Sample Manipulation Research Articles

Evolution of sample-based music authorship network

Sample-based music—characterized by the adoption of extant audio fragments (sampling) in its creation process—plays an essential role in contemporary popular music, fostering inter-generational connections between the creators that have resulted in a rich and diverse sonic landscape. The selection, manipulation, and adoption of samples heavily impact the genre, mood, texture, and finally the distinctive identity of a new musical composition. One could call the samples “cultural genes” of sorts, continually incorporated into new music to contribute to its characteristics. One can then ask how this process has taken place in history and shaped the history of contemporary popular music, which we study in this work. We specifically study the evolution of sample-based music between the years 1980 and 2019, taking cue from the citation network analysis of academic literature that intuitively follows a similar dynamic of the flow of ideas and material from the old works into new ones. First, the community structure in the artist–sample network is identified, and its relationship to distinctive musical styles and flavors is verified. A longitudinal analysis of the passing down of musical styles is then performed based on similarity between communities of distinct eras, identifying continuous temporal developments in music as well as instances of the revival of styles dormant across multiple generations, akin to “genetic atavism.” This study demonstrates the complex nature of cultural evolution using a network framework that is also generally applicable to other creative enterprises.

Combining Top-Down and Bottom-Up: An Open Microfluidic Microtumor Model for Investigating Tumor Cell-ECM Interaction and Anti-Metastasis.

Using a combined top-down (i.e., operator-directed) and bottom-up (i.e., cell-directed) strategy, an Under-oil Open Microfluidic System (UOMS)-based microtumor model is presented for investigating tumor cell migration and anti-metastasis drug test. Compared to the mainstream closed microfluidics-based microtumor models, the UOMS microtumor model features: i) micrometer-scale lateral resolution of surface patterning with open microfluidic design for flexible spatiotemporal sample manipulation (i.e., top-down); ii) self-organized extracellular matrix (ECM) structures and tumor cell-ECM spontaneous remodeling (i.e., bottom-up); and iii) free physical access to the samples on a device with minimized system disturbance. The UOMS microtumor model - allowing a controlled but also self-organized, cell-directed tumor-ECM microenvironment in an open microfluidic configuration - is used to test an anti-metastasis drug (incyclinide, aka CMT-3) with a triple-negative breast cancer cell line (MDA-MB-231). The in vitro results show a suppression of tumor cell migration and ECM remodeling echoing the in vivo mice metastasis results.

Multiplexed Dilute-and-Shoot Liquid Chromatography-Multiple-Reaction Monitoring Mass Spectrometry Clinical Assay for Metanephrines and Catecholamines in Human Urine.

Background: Quantifying urinary catecholamines and metanephrines is essential for the clinical screening and diagnosis of neuroendocrine tumours. HPLC with electrochemical detection (HPLC-ECD) is commonly used for this type of analysis but requires extensive sample cleanup. Simple and rapid dilute-and-shoot LC-multiple-reaction monitoring (MRM)-MS assays have been developed for quantitating these analytes in urine but have not yet been validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Methods: A simple dilute-and-shoot sample preparation without derivatization was used. C18 RP-UPLC-MRM-MS and positive-ion ESI were used, usually with two transitions per analyte being monitored. Certified deuterated internal standards were used for each analyte. Results: This assay was validated according to the CLSI C62-A guidelines, including accuracy/trueness, imprecision, sensitivity, specificity, carryover, stability, and linearity. The final MRM-MS method was compared to the established HPLC-ECD clinical chemistry reference method. The run time was reduced from 25 min to 5 min. Conclusions: A simple, robust, rapid, and cost-effective LC-MRM-MS assay for measuring urinary catecholamines and metanephrines was developed and validated according to the CLSI guidelines. This validated method requires minimal sample manipulation before analysis and provides sensitivity, specificity, and improved precision. The implementation of this assay in clinical laboratories will facilitate early and accurate diagnosis.

Micro- and milli-fluidic sample environments for in situ X-ray analysis in the chemical and materials sciences.

X-ray-based methods are powerful tools for structural and chemical studies of materials and processes, particularly for performing time-resolved measurements. In this critical review, we highlight progress in the development of X-ray compatible microfluidic and millifluidic platforms that enable high temporal and spatial resolution X-ray analysis across the chemical and materials sciences. With a focus on liquid samples and suspensions, we first present the origins of microfluidic sample environments for X-ray analysis by discussing some alternative liquid sample holder and manipulator technologies. The bulk of the review is then dedicated to micro- and milli-fluidic devices designed for use in the three main areas of X-ray analysis: (1) scattering/diffraction, (2) spectroscopy, and (3) imaging. While most research to date has been performed at synchrotron radiation facilities, the recent progress made using commercial and laboratory-based X-ray instruments is then reviewed here for the first time. This final section presents the exciting possibility of performing in situ and operando X-ray analysis in the 'home' laboratory and transforming microfluidic and millifluidic X-ray analysis into a routine method in physical chemistry and materials research.

A novel method for detecting genetic biomarkers in blood-based liquid biopsies using surface plasmon resonance imaging and magnetic beads shows promise in cancer diagnosis and monitoring.

Directly detecting biomarkers in liquid biopsy for diagnosis and personalized treatment plays a crucial role in managing cancer relapse and increasing survival rates. Typically, the standard analysis of circulating tumour DNA requires lengthy isolation, extraction, and amplification steps, leading to sample contamination, longer turnaround time and higher assay costs. Surface plasmon resonance is an emerging and promising technology for rapid and real-time dynamic biomarker monitoring in liquid biopsy. Here, we propose a new SPR imaging biosensing approach to detect tumour DNA circulating in the blood of colorectal cancer patients by exploiting the unique properties of superparamagnetic particles. Micrometer beads functionalized with a biotinylated oligonucleotide can directly capture DNA target sequences bearing single-nucleotide variations of KRAS oncogene in human blood plasma. Mutated and wild-type peptide nucleic acid probes immobilized on an SPR gold surface recognize complementary and non-complementary DNA targets by discriminating a single nucleotide mismatch. The new assay allows for detecting p.G13D mutated DNA in buffer and spiked human plasma at attomolar level (down to 300 copies mL-1) with minimal sample manipulation and in just a few microliters. The assay was validated using plasma samples from colorectal cancer patients and healthy donors, by discriminating mutated DNA circulating in patients and wild-type DNA found in healthy blood donors. This feature underscores the potential of the liquid biopsy assay as a valuable tool for the diagnosis and monitoring of cancer.

An advanced light scattering apparatus for investigating soft matter onboard the International Space Station

Colloidal solids (COLIS) is a state-of-the-art light scattering setup developed for experiments onboard the International Space Station (ISS). COLIS allows for probing the structure and dynamics of soft matter systems on a wide range of length scales, from a few nm to tens of microns, and on time scales from 100 ns to tens of hours. In addition to conventional static and dynamic light scattering, COLIS includes depolarized dynamic light scattering, a small-angle camera, photon correlation imaging, and optical manipulation of thermosensitive samples through an auxiliary near-infrared laser beam, thereby providing a unique platform for probing soft matter systems. We demonstrate COLIS through ground tests on standard Brownian suspensions, and on protein, colloidal glasses, and gel systems similar to those to be used in future ISS experiments.

Ultrastructure Expansion Microscopy (U-ExM) of the Fission Yeast Schizosaccharomyces pombe.

Among widely used super-resolution microscopy techniques, including stimulated emission depletion (STED), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), expansion microscopy (ExM) is unique in achieving increased resolution through a physical manipulation of the actual sample rather than optics or postprocessing. Originally developed for applications in neuroscience, ExM now has a solid foothold across many fields and model systems, and has been adapted to work for organisms with cell walls, including budding and fission yeasts, through the inclusion of a pre-expansion enzymatic digestion step. A variant of the ExM technique optimized for preserving the architecture of protein complexes, ultrastructure expansion microscopy (U-ExM), enables super-resolution imaging of full 3D volumes at increased throughput using conventional microscopes and can be readily combined with commonly used antibodies, dyes, and stains. Here, we present its application to the fission yeast Schizosaccharomyces pombe.

Biodegradable microplastics induce profound changes in lettuce (Lactuca sativa) defense mechanisms and to some extent deteriorate growth traits

The development of agricultural technologies has intensified the use of plastic in this sector. Products of plastic degradation, such as microplastics (MPs), potentially threaten living organisms, biodiversity and agricultural ecosystem functioning. Thus, biodegradable plastic materials have been introduced to agriculture. However, the effects of biodegradable plastic substitutes on soil ecosystems are even less known than those of traditional ones. Here, we studied the effects of environmentally relevant concentrations of MPs prepared from a biodegradable plastic (a starch-polybutylene adipate terephthalate blend, PBAT-BD-MPs) on the growth and defense mechanisms of lettuce (Lactuca sativa) in CLIMECS system (CLImatic Manipulation of ECosystem Samples). PBAT-BD-MPs in the highest concentrations negatively affected some traits of growth, i.e., dry weight percentage, specific leaf area, and both C and N contents. We observed more profound changes in plant physiology and biochemistry, as PBAT-BD-MPs decreased chlorophyll content and triggered a concerted response of plant defense mechanisms against oxidative stress. In conclusion, exposure to PBAT-BD-MPs induced plant oxidative stress and activated plant defense mechanisms, leading to oxidative homeostasis that sustained plant growth and functioning. Our study highlights the need for in-depth understanding of the effect of bioplastics on plants.

Near-Infrared Spectroscopy for Growth Estimation of Spirulina platensis Cultures.

The present study proposes the use of Near-Infrared (NIR) spectroscopy as a rapid method for estimating the growth of Spirulina platensis cultures, avoiding any sample manipulation or pretreatment. NIR spectroscopy in diffuse reflectance mode was used on culture volumes as received, with Principal Component Analysis (PCA) and Partial Least Squares (PLS) linear regression, for developing the calibration model in the wavelength range of 1000-2500 nm, in order to choose the appropriate wavelength to estimate the growth of the microalga. The local reflectance maximum at 1062.6 nm, connected with reduced water absorption and scattering effects by the microalga, was identified from PCA as the positive peak in the first loading plot, correlating diffuse reflectance with dilution levels. The calibration curve of diffuse reflectance at 1062.6 nm in response to dilution presented strong linearity, supported by a coefficient of determination (R2) of 0.995. Cross-validation of NIR spectra with a S. platensis culture confirmed the method's reliability, showing that the growth follows an exponential pattern. The study shows that diffuse reflectance NIR spectroscopy can be used for the rapid monitoring of Spirulina platensis growth.

Non-destructive and rapid identification of yeasts by nearinfrared spectroscopy and machine learning

This study aimed to apply near-infrared (NIR) spectroscopy combined with machine learning techniques to identify yeast strains rapidly and practically, comparing the results with traditional molecular identification methods. Yeasts were isolated from the digestive tracts of aquatic mining insects collected in the extreme north of the Western Amazon (Roraima), Brazil, and preserved through cryopreservation and mineral oil methods. Molecular identification involved PCR amplification and sequencing of ribosomal DNA regions. NIR spectroscopy, coupled with multivariate analysis and machine learning algorithms such as principal component analysis (PCA), hierarchical cluster analysis (HCA), k-nearest neighbor (KNN), and soft independent modeling by class analogy (SIMCA), was used to analyze and classify the yeast samples, accurately identified yeast strains at the genus and species levels, achieving 100% accuracy in both the calibration and validation sets. The results indicate that this method provides a rapid, non-destructive, and environmentally friendly alternative to traditional molecular techniques, making it suitable for real-time, in situ analysis with minimal sample manipulation.

Transdermal Minimally Invasive Optical Multiplex Detection of Protein Biomarkers by Nanopillars Array-Embedded Microneedles.

Biomarkers detection has become essential in medical diagnostics and early detection of life-threatening diseases. Modern-day medicine relies heavily on painful and invasive tests, with the extraction of large volumes of venous blood being the most common tool of biomarker detection. These tests are time-consuming, complex, expensive and require multiple sample manipulations and trained staff. The application of "intradermal" biosensors utilizing microneedles as minimally invasive sensing elements for capillary blood biomarkers detection has gained extensive interest in the past few years as a central point-of-care (POC) detection platform. Herein, we present a diagnosis paradigm based on vertically aligned nanopillar array-embedded microneedles sampling-and-detection elements for the direct optical detection and quantification of biomarkers in capillary blood. We present here a demonstration of the simple fabrication route for the creation of a multidetection-zone silicon nanopillar array, embedded in microneedle elements, followed by their area-selective chemical modification, toward the multiplex intradermal biomarkers detection. The utilization of the rapid and specific antibody-antigen binding, combined with the intrinsically large sensing area created by the nanopillar array, enables the simultaneous efficient ultrafast and highly sensitive intradermal capillary blood sampling and detection of protein biomarkers of clinical relevance, without requiring the extraction of blood samples for the ex vivo biomarkers analysis. Through preliminary in vitro and in vivo experiments, the direct intradermal in-skin blood extraction-free platform has demonstrated excellent sensitivity (low pM) and specificity for the accurate multiplex detection of protein biomarkers in capillary blood.

End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

Contactless Manipulation and Raman Analysis of Cometary Analogs and Micrometeorites by Acoustic Levitation

Extraterrestrial material collected during space missions is highly exposed to contamination issues during on-Earth analysis. Although high-protection-level protocols were developed, to minimize the contamination due to sample manipulation and the substrate contribution an optimal strategy is to perform in situ analysis with contactless techniques. Optical and acoustic trapping represent ideal candidates for contactless manipulation and analysis of nanometer-to-millimeter-sized particles. Here, we show results of the manipulation of cometary analogs and micrometeorite samples using a single-axis acoustic levitator. The investigation of the particle dynamics in the trap allows the calculation of the trap spring constants that are found in the mN/m range. In addition, we collect the Raman spectra of two levitated fragments of Saratov meteorite, demonstrating that acoustic levitation can be effectively used for the contactless and low-contamination characterization of samples of interest in astrophysics.

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7min over ten 88×88µm2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220nm in diameter with a dual 440 and 740nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100nm-in-diameter EVs with fluorescently tagged membrane proteins.

Extracellular RNAs from Whole Urine to Distinguish Prostate Cancer from Benign Prostatic Hyperplasia.

RNAs, especially non-coding RNAs (ncRNAs), are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Altered expression patterns of ncRNAs have been observed in prostate cancer (PCa), contributing to the disease's initiation, progression, and treatment response. This study aimed to evaluate the ability of a specific set of RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and mRNAs, to discriminate between PCa and the non-neoplastic condition benign prostatic hyperplasia (BPH). After selecting by literature mining the most relevant RNAs differentially expressed in biofluids from PCa patients, we evaluated their discriminatory power in samples of unfiltered urine from 50 PCa and 50 BPH patients using both real-time PCR and droplet digital PCR (ddPCR). Additionally, we also optimized a protocol for urine sample manipulation and RNA extraction. This two-way validation study allowed us to establish that miRNAs (i.e., miR-27b-3p, miR-574-3p, miR-30a-5p, and miR-125b-5p) are more efficient biomarkers for PCa compared to long RNAs (mRNAs and lncRNAs) (e.g., PCA3, PCAT18, and KLK3), as their dysregulation was consistently reported in the whole urine of patients with PCa compared to those with BPH in a statistically significant manner regardless of the quantification methodology performed. Moreover, a significant increase in diagnostic performance was observed when molecular signatures composed of different miRNAs were considered. Hence, the abovementioned circulating ncRNAs represent excellent potential non-invasive biomarkers in urine capable of effectively distinguishing individuals with PCa from those with BPH, potentially reducing cancer overdiagnosis.

Complete resolution across the neodymium/samarium isotopic envelope with a liquid sampling-atmospheric pressure glow discharge - Orbitrap mass spectrometer.

Nd and Sm isotope ratios play an important role in geological dating and as nuclear forensic signatures; however, the overlap of the respective 144, 148, 150 Nd/Sm isobars requires prior separations to be performed before analysis on typical MS platforms. The work presented here overcomes these isobaric interferences using ultrahigh-mass resolution to alleviate interference without prior chemical separations. A liquid sampling-atmospheric pressure glow discharge ion source was coupled to a standard, QExactive Focus Orbitrap mass spectrometer, providing a mass resolution of ~80 k. A Spectroswiss FTMS booster X2 data acquisition package was used to collect extended transients, providing much higher mass resolution; ~230 k and ~600 k are employed here for Nd and Sm isotopes. While the standard Orbitrap resolution is far greater than typical "atomic" MS platforms, it was insufficient to alleviate all isobars. The use of a resolution of ~230 k resulted in baseline separation across the entire isotopic envelope for both Nd and Sm. Isotope ratios obtained from Nd:Sm mixtures using high-resolution were equivalent to those found for individual-element solutions, while isotope ratios obtained at a resolution of ~80 k (standard for the OEM data system) showed large deviations. Use of ultrahigh-resolution is an attractive alternative to extensive chemical separations to alleviate severe isobaric interferences. Sufficient mass resolution greatly reduces/eliminates the need for sample manipulations (separations) before analysis while reducing costs and total analysis times.

Electronic detection of apoptotic cells on a microchip

Robust and rapid detection of apoptosis in cells is crucially needed for diagnostics, drug discovery, studying pathogenic mechanisms and tracking patient response to medical interventions and treatments. Traditionally, the methods employed to detect apoptosis rely on complex instrumentation like flow cytometers and fluorescence microscopes, which are both expensive and complex-to-operate except in centralized laboratories with trained labor. In this work, we introduce a microfluidic device that can screen cells in a suspension for apoptosis markers and report the assays results as electronic data. Specifically, our device identifies apoptotic cells by detecting externalized phosphatidylserine on a cell membrane – a well-established biomarker that is also targeted by fluorophore-based labeling in conventional assays. In our device, apoptotic cells are discriminated from others through biochemical capture followed by transduction of individual capture events into electrical signals via integrated electrical sensors. The developed technology was tested on simulated samples containing controlled amounts of cells with artificially-induced apoptosis and validated by benchmarking against conventional flow cytometry. Combining sample manipulation and electronic detection on a disposable microfluidic chip, our cell apoptosis assay is amenable to be implemented in a variety of settings and therefore has the potential to create new opportunities for cell-based diagnostics and therapeutics and contribute to improved healthcare outcomes on a large scale.

DESIGN OF A DEFERRED LABORATORY IN RADIOACTIVITY

This article describes the design of a Remote Laboratory (LR) for nuclear measurements. The use of LR is presented as an opportunity since it allows the process of manipulation and characterization of irradiated samples, as is common in experimentation, to be replicated more times than in a Traditional Laboratory (LT). The rapid technological development in the demanding and changing work environment and the difficult access or availability on the part of students to the infrastructure necessary to carry out specific practices, among other reasons, make it necessary to incorporate these new tools to facilitate the learning of the technology. The research and development presented here are being carried out in the RA-4 experimental reactor of the Institute of Nuclear Studies and Ionizing Radiations (IENRI) of Argentina, which will allow us to carry out student practices at any time, without having to the three people with the corresponding authorizations for handling the irradiated samples must be present: the reactor manager, an operator, and a radioprotection officer. Considering also that the sources to irradiate are scarce and the instrumentation is expensive. Keywords: Remote Laboratory, Radioactivity.

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Association of microbial community structure with gill disease in marine-stage farmed Atlantic salmon (Salmo salar); a yearlong study

BackgroundUnderstanding the relationship between resident microbiota and disease in cultured fish represents an important and emerging area of study. Marine gill disorders in particular are considered an important challenge to Atlantic salmon (Salmo salar) aquaculture, however relatively little is known regarding the role resident gill microbiota might play in providing protection from or potentiating different gill diseases. Here, 16S rRNA sequencing was used to examine the gill microbiome alongside fish health screening in farmed Atlantic salmon. Results were used to explore the relationship between microbial communities and gill disease.ResultsMicrobial community restructuring was observed throughout the sampling period and linked to varied drivers of change, including environmental conditions and severity of gill pathology. Taxa with significantly greater relative abundance on healthier gills included isolates within genus Shewanella, and taxa within family Procabacteriaceae. In contrast, altered abundance of Candidatus Branchiomonas and Rubritalea spp. were associated with damaged gills. Interestingly, more general changes in community richness and diversity were not associated with altered gill health, and thus not apparently deleterious to fish. Gross and histological gill scoring demonstrated seasonal shifts in gill pathology, with increased severity of gill damage in autumn. Specific infectious causes that contributed to observed pathology within the population included the gill disorder amoebic gill disease (AGD), however due to the uncontrolled nature of this study and likely mixed contribution of various causes of gill disease to observed pathology results do not strongly support an association between the microbial community and specific infectious or non-infectious drivers of gill pathology.ConclusionsResults suggest that the microbial community of farmed Atlantic salmon gills undergo continual restructuring in the marine environment, with mixed influences upon this change including environmental, host, and pathogenic factors. A significant association of specific taxa with different gill health states suggests these taxa might make meaningful indicators of gill health. Further research with more frequent sampling and deliberate manipulation of gills would provide important advancement of knowledge in this area. Overall, although much is still to be learnt regarding what constitutes a healthy or maladapted gill microbial community, the results of this study provide clear advancement of the field, providing new insight into the microbial community structure of gills during an annual production cycle of marine-stage farmed Atlantic salmon.