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Premise: A novel protocol for rapid plant DNA extraction using microneedles is proposed, which supports botanic surveys, taxonomy, and systematics. This protocol can be conducted in the field with limited laboratory skills and equipment. The protocol is validated by sequencing and comparing the results with QIAGEN spin‐column DNA extractions using BLAST analyses. Methods and Results: Two sets of DNA extractions were conducted on 13 species spanning various leaf anatomies and phylogenetic lineages: (i) fresh leaves were punched with custom polymeric microneedle patches to recover genomic DNA, or (ii) QIAGEN DNA extractions. Three plastid (matK, rbcL, and trnH‐psbA) and one nuclear ribosomal (ITS) DNA regions were amplified and sequenced using Sanger or nanopore technology. The proposed method reduced the extraction time to 1 min and yielded the same DNA sequences as the QIAGEN extractions. Conclusions: Our drastically faster and simpler method is compatible with nanopore sequencing and is suitable for multiple applications, including high‐throughput DNA‐based species identifications and monitoring. [ABSTRACT FROM AUTHOR]

Objectives: We evaluate the diagnostic performance of dried blood microsampling combined with a high-throughput microfluidic nano-immunoassay (NIA) for the identification of anti-SARS-CoV-2 Spike IgG seropositivity. Methods: We conducted a serological study among 192 individuals with documented prior SARS-CoV-2 infection and 44 SARS-CoV-2 negative individuals. Participants with prior SARS-CoV-2 infection had a long interval of 11 months since their qRT-PCR positive test. Serum was obtained after venipuncture and tested with an automated electrochemiluminescence anti-SARS-CoV-2 S total Ig reference assay, a commercial ELISA anti-S1 IgG assay, and the index test NIA. In addition, 109 participants from the positive cohort and 44 participants from the negative cohort participated in capillary blood collection using three microsampling devices: Mitra, repurposed glucose test strips, and HemaXis. Samples were dried, shipped by regular mail, extracted, and measured with NIA. Results: Using serum samples, we achieve a clinical sensitivity of 98·33% and specificity of 97·62% on NIA, affirming the high performance of NIA in participants 11 months post infection. Combining microsampling with NIA, we obtain a clinical sensitivity of 95·05% using Mitra, 61·11% using glucose test strips, 83·16% using HemaXis, and 91·49% for HemaXis after automated extraction, without any drop in specificity. Discussion: High sensitivity and specificity was demonstrated when testing micro-volume capillary dried blood samples using NIA, which is expected to facilitate its use in large-scale studies using home-based sampling or samples collected in the field. [ABSTRACT FROM AUTHOR]

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the Saccharomyces cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi . However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single-cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we identified an intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control but by simple transport biophysics. The ubiquity of multitransporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well. [ABSTRACT FROM AUTHOR]

Objectives: To report a prospective epidemiological, virological and serological investigation of a SARS-CoV-2 outbreak in a primary school. Methods: As part of a longitudinal, prospective, school-based surveillance study, this investigation involved repeated testing of 73 pupils, 9 teachers, 13 non-teaching staff and 26 household members of participants who tested positive, with rapid antigen tests and/or RT-PCR (Day 0–2 and Day 5–7), serologies on dried capillary blood samples (Day 0–2 and Day 30), contact tracing interviews and SARS-CoV-2 whole genome sequencing. Results: We identified 20 children (aged 4 to 6 years from 4 school classes), 2 teachers and a total of 4 household members who were infected by the Alpha variant during this outbreak. Infection attack rates were between 11.8 and 62.0% among pupils from the 4 school classes, 22.2% among teachers and 0% among non-teaching staff. Secondary attack rate among household members was 15.4%. Symptoms were reported by 63% of infected children, 100% of teachers and 50% of household members. All analysed sequences but one showed 100% identity. Serological tests detected 8 seroconversions unidentified by SARS-CoV-2 virological tests. Conclusions: This study confirmed child-to-child and child-to-adult SARS-CoV-2 transmission and introduction into households. Effective measures to limit transmission in schools have the potential to reduce the overall community circulation. [ABSTRACT FROM AUTHOR]

We designed a microfluidic module that generates complex and dynamic concentration profiles of multiple molecules over a large concentration range using pulse-width modulation (PWM). Our PWM module can combine up to six different inputs and select among three downstream mixing channels, as required by the application. The module can produce concentrations with a dynamic range of three decades. We created complex, temporal concentration profiles of two molecules, with each concentration independently controllable, and show that the PWM module can execute rapid concentration changes as well as long-time scale pharmacokinetic profiles. Concentration profiles were generated for molecules with molecular weights ranging from 560 Da to 150 kDa. Our PWM module produces robust and precise concentration profiles under a variety of operating conditions, making it ideal for integration with existing microfluidic devices for advanced cell and pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. [ABSTRACT FROM AUTHOR]

We present a platform that combines microarrays and microfluidic techniques to measure four protein biomarkers in 1024 serum samples for a total of 4096 assays per device. Detection is based on a surface fluorescence sandwich immunoassay with a limit of detection of ~1 pM for most of the proteins measured: PSA, TNF-α, IL-1β, and IL-6. To validate the utility of our platform, we measured these four biomarkers in 20 clinical human serum samples, 10 from prostate cancer patients and 10 female and male controls. We compared the results of our platform to a conventional ELISA and found a good correlation between them. However, compared to a classical ELISA, our device reduces the total cost of reagents by 4 orders of magnitude while increasing throughput by 2 orders of magnitude. Overall, we demonstrate an integrated approach to perform low-cost and rapid quantification of protein biomarkers from over one thousand serum samples. This new high-throughput technology will have a significant impact on disease diagnosis and management. [ABSTRACT FROM AUTHOR]

Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages. [ABSTRACT FROM AUTHOR]

The precise tuning of gene expression levels is essential for the optimal performance of transcriptional regulatory networks. We created 209 variants of the Saccharomyces cerevisiae PHO5 promoter to quantify how different binding sites for the transcription factor Pho4 affect its output. We found that transcription-factor binding affinities determined in vitro could quantitatively predict the output of a complex yeast promoter. Promoter output was precisely tunable by subtle changes in binding-site affinity of less than 3 kcal mol−1, which are accessible by modifying 1-2 bases. Our results provide insights into how transcription-factor binding sites regulate gene expression, their possible evolution and how they can be used to precisely tune gene expression. More generally, we show that in vitro binding-energy landscapes of transcription factors can precisely predict the output of a native yeast promoter, indicating that quantitative models of transcriptional regulatory networks are feasible. [ABSTRACT FROM AUTHOR]

During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a ''dimer cloud'' that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks. [ABSTRACT FROM AUTHOR]

Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA--binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable. [ABSTRACT FROM AUTHOR]

We developed an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidic affinity assay, and used it for 14,792 on-chip experiments, which exhaustively measured the protein-protein interactions of 43 Streptococcus pneumoniae proteins in quadruplicate. The resulting network of 157 interactions was denser than expected based on known networks. Analysis of the network revealed previously undescribed physical interactions among members of some biochemical pathways. [ABSTRACT FROM AUTHOR]

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ∼3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development. [ABSTRACT FROM AUTHOR]