High-throughput Microfluidics Research Articles

SELEX- Aptamer Technology to Combat Diseases of Animals : Recent Advancement and Future Application: A Review

Aptamers are ‘nucleic acid antibodies’ that bind to target molecules with high affinity and specificity. The process by which aptamers are selected is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). A plethora of aptamers have been selected against various targets, such as proteins, cells, microorganisms, chemical compounds, etc. Technical progress in the aspects of high throughput sequencing (HTS) technologies, bioinformatics, microfluidics and nanodevices have widened the scope of aptamers to contribute significantly in human as well as veterinary medicine. We have searched the Pubmed database and Google Scholar with the search term ‘aptamer’, ‘SELEX’, ‘animal diseases’. The search results have been categorized into bacterial, viral and protozoal diseases of veterinary importance. Here we have systematically described the strategy used to design aptamers for each disease and also shed light on the future research scope in this area. This article presents a comprehensive and up-to-date review of SELEX-aptamer technology, its modifications, aptamers selected against various animal pathogens, their potential to be applied in the future for diagnostic and therapeutic purposes.

A new-to-nature carboxylation module to improve natural and synthetic CO2 fixation

The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

Abstract 2506: Simultaneous DNA, RNA and protein analysis from single cells using a high-throughput microfluidic workflow for resolution of genotype-to-phenotype modalities

Abstract The TCGA, ICGC and other research consortiums has shown that an integral, multi-omic analysis of tumors is necessary in order to understand a more complete picture of the signaling pathways involved in development, evolution, prognosis and treatment resistance of tumors. However, in such studies, because different data modalities (“omics”) are analyzed separately, the information on how the co-existence of in the same cell is lacking. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using high-throughput droplet microfluidics on the Tapestri platform, paired with next generation sequencing. Our triomic methodology evaluates targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations (SNV & CNV) from the same cell. We employ oligonucleotide-conjugated antibody panels to probe cellular surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with cocktails of oligonucleotide-antibodies. These cells are loaded onto the Mission Bio Tapestri® microfluidic cartridge for generation of the first droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA from chromatin scaffolding. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced and demultiplexed bioinformatically yielding a multi-omics readout from the single-cells. Six protein surface markers, 35 transcripts and 88 genomic regions in 68 genes were interrogated with a 50:50 cell mixture of MCF7 and GM12878 cells. These targets play key roles in cellular migration, stemness, differentiation and proliferation. We intend to further explore the relationship between genotype-to-phenotype in breast carcinoma cell lines cultured in patient-derived scaffolds mimicking tumor microenvironments under different drug regimen conditions. We report the dynamic multimodal resolution and demonstrate utility of triomics in further illuminating cellular states and biological responses. Citation Format: Pedro Mendez, Dalia Dhingra, Aik Ooi, Shu Wang, Saurabh Gulati, Nigel Beard, Manimozhi Manivannan, Jens Björkman, Mikael Kubista, Göran Landberg, David Ruff, Anders Ståhlberg. Simultaneous DNA, RNA and protein analysis from single cells using a high-throughput microfluidic workflow for resolution of genotype-to-phenotype modalities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2506.

Abstract LB-316: Innovative multimodal single-cell workflow to interrogate cellular responses to cancer therapy

Abstract Elucidation of biological processes that drive cellular migration, proliferation, stemness and differentiation have powered our view of tumor initiation and promotion. These avenues of research are reliant on engaging key biomarkers to measure and quantify cell systems. Widely practiced methodologies include metabolomic assays, immunoassays, imaging platforms and genomic tools. Understanding the genomic landscape of cancer has proven valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. For sampling convenience, genomic measurements are typically applied to bulk cell samples. Bulk samples often obscure important rare cell types that define disease progression. Single-cell genomic analysis can highlight rare cells. Such efforts commonly generate one “modal” of information, most often next generation sequencing datasets containing RNA transcriptomic expression profiles. While transcriptomic information has great utility for plotting global patterns of response, correlating RNA dynamics to genomic variation and protein levels has proven difficult, especially at the single-cell level. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using high-throughput droplet microfluidics paired with next generation sequencing readouts. Our procedure determines targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts and DNA amplicons. We employ oligo-conjugated antibody panels to probe cell surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with cocktails of oligonucleotide-antibodies. Cells are loaded onto the Mission Bio Tapestri microfluidic cartridge for generation of a droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA/mRNA. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced yielding a multimodal readout. We applied this technique to several dynamic immunology and oncology in vitro models. We explored the relationship between genotype-to-phenotype in breast carcinoma cell lines cultured under different drug regimen conditions, PBMC cell population responses to mitogenic perturbations and imatinib impact on retinoblastoma and BCR-ABL driven leukemic cells. We note CNV influences (such as ERRB) on gene and protein expression as well as RNA-protein concordances. Our pilot studies demonstrate the utility of multimodal resolution in further illuminating cellular states and biological responses in cancer cells. Citation Format: David Ruff, Dalia Dhingra, Pedro Mendez, Aik Ooi, Adam Sciambi, Shu Wang. Innovative multimodal single-cell workflow to interrogate cellular responses to cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-316.

Elongation Index as a Sensitive Measure of Cell Deformation in High-Throughput Microfluidic Systems

One of the promising approaches for high-throughput screening of cell mechanotype is microfluidic deformability cytometry (mDC), in which the apparent deformation index (DI) of the cells stretched by extensional flow at the stagnation point of a cross-slot microchannel is measured. The DI is subject to substantial measurement errors due to cell offset from the flow centerline and velocity fluctuations in inlet channels, leading to artificial widening of DI versus cell size plots. Here, we simulated an mDC experiment using a custom computational algorithm for viscoelastic cell migration. Cell motion and deformation in a cross-slot channel was modeled for fixed or randomized values of cellular mechanical properties (diameter, shear elasticity, cortical tension) and initial cell placement, with or without sinusoidal fluctuations between the inlet velocities. Our numerical simulation indicates that mDC loses sensitivity to changes in shear elasticity when the offset distance exceeds 5 μm, and just 1% velocity fluctuation causes an 11.7% drop in the DI. The obtained relationships between the cell diameter, shear elasticity, and offset distance were used to establish a new measure of cell deformation, referred to as the “elongation index” (EI). In the randomized study, the EI scatter plots were visibly separated for the low- and high-elasticity populations of cells, with a mean of 300 and 3500 Pa, whereas the standard DI output was unable to distinguish between these two groups of cells. The successful suppression of the offset artifacts with a narrower data distribution was shown for the EI output of MCF-7 cells.

High-throughput microfluidics for evaluating microbubble enhanced delivery of cancer therapeutics in spheroid cultures

Drug penetration into solid tumours remains a major challenge in the effective treatment of cancer. Microbubble (MB) mediated sonoporation offers a potential solution to this by enhancing the uptake of drugs into cells. Additionally, in using an ultrasound (US) trigger, drug delivery can be localised to the tumour, thus reducing the off-site toxicity associated with systemic delivery. The majority of in vitro studies involving the observation of MB-enhanced drug efficacy have been conducted on 2D monolayer cell cultures, which are known to be poor models for in vivo tumours. 3D spheroid cultures allow for the production of multicellular cultures complete with extracellular matrix (ECM) components. These cultures effectively recreate many of the physiological features of the tumour microenvironment and have been shown to be far superior to previous 2D monolayer models. However, spheroids are typically handled in well-plates in which the fluid environment is static, limiting the physiological relevance of the model. The combination of 3D cultures and microfluidics would allow for the production of a dynamic system in which spheroids are subjected to in vivo like fluid flow and shear stresses. This study presents a microfluidic device containing an array of spheroid traps, into which multiple pre-grown colorectal cancer (CRC) spheroids were loaded. Reservoirs interfaced with the chip use hydrostatic pressure to passively drive flow through the system and subject spheroids to capillary like flow velocities. The use of reservoirs also enabled multiple chips to be run in parallel, allowing for the screening of multiple therapeutic treatments (n = 690 total spheroids analysed). This microfluidic platform was used to investigate MB enhanced drug delivery and showed that co-delivery of 3 μM doxorubicin (DOX) + MB + US reduced spheroid viability to 48 ± 2%, compared to 75 ± 5% observed with 3 μM DOX alone. Delivery of drug loaded MBs (DLMBs), in which DOX-loaded liposomes (DOX-LS) were conjugated to MBs, reduced spheroid viability to 62 ± 3%, a decrease compared to the 75 ± 3% viability observed with DOX-LS in the absence of MBs + US.

Tumor-Adhesive and pH-Degradable Microgels by Microfluidics and Photo-Cross-Linking for Efficient Antiangiogenesis and Enhanced Cancer Chemotherapy

Tumor angiogenesis with the vascular network formation provides nutrition and oxygen for cancer cells, promoting the proliferation and metastasis of malignant tumors. Bevacizumab (Bev) as an efficient antiangiogenic antibody is able to normalize the tumor vasculature with better blood flow and reduced interstitial fluid pressure, allowing drugs with more uniform distribution and deeper penetration into the tumor; however, it is highly difficult to realize the simultaneous delivery of Bev and anticancer drugs localized at the tumor tissue. Here, we prepared tumor-adhesive and pH-degradable poly(vinyl alcohol) (PVA) microgels for tumor-localized delivery of Bev and docetaxel (DTX), to achieve efficient antiangiogenesis and enhanced cancer chemotherapy. PVA microgels (∼200 μm) decorated with tissue-adhesive dopamine (DA) moieties were fabricated by a combination of high-throughput microfluidics technology and photo-cross-linking chemistry with a considerable coencapsulation efficiency for Bev and DTX. PVA microgels exhibited sustained drug release at the tumoral acidic conditions as the microgel degradation, and DA moieties on the microgels facilitated Bev with long retention at the tumor tissue, highly blocking the vascular endothelial growth factor (VEGF) and inhibiting tumor angiogenesis, as compared to free Bev or no DA-decorated microgels. In addition, the antitumor activity on the 4T1-Luc breast tumor mouse model treated with Bev/DTX-coloaded microgels showed obviously superior tumor growth inhibition than the other treatment groups, in which the combinational therapy efficacy of Bev and DTX mediated by the tumor-adhesive microgels was further confirmed by the immunohistochemistry (IHC) analysis. These PVA microgels with efficient antiangiogenesis and enhanced cancer chemotherapy provide a highly potential platform to treat different malignant tumors as well as the recurrent and metastatic tumors.

A High-Throughput Assay Platform for Next-Generation Mechanistic Enzymology and Applications

Twentieth-century biochemistry dissected cellular metabolism, identified enzymes responsible for its regulation, and revealed reaction pathways. Nevertheless, enhanced understanding is needed to dissect evolutionary pathways, rationally engineer novel catalysts, and predict disease effects of human alleles. Deep mechanistic studies of active site residues have revealed much about how they contribute to catalysis, but the enzyme scaffold is required for these residues to participate in catalysis. Deep mutational scanning and other current high-throughput approaches have revealed functional effects throughout enzyme scaffolds, but these studies intrinsically lack mechanistic depth. We have developed HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a technology for expression, purification, and assay of >1000 rationally-selected enzyme variants in a single experiment, thereby merging the power of traditional and recent high-throughput technologies. HT-MEK yields quantitative measurements of multiple kinetic and thermodynamic parameters for each variant. As a first demonstration of HT-MEK, we performed enzyme-wide mutational scans of a well-characterized Alkaline Phosphatase superfamily member, PafA. We made thousands of kinetic and thermodynamic measurements for over 1000 PafA variants with a battery of substrates and inhibitors to probe specific catalytic features. Consistent with the simplest expectation, the affecting mutations cluster around the active site regions previously shown to be directly involved in each mechanism. Nevertheless, the extent and direction of these clusters was not predictable, nor was the observation that several clusters extend to the protein surface, demonstrating that HT-MEK can be used to identify surfaces that may allow allosteric control of particular functions. Parallel approaches provide analogous quantitative stability measurements, some of which we will also present. The efficient and systematic dissection of any enzyme or system of enzymes allowed by HT-MEK enables next-generation applications to engineer allostery, novel catalysts, and efficient biosynthetic pathways for use in industry and medicine.

Versatile and High-throughput Force Measurement Platform for Dorsal Cell Mechanics

We present a high-throughput microfluidics technique facilitating in situ measurements of cell mechanics parameters at the dorsal side of the cell, including molecular binding strengths, local traction forces, and viscoelastic properties. By adjusting the flow rate, the force magnitude exerted on the cell can be modulated ranging from ~14 pN to 2 nN to perturb various force-dependent processees in cells. Time-lapse images were acquired to record events due to such perturbation. The values of various mechanical parameters are subsequently obtained by single particle tracking. Up to 50 events can be measured simultaneously in a single experiment. Integrating the microfluidic techniques with the analytic framework established in computational fluid dynamics, our method is physiologically relevant, reliable, economic and efficient.

Splendid Isolation for Cells Screened via High-Throughput Microfluidics

Genetic Engineering & Biotechnology NewsVol. 39, No. 7 DepartmentsSplendid Isolation for Cells Screened via High-Throughput MicrofluidicsSphere Fluidics uses picodroplet technology to expedite single-cell analysisGail DuttonGail DuttonSearch for more papers by this authorPublished Online:28 Jun 2019https://doi.org/10.1089/gen.39.07.04AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 39Issue 7Jul 2019 InformationCopyright © by GEN PublishingTo cite this article:Gail Dutton.Splendid Isolation for Cells Screened via High-Throughput Microfluidics.Genetic Engineering & Biotechnology News.Jul 2019.10-12.http://doi.org/10.1089/gen.39.07.04Published in Volume: 39 Issue 7: June 28, 2019PDF download

Passive microinjection within high-throughput microfluidics for controlled actuation of droplets and cells

Microinjection is an effective actuation technique used for precise delivery of molecules and cells into droplets or controlled delivery of genes, molecules, proteins, and viruses into single cells. Several microinjection techniques have been developed for actuating droplets and cells. However, they are still time-consuming, have shown limited success, and are not compatible with the needs of high-throughput (HT) serial microinjection. We present a new passive microinjection technique relying on pressure-driven fluid flow and pulsative flow patterns within an HT droplet microfluidic system to produce serial droplets and manage rapid and highly controlled microinjection into droplets. A microneedle is secured within the injection station to confine droplets during the microinjection. The confinement of droplets on the injection station prevents their movement or deformation during the injection process. Three-dimensional (3D) computational analysis is developed and validated to model the dynamics of multiphase flows during the emulsion generation. We investigate the influence of pulsative flows, microneedle parameters and synchronization on the efficacy of microinjection. Finally, the feasibility of implementing our microinjection model is examined experimentally. This technique can be used for tissue engineering, cells actuation and drug discovery as well as developing new strategies for drug delivery.

A Special Connection between Nanofabrication and Analytical Devices: Chemical Lift-Off Lithography

Abstract Chemical lift-off lithography (CLL) is a chemo-topographical nanopatterning technique developed in 2012 which provides not only precise geometry control with sub-20 nm resolution, but also sharp material interface environment adjustments. Unlike most fabrication techniques, this approach builds a straightforward bridge between structure generation and analytical device designs. This property lies on a direct molecular pattern creation on a metal surface, where the produced unique interface environment acts as a supporting matrix for diverse analytical tools. Operation of CLL includes stamp activation, self-assembled monolayer formation, conformal sealing, and finally interface molecule lift-off. Creation of molecular patterns is achieved by using stamps casting with structures, and a featureless master generates active surfaces capable of arbitrary functionalization. The post lift-off areas present an environment distributed with residual molecules, and its composition can be well-tuned by interface reaction conditions and monolayer selection. Connections between CLL and analytical devices initiate from this point, where tools such as functional probe anchoring, microscopy, high-throughput microfluidics, and spectroscopy can all be integrated. This paper outlines the ideas behind CLL and connections to analytical devices, where invention background introduction, unique molecular environment discussion, modern platform demonstration, challenges in the future, and exciting perspective work in coming years are all included.

A Microfluidics-Based Assay for Mapping Connectivity in Highly Proficient Enzymes Reveals Functional Modularity

Enzymes are some of the most proficient and most specific catalysts known; however, a complete understanding of how these enormous rate enhancements are achieved is lacking. Enzymes are large and highly cooperative, containing functional connections throughout their entire structures. However, due to the cost and labor required to prepare and assay the number of mutants required to characterize this connectivity, it has been difficult to obtain the large sets of data necessary for improving rational enzyme design; as a result, the proficiencies of designed enzymes fall well short of their natural counterparts. To address these experimental limitations, we have developed a microfluidic assay, HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), in which we express, purify, and quantitatively characterize >1000 enzyme mutants in a single experiment across multiple kinetic parameters. This platform greatly reduces both time and cost compared to traditional assays, while providing data of comparable precision. Using this technology, we performed systematic mutagenesis over the entire scaffold of the highly proficient phosphatase PafA. We measured the activity of each mutant against multiple substrates and inhibitors, obtaining the same quantitative biochemical parameters as obtained from traditional assays: kcat, KM, kcat/KM, and KI. Together, these measurements define a quantitative functional ‘fingerprint’ for each mutation. Mutants having similar fingerprints distinctly cluster around the specific active site residues that display similar mutational fingerprints when themselves mutated. These comparisons reveal functional connections between individual active site residues and those throughout the structure, and suggest a high degree of modularity within the enzyme. The ability to rapidly characterize thousands of mutants with the quantitative precision of traditional assays will allow us to efficiently test models from computation and phylogeny, while providing the necessary data to improve future design efforts.

Bringing Enzymology into the Genomic Era: Developing and Deploying New Tools to Quantitatively Map Functional Connections Throughout an Enzyme

Despite enormous advances in our understanding of enzymes and their mechanisms over the past decades, how they achieve their enormous rate enhancements and exquisite specificities remains elusive, as evidenced by our limited ability to engineer new enzymes. A fundamental reason for this lack of understanding is the large number of interconnected residues and long-range interactions that cannot be revealed via traditional, low-throughput quantitative assays. While high-throughput screens and selections can identify enzymes with desired changes, the improvements obtained are typically modest and the qualitative or limited quantitative data obtainable by these approaches is insufficient to reveal functional interconnections between remote and active site residues. To meet this unmet need, we have developed a new technology, HT-MEK (High-throughput Microfluidic Enzyme Kinetics), that allows measurement of myriad quantitative biochemical parameters for 1000 rationally chosen enzyme variants in a single experiment. Using this platform, we carried out deep functional analysis of multiple substitutions at each position within a single enzyme, the Alkaline Phosphatase superfamily member PafA. For each substitution, we measured multiple biochemical parameters, including Michaelis-Menten constants for cognate and non-cognate substrates and inhibition constants for multiple inhibitors and transition state analogs. The results provide a detailed functional signature for each position within PafA. We observe distinct physically contiguous ‘functional units’ throughout the enzyme that tune specific aspects of function, yielding blueprints for future rational design efforts. HT-MEK is applicable to enzyme function and stability, provides data that rivals or exceeds the precision typically obtained from standard assays, and reduces the time, effort, and cost to quantitatively profile the effects of individual substitutions on enzyme function by orders of magnitude, providing a technology to access previously inaccessible questions across multiple systems and disciplines.

Versatile and High-Throughput Microfluidics Platform for Dorsal Cell Mechanics

Versatile and High-Throughput Microfluidics Platform for Dorsal Cell Mechanics

A microfluidic platform for the characterisation of membrane active antimicrobials.

The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favour attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiologically relevant buffers on demand. Hundreds of these GUVs are individually immobilised downstream in physical traps connected to separate perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quantitative, high-throughput microfluidics devices for screening antibiotics.

Detection and Assessment of the Distribution of Infectious Agents in Juvenile Fraser River Sockeye Salmon, Canada, in 2012 and 2013.

Infectious diseases may contribute to declines in Fraser River Sockeye salmon (Oncorhynchus nerka) stocks, but a clear knowledge gap exists around which infectious agents and diseases are important. This study was conducted to: (1) determine the presence and prevalence of 46 infectious agents in juvenile Fraser River Sockeye salmon, and (2) evaluate spatial patterns in prevalence and burden over initial seaward migration, contrasting patterns between 2 years of average and poor productivity. In total, 2,006 out-migrating Sockeye salmon were collected from four regions along their migration trajectory in British Columbia, in 2012 and 2013. High-throughput microfluidics quantitative PCR was employed for simultaneous quantitation of 46 different infectious agents. Twenty-six agents were detected at least once, including nine with prevalence >5%. Candidatus Brachiomonas cysticola, Myxobolus arcticus, and Pacific salmon parvovirus were the most prevalent agents. Infectious agent diversity and burden increased consistently upon smolts entry into the ocean, but they did not substantially change afterwards. Notably, both freshwater- and saltwater-transmitted agents were more prevalent in 2013 than in 2012, leading to an overall higher infection burden in the first two sampling regions. A reduction in the prevalence of two agents, erythrocytic necrosis virus and Paraneuclospora theridion, was observed between regions 2 and 3, which was speculated to be associated with mortality during the 1st month at sea. The most prevalent infectious agents were all naturally occurring. In a small number of samples (0.9%), seven agents were only detected around and after salmon farming regions, including four important pathogens: piscine orthoreovirus, Piscirickettsia salmonis, Tenacibaculum maritimum, and Moritella viscosa. As the first synoptic survey of infectious agents in juvenile Sockeye salmon in British Columbia, this study provides the necessary baseline for further research on the most prevalent infectious agents and their potential pathogenicity, which may adversely affect the productivity of valuable Sockeye salmon stocks. In addition, our findings are informative to the decision makers involved in conservation programs.

A comparison of infectious agents between hatchery-enhanced and wild out-migrating juvenile chinook salmon (Oncorhynchus tshawytscha) from Cowichan River, British Columbia

Infectious diseases are likely contributing to large-scale declines in chinook salmon stocks in the Pacific Northwest, but the specific agents and diseases involved, and the prevalences in migratory salmon, are mostly unknown. We applied a high-throughput microfluidics platform to screen for 45 infectious agents in 556 out-migrating juvenile chinook salmon, collected from freshwater (FW) and saltwater (SW) locations in the Cowichan River system on Vancouver Island, Canada, during 2014. Nineteen agents (5 bacterial, 2 viral, and 12 parasitic) were detected, with prevalences ranging from 0.2% to 57.6%. Co-infections between Candidatus Branchiomonas cysticola Toenshoff, Kvellestad, Mitchell, Steinum, Falk, Colquhoun & Horn, 2012, Paranucleospora theridion Nylund, Nylund, Watanabe, Arnesen & Kalrsbakk, 2010, and gill chlamydia, all associated with gill disease, were observed in SW samples. We detected agents known to cause large-scale mortalities in Pacific salmon ( Ceratonova shasta (Noble, 1950), Parvicapsula minibicornis Kent, Whitaker & Dawe, 1977), and agents only recently reported in Pacific salmon in BC ( Ca. B. cysticola, P. theridion, Facilispora margolisi Jones, Prosperi-Porta & Kim, 2012 and Parvicapsula pseudobranchicola Karlsbakk, Saether, Hostlund, Fjellsoy & Nylund, 2002). Wild and hatchery fish were most divergent in agent profiles in FW, with higher agent diversity in wild fish. Differences in prevalence largely dissipated once they converged in the marine environment, although hatchery fish may be infected by a greater diversity of agents sooner after ocean entry by virtue of their more rapid migration from nearshore to offshore environments.

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches.We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%–91% versus 44%–61% for single region QIIME analyses (p < .01, p < .001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively.MVRSION represents a generally applicable tool for taxonomic classification that is superior to single-region 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Microengineering double layer hydrogel structures towards the recapitulation of the hematopoietic stem cell niche

Adult stem cells, especially from bone marrow, have a tremendous therapeutic potential, and its application in regenerative medicine has been increasingly studied. It is well known that three-dimensional (3D) cell culture matrix promotes many biological relevant functions not observed in two-dimensional (2D) monolayer cell culture and therefore, engineering 3D cell culture platforms to mimic the native in vivo biological systems represents a realistic approach for studies of bone marrow stem cells. High-throughput microfluidics constitutes a promising technology to generate hydrogel three dimensional constructs, however, its application to host adult stem cells remains still a challenge.