use of exogenous osteoinductive factors.

bioengineering↗

bioRxiv2017

Taxon disappearance from microbiome analysis indicates need for mock communities as a standard in every sequencing run

Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification, sequencing, and to optimize pipeline outputs. Nevertheless, the necessity of routine mock community analysis beyond initial method development is rarely, if ever, considered. Here we report that our routine use of mock communities as internal standards allowed us to discover highly aberrant and strong biases in the relative proportions of multiple taxa in a single Illumina HiSeqPE250 run. In this run, an important archaeal taxon virtually disappeared from all samples, and other mock community taxa showed >2-fold high or low abundance, whereas a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded \"normal\" results. Although obvious from the strange mock community results, due to natural variation of microbiomes at our site, we easily could have missed the problem had we not used the mock communities. The \"normal\" results were validated over 4 MiSeqPE300 runs and 3 HiSeqPE250 runs, and run-to-run variation was usually low (Bray-Curtis distance was 0.12{+/-}0.04). While validating these \"normal\" results, we also discovered some mock microbial taxa had relatively modest, but consistent, differences between sequencing platforms. We suggest that using mock communities in every sequencing run is essential to distinguish potentially serious aberrations from natural variations. Such mock communities should have more than just a few members and ideally at least partly represent the samples being analyzed, to detect problems that show up only in some taxa, as we observed.\n\nImportanceDespite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of \"control\"), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs, and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be avoided by the use of suitable mock communities.

microbiology↗