Digital Isothermal Quantification of Nucleic Acids via Simultaneous Chemical Initiation of Recombinase Polymerase Amplification Reactions on SlipChip

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for “yes or no” digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37−42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications

Nanoliter Multiplex PCR Arrays on a SlipChip

The SlipChip platform was tested to perform highthroughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and highthroughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays

Early mobilisation in critically ill COVID-19 patients: a subanalysis of the ESICM-initiated UNITE-COVID observational study

Background Early mobilisation (EM) is an intervention that may improve the outcome of critically ill patients. There is limited data on EM in COVID-19 patients and its use during the first pandemic wave. Methods This is a pre-planned subanalysis of the ESICM UNITE-COVID, an international multicenter observational study involving critically ill COVID-19 patients in the ICU between February 15th and May 15th, 2020. We analysed variables associated with the initiation of EM (within 72 h of ICU admission) and explored the impact of EM on mortality, ICU and hospital length of stay, as well as discharge location. Statistical analyses were done using (generalised) linear mixed-effect models and ANOVAs. Results Mobilisation data from 4190 patients from 280 ICUs in 45 countries were analysed. 1114 (26.6%) of these patients received mobilisation within 72 h after ICU admission; 3076 (73.4%) did not. In our analysis of factors associated with EM, mechanical ventilation at admission (OR 0.29; 95% CI 0.25, 0.35; p = 0.001), higher age (OR 0.99; 95% CI 0.98, 1.00; p ≤ 0.001), pre-existing asthma (OR 0.84; 95% CI 0.73, 0.98; p = 0.028), and pre-existing kidney disease (OR 0.84; 95% CI 0.71, 0.99; p = 0.036) were negatively associated with the initiation of EM. EM was associated with a higher chance of being discharged home (OR 1.31; 95% CI 1.08, 1.58; p = 0.007) but was not associated with length of stay in ICU (adj. difference 0.91 days; 95% CI − 0.47, 1.37, p = 0.34) and hospital (adj. difference 1.4 days; 95% CI − 0.62, 2.35, p = 0.24) or mortality (OR 0.88; 95% CI 0.7, 1.09, p = 0.24) when adjusted for covariates. Conclusions Our findings demonstrate that a quarter of COVID-19 patients received EM. There was no association found between EM in COVID-19 patients' ICU and hospital length of stay or mortality. However, EM in COVID-19 patients was associated with increased odds of being discharged home rather than to a care facility. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021)

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a 5-bp stem–3 nt loop hairpin structure. Here, we describe the 2.0-Å resolution x-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the full-length vRNAP. The structure resembles a “fisted right hand” with Fingers, Palm and Thumb subdomains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with W129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription

Towards DNA sensing polymers: interaction between acrylamide/3-(N,N-dimethylaminopropyl)-acrylamide and DNA phage λ at various N/P ratios

The present study strongly relates to ongoing research on the development of cationic polymers which are of great interest due to their enormous potential for biomedical applications, especially as non-viral vectors for gene therapy, antimicrobial agents and active components in DNA sensing devices. The current paper demonstrates that a functional group approach can be successfully realized in a free-radical copolymerization process to prepare cationic copolymers with a desired composition of amine groups, which can be protonated in water thus, providing electrostatic interactions between a polycation and DNA. Three replicas of the cationic copolymer, acrylamide/3-(N,N-dimethylaminopropyl)-acrylamide (AADMAPA), were synthesized using this strategy. The values of average molecular mass and polydispersity index, are similar for the replicas, averaged to 24 000 2000 g mol1 and 1.5 0.1, respectively. The copolymer composition according to 1 H-NMR (D2O), was corresponded to the molar ratio of initial monomers. The dynamic light scattering studies and zeta potential measurements confirmed that in water positively charged AADMAPA/DNA polyplexes are formed at N/P > 2.2: the formed particles have bimodal distributions with the average diameters of 70 and 700 nm. Zeta potential measurements indicated that the point of zero charge (isoelectric point) is close to N/P y 2.2. According to the atomic force microscopy positively charged AADMAPA/DNA polyplexes have axially symmetric shapes.The authors are grateful to Prof. Babushkina T. A., Mrs Klimova T. P. for helpful discussion and NMR spectral data. The authors also acknowledge Mrs Klemenkova Z. S. for IR spectral data. E. Laukhina acknowledges the support from Instituto de Salud Carlos III, through “Acciones CIBER”. The Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010.Peer reviewe

Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes*

Bacteriophage N4 mini-virion RNA polymerase (mini-vRNAP), the 1106-amino acid transcriptionally active domain of vRNAP, recognizes single-stranded DNA template-containing promoters composed of conserved sequences and a 3-base loop–5-base pair stem hairpin structure. The major promoter recognition determinants are a purine located at the center of the hairpin loop (–11G) and a base at the hairpin stem (–8G). Mini-vRNAP is an evolutionarily highly diverged member of the T7 family of RNAPs. A two-plasmid system was developed to measure the in vivo activity of mutant mini-vRNAP enzymes. Five mini-vRNAP derivatives, each containing a pair of cysteine residues separated by ∼100 amino acids and single cysteine-containing enzymes, were generated. These reagents were used to determine the smallest catalytically active polypeptide and to map promoter, substrate, and RNA-DNA hybrid contact sites to single amino acid residues in the enzyme by using end-labeled 5-iododeoxyuridine- and azidophenacyl-substituted oligonucleotides, cross-linkable derivatives of the initiating nucleotide, and RNA products with 5-iodouridine incorporated at specific positions. Localization of functionally important amino acid residues in the recently determined crystal structures of apomini-vRNAP and the mini-vRNAP-promoter complex and comparison with the crystal structures of the T7 RNAP initiation and elongation complexes allowed us to predict major rearrangements in mini-vRNAP in the transition from transcription initiation to elongation similar to those observed in T7 RNAP, a task otherwise precluded by the lack of sequence homology between N4 mini-vRNAP and T7 RNAP

Role of Fe in the hydrogen oxidation reaction in a NiFe-based catalyst: An in situ Mössbauer spectroscopic investigation

Nickel-based catalysts reach a high activity for the hydrogen oxidation reaction (HOR) in anion exchange membrane fuel cells. While incorporation of iron significantly decreases the HOR overpotential on NiFe-based catalysts, the reason for the enhanced activity remains only partially understood. For the first time, in situ 57Fe Mössbauer spectroscopy is used to gain insights into the iron-related composition at different potentials. The aim is to evaluate which changes occur on iron at potentials relevant for the HOR on the active Ni sites. It is found that different pre-conditionings at low potentials stabilize the iron at a low oxidation state as compared to the as-prepared catalyst powder. It is likely that the lower average oxidation state enables a higher exchange current density and a more efficient OH adsorption, which make the Volmer step much faster in the HOR. Insights from in situ Mössbauer spectroscopy enlighten the role of iron in the nickel-iron catalyst, paving the way for developing improved Ni-based catalysts for HOR catalysis