Multiple Samples In Parallel Research Articles

Portable Microfluidic Viscometer for Formulation Development and in Situ Quality Control of Protein and Antibody Solutions.

Viscosity of protein solutions is a critical product quality attribute for protein therapeutics such as monoclonal antibodies. Here we introduce a portable single-use analytical chip-based viscometer for determining the viscosity of protein solutions using low sample volumes of 10 μL. Through the combined use of a microfluidic viscometer, a smartphone camera for image capture, and an automated data processing algorithm for the calculation of the viscosity of fluids, we enable measurement of viscosity of multiple samples in parallel. We first validate the viscometer using glycerol-water mixtures and subsequently demonstrate the ability to perform rapid characterization of viscosity in four different monoclonal antibody formulations in a broad concentration (1 to 320 mg/mL) and viscosity (1 to 600 cP) range, showing excellent agreement with values obtained by a conventional cone-plate rheometer. Not only does the platform offer benefits of viscosity measurements using minimal sample volumes, but enables higher throughput compared to gold-standard methodologies owing to multiplexing of the measurement and single-use characteristics of the viscometer, thus showing great promise in developability studies. Additionally, as our platform has the capability of performing viscosity measurements at the point of sample collection, it offers the opportunity to employ viscosity measurement as an in situ quality control of therapeutic proteins and antibodies.

Riboseq-flow: A streamlined, reliable pipeline for ribosome profiling data analysis and quality control

Ribosome profiling is a powerful technique to study translation at a transcriptome-wide level. However, ensuring good data quality is paramount for accurate interpretation, as is ensuring that the analyses are reproducible. We introduce a new Nextflow DSL2 pipeline, riboseq-flow, designed for processing and comprehensive quality control of ribosome profiling experiments. Riboseq-flow is user-friendly, versatile and upholds high standards in reproducibility, scalability, portability, version control and continuous integration. It enables users to efficiently analyse multiple samples in parallel and helps them evaluate the quality and utility of their data based on the detailed metrics and visualisations that are automatically generated. Riboseq-flow is available at https://github.com/iraiosub/riboseq-flow.

Drug Screening Using Normal Cell and Cancer Cell Mixture in an Automated 3D Cell Culture System.

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

Automated and parallel microfluidic DNA extraction with integrated pneumatic microvalves/pumps and reusable open-channel columns.

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.

Optics-Free, In Situ Swelling Monitoring of Articular Cartilage with Graphene Strain Sensors.

Articular cartilage derives its load-bearing strength from the mechanical and physiochemical coupling between the collagen network and negatively charged proteoglycans, respectively. Current disease modeling approaches and treatment strategies primarily focus on cartilage stiffness, partly because indentation tests are readily accessible. However, stiffness measurements via indentation alone cannot discriminate between proteoglycan degradation versus collagen degradation, and there is a lack of methods to monitor physiochemical contributors in full-stack tissue. To decouple these contributions, here, we developed a platform that measures tissue swelling in full-depth equine cartilage explants using piezoresistive graphene strain sensors. These piezoresistive strain sensors are embedded within an elastomer bulk and have sufficient sensitivity to resolve minute, real-time changes in swelling. By relying on simple DC resistance measurements over optical techniques, our platform can analyze multiple samples in parallel. Using these devices, we found that cartilage explants under enzymatic digestion showed distinctive swelling responses to a hypotonic challenge and established average equilibrium swelling strains in healthy cartilage (4.6%), cartilage with proteoglycan loss (0.5%), and in cartilage with both collagen and proteoglycan loss (-2.6%). Combined with histology, we decoupled the pathologic swelling responses as originating either from reduced fixed charge density or from loss of intrinsic stiffness of the collagen matrix in the superficial zone. By providing scalable and in situ monitoring of cartilage swelling, our platform could facilitate regenerative medicine approaches aimed at restoring osmotic function in osteoarthritic cartilage or could be used to validate physiologically relevant swelling behavior in synthetic hydrogels.

Application of approach for development of hptlc identification and quantification methods for determination of phenolic compounds and terpenoids of several Thymus L. species

The aim of this work was to improve the approaches for standardization of Thymus L. species by development of HPTLC identification of phenolic compounds (flavonoids and phenylpropanoids) and terpenoids (essential oils and triterpenoids) and quantitative determination of rosmarinic acid and flavonoids for six Thymus L. species of Ukraine flora, as well as determination of chromatographic profiles of Thymus L. species’ extracts obtained using different extraction methods.
 Materials and methods. Objects were: samples of dry herb of Th.serpyllum, Th.vulgaris, Th.Marschallianus, Th.Pallasianus, Th.calcareus, and Th.moldavicus of Ukraine origin collected in 2019-2021 years or acquired commercially on Ukraine market. Instruments: CAMAG HPTLC Herbal System, visionCats 2.5. The analytical grade reagents were used. Reference substances were purchased from Extrasynthese, Sigma Aldrich. Chromatography was performed on HPTLC plates Si 60 F254, Merck according to the developed methods.
 Results. A new approach for quality control of different Thymus L. species of Ukraine flora included the development of HPTLC methods for identification of main groups of bioactive substances of these species, such as flavonoids and phenylpropanoids, essential oils, triterpenoids; development of quantification method of rosmarinic acid and assay of total flavonoids, expressed as luteolin-7-O-glucoside. The characteristic HPTLC fingerprints of six Thymus L. species in three mobile phases of different polarities that cover a wide range of bioactive substances were established. The content of rosmarinic acid in different Thymus L. species samples was in the range of 0,11-0,72 %: Th.moldavicus – 0,11 %; Th.Marschallianus – 0,19-0,27 %; Th.serpyllum – 0,38 %; Th.vulgaris – 0,51 %; Th.calcareus – 0,56 %; T.Pallasianus – 0,72 %. The total flavonoids content, expressed as luteolin-7-O-glucoside, was in the range of 0,8-2,72 %: Th.moldavicus – 0,8 %; Th.serpyllum – 0,87 %; Th.vulgaris – 1,06 %; Th.Pallasianus – 1,28 %; Th.Marschallianus – 1,89 %; Th.calcareus – 2,72 %.
 Conclusions. The proposed scientific approach for quality evaluation of Thymus L. species using HPTLC allows to determine comprehensive information of chemical composition and content of active substances of multiple samples in parallel, in a cost and time-efficient manner

Automated and Continuous-Flow Platform to Analyze Semiconductor–Metal Complex Hybrid Systems for Photocatalytic CO2 Reduction

Sunlight-driven CO2 reduction is increasingly considered as a promising approach to contribute toward a carbon-neutral fuel cycle, but most photocatalyst systems are currently studied individually under batch conditions with manual, labor-intensive analytical procedures. Here, we present the advantages of a continuous-flow setup to study photocatalytic CO2 to CO reduction systems, which also benefits from aspects of automation (using programmed in-line gas quantification of multiple samples in parallel). The capabilities of the methodology are demonstrated using a state-of-the-art light absorber platform based on ZnSe quantum dots (QDs) in combination with a series of molecular co-catalysts based on Ni and Co for visible-light-driven CO2 reduction in aqueous ascorbate solution. A newly synthesized Co-tetraphenylporphyrin featuring three sulfonate groups and one amine group (Co(tppS3N1)) is identified to exhibit a benchmark photocatalytic activity (18.6 μmol of CO, 79.7 mmol of CO gZnSe–1, TONCo (CO) of 619, external quantum efficiency (EQE) >5%). The utility of our methodology is further shown by applying the setup to study the photocatalyst systems under lower light intensities, low CO2 concentration, and aerobic conditions, which impact the photocatalytic activity and selectivity. Overall, this work reports an improved methodology for studying photocatalytic CO2 reduction alongside advancing the understanding of QD molecular co-catalyst hybrids using ZnSe QDs as a versatile light absorber based on earth-abundant components that operate under fully aqueous conditions.

A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings.

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.

Sample Tracking Using Unique Sequence Controls

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.

Nanofluidic and monolithic environmental cells for cryogenic microscopy

We present a device capable of combining nanofluidics and cryogenic transmission electron microscopy (cryo-TEM) to allow inspection of water-soluble samples under near-native conditions. The devices can be produced in a multitude of designs, but as a general rule, they consist of channels or chambers enclosed between two electron-transparent silicon nitride windows. With the appropriate design, those devices can allow screening of multiple samples in parallel and remove the interaction between the sample and the environment (no air–water interface). We demonstrate channel sizes from 80 to 500 nm in height and widths from 100 to 2000 μm. The presented fabrication flow allows producing hollow devices on a single wafer eliminating the need of aligning or bonding two half-cavities from separate wafers, which provides additional resistance to thermal stress. Taking advantage of a single-step through-membrane exposure with a 100 keV electron beam, we introduced arrays of thin (10–15 nm) electron-transparent silicon nitride membrane windows aligned between top and bottom (200–250 nm) carrier membranes. Importantly, the final devices are compatible with standard TEM holders. Furthermore, they are compatible with rapid freezing of samples, which is crucial for the formation of vitreous water, hence avoiding the formation of crystalline ice, that is detrimental for TEM imaging. To demonstrate the potential of this technology, we tested those devices in imaging experiments verifying their applicability for cryo-TEM applications and proved that vitreous water could be prepared through conventional plunge freezing of the chips.

Sieve-through vertical flow platform for efficient liquid exchange in particle-based assays

Particle-based assays are widely used in many biomedical applications. However, the performance of particle-based systems is often compromised by the carry-over contamination caused by the residual reagents during the liquid-exchange process. We have developed a sieve-through platform that utilizes a porous membrane to sieve out the particles, and an absorbent pad to remove the waste liquid by capillary force. The porous membrane is able to contain the liquid in the reaction chamber, and allows the waste liquid to flow through when it is brought into contact with the absorbent pad. The sieve-through platform is able to effectively remove the waste liquid, thereby achieving a more efficient liquid exchange as compared to the conventional process, and minimizing the carry-over contamination. In this study, we have determined the factors that affect the flow characteristics through the porous membrane on the sieve-through platform. We have shown that the sieve-through platform effectively reduces the carry-over contamination. In addition, we have shown particle-based ELISA on the sieve-through platform for the analysis of proteins and cells. We have further demonstrated the potential of the sieve-through platform for high-throughput analysis by presenting a sieve-array, which allows concurrent analysis of multiple samples in parallel. The sieve-through platform can significantly improve the performance of particle-based systems.

Magnitude Assessment of Adult Neurogenesis in the Octopus vulgaris Brain Using a Flow Cytometry-Based Technique

Adult neurogenesis is widespread among metazoans, it occurs in animals with a network nervous system, as cnidarians, and in animals with a complex and centralized brain, such as mammals, non-mammalian vertebrates, ecdysozoans, and a lophotrochozoan, Octopus vulgaris. Nevertheless, there are important differences among taxa, especially in the number of the regions involved and in cell proliferation rate during the life-cycle. The comparative evaluation of adult neurogenesis among different brain regions is an arduous task to achieve with only stereological techniques. However, in Octopus vulgaris we recently confirmed the presence of active proliferation in the learning-memory centers, multisensory integration centers, and the motor centers of the adult brain. Here, using a flow cytometry technique, we provide a method to quantify the active proliferation in octopus nervous system using a BrdU in vitro administration without exposing the animals to stress or painful injections usually used. This method is in line with the current animal welfare regulations regarding cephalopods, and the flow cytometry-based technique enabled us to measure adult neurogenesis more quickly and reliably than histological techniques, with the additional advantage of processing multiple samples in parallel. Flow cytometry is thus an appropriate technique for measuring and comparing adult neurogenesis in animals that are in a different physiological and/or environmental contexts. A BrdU immunoreactivity distribution, to define the neurogenic areas, and the effective penetration in vitro of the BrdU is also provided.

BARCOSEL: a tool for selecting an optimal barcode set for high-throughput sequencing

BackgroundCurrent high-throughput sequencing platforms provide capacity to sequence multiple samples in parallel. Different samples are labeled by attaching a short sample specific nucleotide sequence, barcode, to each DNA molecule prior pooling them into a mix containing a number of libraries to be sequenced simultaneously. After sequencing, the samples are binned by identifying the barcode sequence within each sequence read.In order to tolerate sequencing errors, barcodes should be sufficiently apart from each other in sequence space. An additional constraint due to both nucleotide usage and basecalling accuracy is that the proportion of different nucleotides should be in balance in each barcode position. The number of samples to be mixed in each sequencing run may vary and this introduces a problem how to select the best subset of available barcodes at sequencing core facility for each sequencing run. There are plenty of tools available for de novo barcode design, but they are not suitable for subset selection.ResultsWe have developed a tool which can be used for three different tasks: 1) selecting an optimal barcode set from a larger set of candidates, 2) checking the compatibility of user-defined set of barcodes, e.g. whether two or more libraries with existing barcodes can be combined in a single sequencing pool, and 3) augmenting an existing set of barcodes.In our approach the selection process is formulated as a minimization problem. We define the cost function and a set of constraints and use integer programming to solve the resulting combinatorial problem. Based on the desired number of barcodes to be selected and the set of candidate sequences given by user, the necessary constraints are automatically generated and the optimal solution can be found. The method is implemented in C programming language and web interface is available at http://ekhidna2.biocenter.helsinki.fi/barcosel.ConclusionsIncreasing capacity of sequencing platforms raises the challenge of mixing barcodes. Our method allows the user to select a given number of barcodes among the larger existing barcode set so that both sequencing errors are tolerated and the nucleotide balance is optimized. The tool is easy to access via web browser.

A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis

Engineered nucleases in genome editing manifest diverse efficiencies at different targeted loci. There is therefore a constant need to evaluate the mutation rates at given loci. T7 endonuclease 1 (T7E1) and Surveyor mismatch cleavage assays are the most widely used methods, but they are labour and time consuming, especially when one must address multiple samples in parallel. Here, we report a surrogate system, called UDAR (Universal Donor As Reporter), to evaluate the efficiency of CRISPR/Cas9 in targeted mutagenesis. Based on the non-homologous end-joining (NHEJ)-mediated knock-in strategy, the UDAR-based assay allows us to rapidly evaluate the targeting efficiencies of sgRNAs. With one-step transfection and fluorescence-activated cell sorting (FACS) analysis, the UDAR assay can be completed on a large scale within three days. For detecting mutations generated by the CRISPR/Cas9 system, a significant positive correlation was observed between the results from the UDAR and T7E1 assays. Consistently, the UDAR assay could quantitatively assess bleomycin- or ICRF193-induced double-strand breaks (DSBs), which suggests that this novel strategy is broadly applicable to assessing the DSB-inducing capability of various agents. With the increasing impact of genome editing in biomedical studies, the UDAR method can significantly benefit the evaluation of targeted mutagenesis, especially for high-throughput purposes.

Proteomics and phosphoproteomics in precision medicine: applications and challenges.

Recent advances in proteomics allow the accurate measurement of abundances for thousands of proteins and phosphoproteins from multiple samples in parallel. Therefore, for the first time, we have the opportunity to measure the proteomic profiles of thousands of patient samples or disease model cell lines in a systematic way, to identify the precise underlying molecular mechanism and discover personalized biomarkers, networks and treatments. Here, we review examples of successful use of proteomics and phosphoproteomics data sets in as well as their integration other omics data sets with the aim of precision medicine. We will discuss the bioinformatics challenges posed by the generation, analysis and integration of such large data sets and present potential reasons why proteomics profiling and biomarkers are not currently widely used in the clinical setting. We will finally discuss ways to contribute to the better use of proteomics data in precision medicine and the clinical setting.

High dimensional analysis of immune signatures in melanoma patients

Abstract Checkpoint inhibitor therapy (CIT) has revolutionized cancer therapy. Notably, inhibition of programmed cell death protein 1 (PD-1) showed benefit in the treatment of metastatic melanoma and has even been promoted to first line therapy in advanced melanoma. Despite an increase in overall survival a minority of patients profits from CIT with anti-PD-1. Therefore, immune components or markers predicting responsiveness are urgently needed. Using phenotypic and functional markers we used high-dimensional single cell cytometry by time of flight (CyTOF), to measure blood samples from anti-PD-1 treated patients before and during therapy. CyTOF uses metal-tagged instead of fluorochrome-tagged antibodies, thus eliminating spectral overlap and, in combination with barcoding, enables the measurement of more than 30 parameters on a single cell in multiple samples in parallel. Due to the complexity of the data set and in order to reduce analytical bias we adapted a custom bioinformatic workflow. Using this approach we find immune signatures which allow us to separate responders from non-responders and might provide insights into the biology of immune therapy.

Cytokines and cytokine networks target neurons to modulate long-term potentiation

Cytokines play crucial roles in the communication between brain cells including neurons and glia, as well as in the brain-periphery interactions. In the brain, cytokines modulate long-term potentiation (LTP), a cellular correlate of memory. Whether cytokines regulate LTP by direct effects on neurons or by indirect mechanisms mediated by non-neuronal cells is poorly understood. Elucidating neuron-specific effects of cytokines has been challenging because most brain cells express cytokine receptors. Moreover, cytokines commonly increase the expression of multiple cytokines in their target cells, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Here, we review evidence on both direct and indirect-mediated modulation of LTP by cytokines. We also describe novel approaches based on neuron- and synaptosome-enriched systems to identify cytokines able to directly modulate LTP, by targeting neurons and synapses. These approaches can test multiple samples in parallel, thus allowing the study of multiple cytokines simultaneously. Hence, a cytokine networks perspective coupled with neuron-specific analysis may contribute to delineation of maps of the modulation of LTP by cytokines.

Preparation of Intracellular Metabolite Extracts from Liquid Schizosaccharomyces pombe Cultures.

The success of metabolomic analysis relies heavily on the sample preparation protocol. Here we present a protocol for intracellular metabolite extraction from liquid fission yeast cultures based on rapid quenching in pure methanol at -40°C, bead-beating in 50% methanol for cell disruption, and 10 kDa cutoff ultrafiltration for removal of proteins. Samples are concentrated by vacuum evaporation and resuspended in 50% acetonitrile for mass spectrometric analysis. This protocol is optimal for extraction of polar metabolites such as amino acids, organic acids, nucleotides, sugars, or sugar-phosphates. Its implementation requires <6 h and allows preparation of multiple samples in parallel.

Use of Impedance Biotesting to Assess the Actions of Pharmaceutical Compounds on the Growth of Microorganisms

A method for the analysis of microbial “growth curves” by real-time recording of changes in the impedance electrical conductance of the medium from multiple samples in parallel is described; these changes arise from metabolic processes taking place in the test microorganisms in the samples. Results obtained from practical application of the impedance biotesting method for analysis of the antibacterial activity of the aseptic agent chlorhexidine bigluconate are presented, along with results obtained using solutions of a catholyte and an anolyte obtained by processing 1% NaCl and Na2SO4 solutions in a specially constructed 2-liter eletrolyzer for 25 min with a current of I = 7 A and a voltage of V = 28 V.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays

In this study, a multicenter evaluation of the Life Technologies TaqMan® Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.