Search

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.

The leaf area index (LAI) is a key parameter in the context of monitoring the development of tree crowns and plants in general. As parameters such as carbon assimilation, environmental stress on carbon, and the water fluxes within tree canopies are correlated to the leaves surface, this parameter is essential for understanding and modeling ecological processes. However, its continuous monitoring using manual state-of-the-art measurement instruments is still challenging. To address this challenge, we present an innovative sensor concept to obtain the LAI based on the cheap and easy to integrate multi-channel spectral sensor AS7341. Additionally, we present a method for processing and filtering the gathered data, which enables very high accuracy measurements with an nRMSE of only 0.098, compared to the manually-operated state-of-the-art instrument LAI-2200C (LiCor). The sensor that is embedded on a sensor node has been tested in long-term experiments, proving its suitability for continuous deployment over an entire season. It permits the estimation of both the plant area index (PAI) and leaf area index (LAI) and provides the first wireless system that obtains the LAI solely powered by solar cells. Its energy autonomy and wireless connectivity make it suitable for a massive deployment over large areas and at different levels of the tree crown. It may be upgraded to allow the parallel measurement of photosynthetic active radiation (PAR) and light quality, relevant parameters for monitoring processes within tree canopies.

RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g., dendriplexes. siRNA quantification inside carriers is essential in drug delivery system development. However, current siRNA measuring methods either are not very sensitive, only semi-quantitative or not specific towards intact target siRNA sequences. We present a novel reverse transcription real-time PCR (RT-qPCR)-based application for siRNA quantification in drug formulations. It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. The limit of detection (LOD) was 4.2 pM (±0.2 pM) and the limit of quantification (LOQ) 77.8 pM (±13.4 pM) for uncomplexed siRNA. LOD and LOQ of dendriplex samples were 31.6 pM (±0 pM) and 44.4 pM (±9.0 pM), respectively. Unspecific non-target siRNA sequences did not decrease quantification accuracy when present in samples. As an example of use, we assessed siRNA complexation inside dendriplexes with varying nitrogen-to-phosphate ratios. Further, protection of siRNA inside dendriplexes from RNase A degradation was quantitatively compared to degradation of uncomplexed siRNA. This novel application for quantification of siRNA in drug delivery systems is an important tool for the development of new siRNA-based drugs and quality checks including drug stability measurements.

In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.

The generation of artificial human tissue by 3D-bioprinting has expanded significantly as a clinically relevant research topic in recent years. However, to produce a complex and viable tissue, in-depth biological understanding and advanced printing techniques are required with a high number of process parameters. Here, we systematically evaluate the process parameters relevant for a hybrid bioprinting process based on fused-deposition modeling (FDM) of thermoplastic material and microextrusion of a cell-laden hydrogel. First, we investigated the effect of the printing temperature of polycaprolactone (PCL), on the junction strength between individual fused filaments and on the viability of immortalized mesenchymal stem cells (iMSC) in the surrounding alginate-gelatin-hydrogel. It was found that a printing temperature of 140 °C and bonds with an angle of 90° between the filaments provided a good compromise between bonding strength of the filaments and the viability of the surrounding cells. Using these process parameters obtained from individual fused filaments, we then printed cubic test structures with a volume of 10 × 10 × 10 mm 3 with different designs of infill patterns. The variations in mechanical strength of these cubes were measured for scaffolds made of PCL-only as well as for hydrogel-filled PCL scaffolds printed by alternating hybrid bioprinting of PCL and hydrogel, layer by layer. The bare scaffolds showed a compressive modulus of up to 6 MPa, close to human hard tissue, that decreased to about 4 MPa when PCL was printed together with hydrogel. The scaffold design suited best for hybrid printing was incubated with cell-laden hydrogel and showed no degradation of its mechanical strength for up to 28 days., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Neurosurgical approaches to the brain often require the mobilization of the temporal muscle. Many patients complain of postoperative pain, atrophy, reduced mouth opening, and masticatory problems. Although the pterional, frontolateral-extended-pterional, and temporal craniotomies are the most frequently used approaches in neurosurgery, a systematic assessment of the postoperative oral health-related quality of life has never been performed so far. This study evaluates the oral health-related quality of life of patients after pterional, frontolateral-extended-pterional, or temporal craniotomy using a validated and standardized dental questionnaire, compares the results with the normal values of the general population, and investigates whether this questionnaire is sensitive to changes caused by surgical manipulation of the temporal muscle., Methods: The "Oral Health Impact Profile" (OHIP14) is a validated questionnaire to assess the oral health-related quality of life. It asks the patients to assess their oral health situation within the past 7 days in 14 questions. Possible answers range from 0 (never) to 4 (very often). Sixty patients with benign intracranial processes operated through a lateral cranial approach were included. The questionnaire was answered before surgery (baseline) and 3 months and 15 months after surgery., Results: Overall, postoperative OHIP scores increase significantly after 3 months and decrease after 15 months, but not to preoperative values. No factors can be identified which show a considerable relationship with the postoperative OHIP score., Conclusions: Postoperative impairment of mouth opening and pain during mastication can be observed 3 to 15 months after surgery and sometimes cause feedback from patients and their dentists. However, in line with existing literature, these complaints decrease with time. The study shows that the OHIP questionnaire is sensitive to changes caused by surgical manipulation of the temporal muscle and can therefore be used to investigate the influence of surgical techniques on postoperative complaints. Postoperatively, patients show worse OHIP scores than the general population, demonstrating that neurosurgical cranial approaches negatively influence the patient's oral health-related wellbeing. Larger studies using the OHIP questionnaire should evaluate if postoperative physical therapy, speech therapy, or specialized rehabilitation devices can improve the masticatory impairment after craniotomy., Trial Registration: Clinical trial register: DRKS00011096., (© 2021. The Author(s).)

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli , Klebsiella pneumonia , and Proteus mirabilis . The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 10 3 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 10 7 bacteria/mL, anda linearity R 2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

Here, we report on the time dependence of a synthesis procedure for generation of both n- and p-type bismuth telluride-based materials. To initiate the reaction, the starting materials were first mechanical pre-reacted. The Rietveld refinements of X-ray diffraction (XRD) data collected after different milling times demonstrate that Bi 2 Te 3 was formed after only 10 min, and longer milling times do not alter the composition. To complete the phase formation, the powders were treated by field-assisted sintering and heat treatment afterwards. The effect of this fast procedure on the structural and thermoelectric properties was investigated. Samples were obtained with relative densities above 99%. A clear preferred orientation of the crystallites in the samples is evidenced by Rietveld refinements of XRD data. The thermoelectric characteristics demonstrate a good performance despite the short milling time. Further, it was demonstrated for this fast synthesis that the physical transport properties can be varied with well-known n- and p-type dopants like CHI 3 or Pb. For these non-optimized materials, a ZT value of 0.7 (n-type) and 0.9 (p-type) between 400 and 450 K was achieved. The long-term stability is demonstrated by repeated measurements up to 523 K showing no significant alteration of the thermoelectric performance., Competing Interests: We have no competing interests., (© 2022 The Authors.)

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

The effect of an externally applied electric field on exciton splitting and carrier transport was studied on 3.5 nm Si nanocrystals embedded in SiO 2 superlattices with barrier oxide thicknesses varied between 2 and 4 nm. Through a series of photoluminescence measurements performed at both room temperature and with liquid N 2 cooling, it was shown that the application of an electric field resulted in a reduction of luminescence intensity due to exciton splitting and charging of nanocrystals within the superlattices. This effect was found to be enhanced when surface defects at the Si/SiO 2 interface were not passivated by H 2 treatment and severely reduced for inter layer barrier oxide thicknesses above 3 nm. The findings point to the surface defects assisting in carrier transport, lowering the energy required for exciton splitting. Said enhancement was found to be diminished at low temperatures due to the freezing-in of phonons. We propose potential device design parameters for photon detection and tandem solar cell applications utilizing the quantum confinement effect based on the findings of the present study., (© 2022. The Author(s).)

Flexible neural implants are extremely favored, as the most successful strategy to promote probe-tissue integration and avoid severe gliosis relies on reducing the mechanical mismatch between probe and brain tissue. But what are the realistic requirements for achieving chronic recording stability? What are the critical dimensions and main factors determining glial scar-free device integration? To answer these questions, two types of hair-sized polyimide-based flexible intracortical (PIXI) arrays were fabricated, differing only in their cross-sectional area. Chronic tissue reaction to both types was evaluated in rats, and in different implantation setups. Interfacial stresses were found to play a critical role in long-term tissue integration. Still, all the devices provided high quality chronic recordings of single units and inflammatory gene expression was not significantly upregulated for larger devices. Our study points out that the most relevant factor in eliciting FBR is played by mechanical probe-tissue interactions, that polyimide is well tolerated by the tissue, and that a holistic design - considering material properties, geometrical dimensions and assembling techniques - is the key towards longevity and long-term performance of intracortical probes. The optimization of only one parameter did not yet lead to the successful translation of research accomplishments into chronic preclinical and clinical applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy., (© 2021 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Computer-guided clinical decision support systems have been finding their way into practice for some time, mostly integrated into electronic medical records. The primary goals are to improve the quality of treatment, save time and avoid errors. These are mostly rule-based algorithms that recognize drug interactions or provide reminder functions. Through artificial intelligence (AI), clinical decision support systems can be disruptively further developed. New knowledge is constantly being created from data through machine learning in order to predict the individual course of a patient's disease, identify phenotypes or support treatment decisions. Such algorithms already exist for rheumatological diseases. Automated image recognition and disease prediction in rheumatoid arthritis are the most advanced; however, these have not yet been sufficiently tested or integrated into existing decision support systems. Rather than dictating the AI-assisted choice of treatment to the doctor, future clinical decision systems are seen as hybrid decision support, always involving both the expert and the patient. There is also a great need for security through comprehensible and auditable algorithms to sustainably guarantee the quality and transparency of AI-assisted treatment recommendations in the long term., (© 2021. The Author(s).)

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types ( KRAS and BRAF ) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R 2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated ( Aggregatibacter actinomycetemcomitans , Campylobacter rectus , Fusobacterium nucleatum , Prevotella intermedia , Porphyromonas gingivalis , Tannerella forsythia , Treponema denticola ) and three caries-associated (oral Lactobacilli , Streptococcus mutans , Streptococcus sobrinus ). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Next generation sequencing is evolving from a research tool into a method applied in diagnostic routine. The complete sequencing workflow includes sample pre-processing, library preparation, sequencing and bioinformatics. High quality in each of these steps is necessary to obtain excellent sequencing results. The tedious and error-prone library preparation poses a significant challenge for smaller laboratories, where high throughput pipetting robots are not cost-effective. Here we present an automated library preparation for whole genome sequencing using centrifugal microfluidics. Two samples can be run per cartridge. Precise metering of reagents allows the required liquid volumes to be reduced by 40% and the amount of sample used by 60%. The functionality of the cartridge is demonstrated with bacteria and DNA extracted from a human FFPE sample. For the bacterial sample, mean sequencing depths from 140 to 183 reads and a coverage of 99.8% of the reference genome were detected. For the human DNA, mean sequencing depths of 4.4-5.7 reads and a coverage of 78.2% of the effective reference genome were observed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.P. and T.H. filed patents on unit operations of centrifugal microfluidics (DE102013210818, DE102012202775, DE10201322025, DE102016207845 and their associated patent families)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

(1) Significance of geometry for bio-inspired hygroscopically actuated bilayer structures is well studied and can be used to fine-tune curvatures in many existent material systems. We developed a material design space to find new material combinations that takes into account unequal effective widths of the layers, as commonly used in fused filament fabrication, and deflections under self-weight. (2) For this purpose, we adapted Timoshenko's model for the curvature of bilayer strips and used an established hygromorphic 4D-printed bilayer system to validate its ability to predict curvatures in various experiments. (3) The combination of curvature evaluation with simple, linear beam deflection calculations leads to an analytical solution space to study influences of Young's moduli, swelling strains and densities on deflection under self-weight and curvature under hygroscopic swelling. It shows that the choice of the ratio of Young's moduli can be crucial for achieving a solution that is stable against production errors. (4) Under the assumption of linear material behavior, the presented development of a material design space allows selection or design of a suited material combination for application-specific, bio-inspired bilayer systems with unequal layer widths.

To boost the performance of vanadium redox flow batteries, modification of the classically used felt electrodes is required to enable higher cycling performance and longer life cycles. Alternative approaches to the standard thermal oxidation procedure such as wet chemical oxidation are promising to reduce the thermal budget and thus the cost of the activation procedure. In this work we report a rapid 1 hour activation procedure in an acidified KMnO 4 solution. We show that the reported modification process of the felt electrodes results in an increase in surface area, density of oxygenated surface functionalities as well as electrolyte wettability, as demonstrated by N 2 -physisorption, XPS, Raman spectroscopy as well as contact angle measurements. The activation process enables battery cycling at remarkably high current densities up to 400 mA cm -2 . Stable cycling at 400 mA cm -2 over 30 cycles confirms promising stability of the reported activation procedure., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In this work, a process chain for the fabrication of dense zirconia parts will be presented covering the individual steps feedstock compounding, 3D printing via Fused Filament Fabrication (FFF) and thermal postprocessing including debinding and sintering. A special focus was set on the comprehensive rheological characterization of the feedstock systems applying high-pressure capillary and oscillation rheometry. The latter allowed the representation of the flow situation especially in the nozzle of the print head with the occurring low-shear stress. Oscillation rheometry enabled the clarification of the surfactant's concentration, here stearic acid, or more general, the feedstocks composition influence on the resulting feedstock flow behavior. Finally, dense ceramic parts (best values around 99 % of theory) were realized with structural details smaller than 100 µm.

3D-bioprinting is a promising technology applicable in areas such as regenerative medicine or in vitro organ model development. Various 3D-bioprinting technologies and systems have been developed and are partly commercially available. Here, we present the construction and characterization of an open-source low-cost 3D-bioprinter that allows the alternated microextrusion of hydrogel and fused deposition modeling (FDM) of thermoplastic filaments. The presented 3D-bioprinter is based on a conventional Prusa i3 MK3 printer and features two independent printheads: the original FDM-head and a syringe-based microextrusion printhead for soft materials. Modifications were designed modularly to fit various syringe formats or heating elements to the device. The bioprinter is the first hybrid DIY 3D-bioprinter that allows switching between materials as often as required during a print run to produce complex multi-material constructs with arbitrary patterns in each layer. For validation of the printer, two designs suitable for relevant bioprinting applications were realized. First, a porous plastic construct filled with hydrogel was printed, serving as a mechanically stable bone replacement tissue model. Second, a plastic chamber, which might be used in organ-on-a-chip applications, was printed with an extruded silicone sealing that enables the liquid-tight attachment of glass slides to the top and bottom of the chamber., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Drought negatively impacts growth and productivity of plants, particularly in arid and semi-arid regions. Although drought events can take place in summer and winter, differences in the impact of drought on physiological processes between seasons are largely unknown. The aim of this study was to elucidate metabolic strategies of date palms in response to drought in summer and winter season. To identify such differences, we exposed date palm seedlings to a drought-recovery regime, both in simulated summer and winter climate. Leaf hydration, carbon discrimination (${\Delta}$13C), and primary and secondary metabolite composition and contents were analyzed. Depending on season, drought differently affected physiological and biochemical traits of the leaves. In summer, drought induced significantly decreased leaf hydration, concentrations of ascorbate, most sugars, primary and secondary organic acids, as well as phenolic compounds, while thiol, amino acid, raffinose and individual fatty acid contents were increased compared with well-watered plants. In winter, drought had no effect on leaf hydration, ascorbate and fatty acids contents, but resulted in increased foliar thiol and amino acid levels as observed in summer. Compared with winter, foliar traits of plants exposed to drought in summer only partly recovered after re-watering. Memory effects on water relations, and primary and secondary metabolites seem to prepare foliar traits of date palms for repeated drought events in summer. Apparently, a well-orchestrated metabolic network, including the anti-oxidative system, compatible solutes accumulation and osmotic adjustment, and maintenance of cell-membrane stability strongly reduces the susceptibility of date palms to drought. These mechanisms of drought compensation may be more frequently required in summer., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Despite the widespread application of point-of-care lateral flow tests, the viscosity dependence of these assay results remains a significant challenge. Here, we employ centrifugal microfluidic flow control through the nitrocellulose membrane of the strip to eliminate the viscosity bias. The key feature is the balancing of the sample flow into the cassette of the lateral flow test with the air flow out of the cassette. A viscosity-independent flow rate of 3.01 ± 0.18 µl/min (±6%) is demonstrated for samples with viscosities ranging from 1.1 mPas to 24 mPas, a factor greater than 20. In a model human IgG lateral flow assay, signal-intensity shifts caused by varying the sample viscosity from 1.1 mPas to 2.3 mPas could be reduced by more than 84%., Competing Interests: Conflict of interestThe authors declare the following competing financial interests: N.P., D.K., and S.F. filed a patent based on these results., (© The Author(s) 2021.)

Neural electrodes are primary functional elements of neuroelectronic devices designed to record neural activity based on electrochemical signals. These electrodes may also be utilized for electrically stimulating the neural cells, such that their response can be simultaneously recorded. In addition to being medically safe, the electrode material should be electrically conductive and electrochemically stable under harsh biological environments. Mechanical flexibility and conformability, resistance to crack formation and compatibility with common microfabrication techniques are equally desirable properties. Traditionally, (noble) metals have been the preferred for neural electrode applications due to their proven biosafety and a relatively high electrical conductivity. Carbon is a recent addition to this list, which is far superior in terms of its electrochemical stability and corrosion resistance. Carbon has also enabled 3D electrode fabrication as opposed to the thin-film based 2D structures. One of carbon's peculiar aspects is its availability in a wide range of allotropes with specialized properties that render it highly versatile. These variations, however, also make it difficult to understand carbon itself as a unique material, and thus, each allotrope is often regarded independently. Some carbon types have already shown promising results in bioelectronic medicine, while many others remain potential candidates. In this topical review, we first provide a broad overview of the neuroelectronic devices and the basic requirements of an electrode material. We subsequently discuss the carbon family of materials and their properties that are useful in neural applications. Examples of devices fabricated using bulk and nano carbon materials are reviewed and critically compared. We then summarize the challenges, future prospects and next-generation carbon technology that can be helpful in the field of neural sciences. The article aims at providing a common platform to neuroscientists, electrochemists, biologists, microsystems engineers and carbon scientists to enable active and comprehensive efforts directed towards carbon-based neuroelectronic device fabrication., (Creative Commons Attribution license.)

Circulating tumor cells (CTCs) that enter the bloodstream play an important role in the formation of metastases. The prognostic significance of CTCs as biomarkers obtained from liquid biopsies is intensively investigated and requires accurate methods for quantification. The purpose of this study was the capture of CTCs on an optically accessible surface for real-time quantification. A filtration device was fabricated from a transparent material so that capturing of cells could be observed microscopically. Blood samples were spiked with stained tumor cells and the sample was filtrated using a porous structure with pore sizes of 7.4 µm. The possible removal of lysed erythrocytes and the retention of CTCs were assessed. The filtration process was observed in real-time using fluorescence microscopy, whereby arriving cells were counted in order to determine the number of CTCs present in the blood. Through optimization of the microfluidic channel design, the cell retention rate could be increased by 13% (from 76% ± 7% to 89% ± 5%). Providing the possibility for real-time detection significantly improved quantification efficiency even for the smallest cells evaluated. While end-point evaluation resulted in a detection rate of 63% ± 3% of the spiked cells, real-time evaluation led to an increase of 21% to 84% ± 4%. The established protocol provides an advantageous and efficient method for integration of fully automated sample preparation and CTC quantification into a lab-on-a-chip system.

Background: Cross-linking and immunoprecipitation followed by next-generation sequencing (CLIP-seq) is the state-of-the-art technique used to experimentally determine transcriptome-wide binding sites of RNA-binding proteins (RBPs). However, it relies on gene expression, which can be highly variable between conditions and thus cannot provide a complete picture of the RBP binding landscape. This creates a demand for computational methods to predict missing binding sites. Although there exist various methods using traditional machine learning and lately also deep learning, we encountered several problems: many of these are not well documented or maintained, making them difficult to install and use, or are not even available. In addition, there can be efficiency issues, as well as little flexibility regarding options or supported features., Results: Here, we present RNAProt, an efficient and feature-rich computational RBP binding site prediction framework based on recurrent neural networks. We compare RNAProt with 1 traditional machine learning approach and 2 deep-learning methods, demonstrating its state-of-the-art predictive performance and better run time efficiency. We further show that its implemented visualizations capture known binding preferences and thus can help to understand what is learned. Since RNAProt supports various additional features (including user-defined features, which no other tool offers), we also present their influence on benchmark set performance. Finally, we show the benefits of incorporating additional features, specifically structure information, when learning the binding sites of an hairpin loop binding RBP., Conclusions: RNAProt provides a complete framework for RBP binding site predictions, from data set generation over model training to the evaluation of binding preferences and prediction. It offers state-of-the-art predictive performance, as well as superior run time efficiency, while at the same time supporting more features and input types than any other tool available so far. RNAProt is easy to install and use, comes with comprehensive documentation, and is accompanied by informative statistics and visualizations. All this makes RNAProt a valuable tool to apply in future RBP binding site research., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations ( KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method ( KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody's Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.

Conductive polymer actuators and sensors rely on controlled ion transport coupled to a potential/charge change. In order to understand and control such devices, it is of paramount importance to understand the factors that determine ion flux at various conditions, including the synthesis potential. In this work, the ion transport in thinner poly-3,4-ethylenedioxythiophene (PEDOT) films during charge/discharge driven by cyclic voltammetry is studied by consideration of the electrochemical quartz crystal microbalance (EQCM) and the results are compared to the actuation responses of thicker films that have been synthesized with the same conditions in the bending and linear expansion modes. The effects of polymerization potentials of 1.0 V, 1.2 V, and 1.5 V are studied to elucidate how polymerization potential contributes to actuation, as well the involvement of the EQCM. In this work, it is revealed that there is a shift from anion-dominated to mixed to cation-dominated activity with increased synthesis potential. Scanning electron microscopy shows a decrease in porosity for the PEDOT structure with increasing synthesis potential. EQCM analysis of processes taking place at various potentials allows the determination of appropriate potential windows for increased control over devices.

CRISPR-Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR-Cas system characterization that offers annotation quality previously available only after manual inspection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 μL/reaction to 9.6 μL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.

For large-scale analysis of complex protein mixtures, liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity and ability to both identify and quantify thousands of proteins in a single measurement. Sample preparation typically comprises site-specific cleavage of proteins into peptides, followed by desalting and concomitant peptide enrichment, which is commonly performed by solid phase extraction. Desalting workflows may include multiple liquid handling steps and are thus error prone and labour intensive. To improve the reproducibility of sample preparation for low amounts of protein, we present a centrifugal microfluidic disk that automates all liquid handling steps required for peptide desalting by solid phase extraction (DesaltingDisk). Microfluidic implementation was enabled by a novel centrifugal microfluidic dosing on demand structure that enabled mapping multiple washing steps onto a microfluidic disk. Evaluation of the microfluidic disk was performed by LC-MS/MS analysis of tryptic HEK-293 eukaryotic cell peptide mixtures desalted either using the microfluidic disk or a manual workflow. A comparable number of peptides were identified in the disk and manual set with 19 775 and 20 212 identifications, respectively. For a core set of 10 444 peptides that could be quantified in all injections, intensity coefficients of variation were calculated based on label-free quantitation intensities. The disk set featured smaller variability with a median CV of 9.3% compared to the median CV of 12.6% for the manual approach. Intensity CVs on protein level were lowered from 5.8% to 4.2% when using the LabDisk. Interday reproducibility for both workflows was assessed by LC-SRM/MS analysis of samples that were spiked with 11 synthetic peptides of varying hydrophobicity. Except for the most hydrophilic and hydrophobic peptides, the average CV was lowered to 3.6% for the samples processed with the disk compared to 7.2% for the manual workflow. The presented centrifugal microfluidic DesaltingDisk demonstrates the potential to improve reproducibility in the sample preparation workflow for proteomic mass spectrometry, especially for application with limited amount of sample material.

Open burning is illegal in Ukraine, yet Ukraine has, on average, 300 times more fire activity per year (2001-2019) than most European countries. In 2016 and 2017, 47% of Ukraine was identified as cultivated area, with a total of 70% of land area dedicated to agricultural use. Over 57% of all active fires in Ukraine detected using space-borne Visible Infrared Imaging Radiometer Suite (VIIRS) during 2016 and 2017 were associated with pre-planting field clearing and post-harvest crop residue removal, meaning that the majority of these fires are preventable. Due to the small size and transient nature of cropland burns, satellite-based burned area (BA) estimates are often underestimated. Moreover, traditional spectral-based BA algorithms are not suitable for distinguishing burned from plowed fields, especially in the black soil regions of Ukraine. Therefore, we developed a method to estimate agricultural BA by calibrating VIIRS active fire data with exhaustively mapped cropland reference areas (42 958 fields). Our study found that cropland BA was significantly underestimated (by 30%-63%) in the widely used Moderate Resolution Imaging Spectroradiometer-based MCD64A1 BA product, and by 95%-99.9% in Ukraine's National Greenhouse Gas Inventory. Although crop residue burns are smaller and emit far less emissions than larger wildfires, reliable monitoring of crop residue burning has a number of important benefits, including (a) improving regional air quality models and the subsequent understanding of human health impacts due to the proximity of crop residue burns to urban locations, (b) ensuring an accurate representation of predominantly smaller fires in regional emission inventories, and (c) increasing awareness of often illegal managed open burning to provide improved decision-making support for policy and resource managers.

We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml-1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 μL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We present a simple and fast one-step heterogeneous immunoassay, with performance characteristics that can enable easy and versatile adaptation to miniaturized, automated point-of-care systems. This novel analytical method uses magnetic and fluorescent beads as capture and detection agents respectively. Its main feature is the measurement of the fluorescent signal in the bound-free phase for (semi-)quantitative detection of analytes. Thus, no washing is required and the workflow consists only of sample and reagent supply, incubation, separation and detection. The immunoassay concept is demonstrated with C-reactive protein (CRP), a systemic inflammation marker. CRP in only 5 μL of undiluted serum was measured in the range 20-140 mg L -1 (includes clinically relevant cut-off values). The limit of detection (LOD) was 22.1 ± 6.3 mg L -1 (incubation 15 min). A CRP certified reference material was measured on five different days. Intra- and inter-assay coefficients of variation were 4.6 ± 1.9% and 5.6% respectively. To demonstrate the compatibility of the assay concept with additional matrices and concentration ranges, three oral inflammation markers, namely matrix metalloproteinases 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), were measured in saliva in the ranges 0.47-30 ng mL -1 for MMP-8 and MMP-9, and 0.69-44 ng mL -1 for TIMP-1. LODs were 0.24 ng mL -1 , 0.38 ng mL -1 and 0.39 ng mL -1 respectively (incubation 20 min). Multiplexing capacity of the assay concept was also shown with these markers. The demonstrated excellent reproducibility of the results, combined with the versatility and low complexity of the introduced immunoassay concept, make it an attractive candidate for applied analytical chemistry and automated point-of-care testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120-131 µ m in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22 µ m). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm -2 driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model., (Creative Commons Attribution license.)

Anion exchange membranes (AEMs) have shown a significant rise in performance and durability within recent years for applications such as electrolysis and fuel cells. However, in vanadium redox-flow batteries, their use is of particular interest to lower costs and self-discharge rates compared to conventional perfluorinated sulfonic acid-based ionomers such as Nafion. In this work we evaluate the properties of two commercial AEMs, Aemion™ and Aemion+™, based on ex situ characterizations, an accelerated stress test degradation study (>1000 hours storage in highly oxidizing VO 2 + electrolyte at 35 °C) and electrochemical battery cycle tests. All membranes feature low ionic resistances of below 320 mΩ cm 2 , enabling battery cycling at 100 mA cm -2 . Aemion shows considerable VO 2+ formation within a VO 2 + stress test, whereas Aemion+ remains almost unaffected in the 1058 h stress test. Evaluating self-discharge data, cycling performance and durability data, Aemion+™ (50 μm thickness) features the best properties for vanadium redox-flow battery operation., Competing Interests: B. Britton and A. Belletti are employees and minor shareholders of Ionomr Innovations Inc., (This journal is © The Royal Society of Chemistry.)

This study investigated the potential of salivary bacterial and protein markers for evaluating the disease status in healthy individuals or patients with gingivitis or caries. Saliva samples from caries- and gingivitis-free individuals ( n = 18), patients with gingivitis ( n = 17), or patients with deep caries lesions ( n = 38) were collected and analyzed for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). The resulting data were subjected to principal component analysis and used as a training set for random forest (RF) modeling. This computational analysis revealed four biomarkers (IL-4, IL-13, IL-2-RA, and eotaxin/CCL11) to be of high importance for the correct depiction of caries in 37 of 38 patients. The RF model was then used to classify 10 subjects (five caries-/gingivitis-free and five with caries), who were followed over a period of six months. The results were compared to the clinical assessments of dental specialists, revealing a high correlation between the RF prediction and the clinical classification. Due to the superior sensitivity of the RF model, there was a divergence in the prediction of two caries and four caries-/gingivitis-free subjects. These findings suggest IL-4, IL-13, IL-2-RA, and eotaxin/CCL11 as potential salivary biomarkers for identifying noninvasive caries. Furthermore, we suggest a potential association between JAK/STAT signaling and dental caries onset and progression.

Oral health is important not only due to the diseases emerging in the oral cavity but also due to the direct relation to systemic health. Thus, early and accurate characterization of the oral health status is of utmost importance. There are several salivary biomarkers as candidates for gingivitis and periodontitis, which are major oral health threats, affecting the gums. These need to be verified and validated for their potential use as differentiators of health, gingivitis and periodontitis status, before they are translated to chair-side for diagnostics and personalized monitoring. We aimed to measure 10 candidates using high sensitivity ELISAs in a well-controlled cohort of 127 individuals from three groups: periodontitis (60), gingivitis (31) and healthy (36). The statistical approaches included univariate statistical tests, receiver operating characteristic curves (ROC) with the corresponding Area Under the Curve (AUC) and Classification and Regression Tree (CART) analysis. The main outcomes were that the combination of multiple biomarker assays, rather than the use of single ones, can offer a predictive accuracy of > 90% for gingivitis versus health groups; and 100% for periodontitis versus health and periodontitis versus gingivitis groups. Furthermore, ratios of biomarkers MMP-8, MMP-9 and TIMP-1 were also proven to be powerful differentiating values compared to the single biomarkers.

The need for tissue contact makes photoacoustic imaging not applicable for special medical applications like wound imaging, endoscopy, or laser surgery. An easy, stable, and contact-free sensing technique might thus help to broaden the applications of the medical imaging modality. In this work, it is demonstrated for the first time that remote photoacoustic sensing by speckle analysis can be performed in the MHz sampling range by tracking a single speckle using a four quadrant photo-detector. A single speckle, which is created by self-interference of surface back-reflection, is temporally analyzed using this photo-detector. Phantoms and skin samples are measured in transmission and reflection mode. The potential for miniaturization for endoscopic application is demonstrated by fiber bundle measurements. In addition, sensing parameters are discussed. Photoacoustic sensing in the MHz sampling range by single speckle analysis with the four quadrant detector is successfully demonstrated. Furthermore, the endoscopic applicability is proven, and the sensing parameters are convenient for photoacoustic sensing. It can be concluded that a single speckle contains all the relevant information for remote photoacoustic signal detection. Single speckle sensing is therefore an easy, robust, contact-free photoacoustic detection technique and holds the potential for economical, ultra-fast photoacoustic sensing. The new detection technique might thus help to broaden the field of photoacoustic imaging applications in the future.

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 10 5 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

Over the past few years, great attention has been given to nonaqueous lithium-air batteries owing to their ultrahigh theoretical energy density when compared with other energy storage systems. Most of the research interest, however, is dedicated to batteries operating in pure or dry oxygen atmospheres, while Li-air batteries that operate in ambient air still face big challenges. The biggest challenges are H 2 O and CO 2 that exist in ambient air, which can not only form byproducts with discharge products (Li 2 O 2 ), but also react with the electrolyte and the Li anode. To this end, recent progress in understanding the chemical and electrochemical reactions of Li-air batteries in ambient air is critical for the development and application of true Li-air batteries. Oxygen-selective membranes, multifunctional catalysts, and electrolyte alternatives for ambient air operational Li-air batteries are presented and discussed comprehensively. In addition, separator modification and Li anode protection are covered. Furthermore, the challenges and directions for the future development of Li-air batteries are presented., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present a proof-of-principle study on automated, highly sensitive and multiplexed qPCR quantification by centrifugal microfluidics. The MRD disk can be used for standardisation of repetitive, longitudinal assays with high requirements on reproducibility and sensitivity, such as cancer monitoring. In contrast to high-throughput qPCR automation by bulky and expensive robotic workstations we employ a small centrifugal microfluidic instrument, addressing the need of low- to mid-throughput applications. As a potential application we demonstrate automated minimum residual disease (MRD) monitoring of prognostic markers in patients with acute lymphoblastic leukaemia (ALL). The disk-workflow covers all aspects of clinical gold standard MRD quantification: generation of standard curves, specificity controls, no template controls and quantification of the ALL patient sample. We integrated a highly sensitive, colorimetric 2-plex analysis of MRD targets, as well as a 2-plex analysis of reference genes, both in parallel and in a single LabDisk cartridge. For this purpose, a systematic procedure for crosstalk- and signal-to-noise-optimisation is introduced, providing a guideline for efficient multiplex readout inside microfluidic platforms. The qPCR standard curves (n = 12/12) generated on-disk reach clinically required linearity (R2 = 98.1% to R2 = 99.8%). In three consecutive MRD disk runs with an ALL patient sample containing the two representative MRD targets VH3D3D5JH3 and VkIkde, we observe high accordance between the on-disk quantifications (48 ± 6 copies/reaction and 69 ± 6 copies/reaction) and the expected concentrations (57 copies/reaction for both targets). In comparison to the clinical gold standard of manually pipetted, singleplex assays, the MRD disk yields comparable limit of quantification (1 × 10-4) in n = 6/6 analyses (vs. n = 4/4 in gold standard) and a limit of detection (1 × 10-5) in n = 6/6 analysis (vs. n = 2/4 in gold standard). The automation reduces the risk of manual liquid handling errors, making the MRD disk an attractive solution to assure reproducibility in moderate-throughput, longitudinal gene quantification applications.

We implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two subvolumes, droplet generation, and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by exemplary droplet digital loop-mediated isothermal amplification (ddLAMP). Within 5 min, the reaction mix is split into subvolumes of 10.5 and 2.5 μL, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 and 213.9 ± 7.5 μm, respectively. After 30 min of incubation, quantification over 5 log steps is demonstrated with a linearity of R 2 ≥ 0.992.

Mesenchymal stem cells (MSCs) play an important role in tissue engineering applications aiming at the regeneration or substitution of damaged tissues. In this context, off-the-shelf allogeneic MSCs would represent an attractive universal cell source. However, immune rejection is a major limitation for the clinical use of allogeneic MSCs. Immune rejection is mediated by the expression of major histocompatibility complexes (MHC)-I and -II on the donor cells. In this study, we eliminated MHC-I and/or MHC-II expression in human MSCs by using the CRISPR/Cas9 technology and investigated the effect of the individual or combined knockout of MHC-I and MHC-II on MSC survival after transplantation into immunocompetent mice. Elimination of MHC-I and/or MHC-II expression did not affect mesenchymal marker gene expression, viability, proliferation and the differentiation potential of MSCs in vitro . However, cell survival of transplanted MSCs was significantly elevated in MHC-I and MHC-II deficient MSCs. A direct side-by-side comparison does not reveal any significant difference in the immunogenicity of MHC-I and MHC-II knockout MSCs. Moreover, double knockout of MHC-I and MHC-II did not further increase in vivo cell survival of transplanted MSCs. Our results demonstrate that knockout of MHC-I and/or MHC-II represents an effective strategy to prevent immune rejection of allogeneic MSCs., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Background: With the advances in next-generation sequencing technologies, it is possible to determine RNA-RNA interaction and RNA structure predictions on a genome-wide level. The reads from these experiments usually are chimeric, with each arm generated from one of the interaction partners. Owing to short read lengths, often these sequenced arms ambiguously map to multiple locations. Thus, inferring the origin of these can be quite complicated. Here we present ChiRA, a generic framework for sensitive annotation of these chimeric reads, which in turn can be used to predict the sequenced hybrids., Results: Grouping reference loci on the basis of aligned common reads and quantification improved the handling of the multi-mapped reads in contrast to common strategies such as the selection of the longest hit or a random choice among all hits. On benchmark data ChiRA improved the number of correct alignments to the reference up to 3-fold. It is shown that the genes that belong to the common read loci share the same protein families or similar pathways. In published data, ChiRA could detect 3 times more new interactions compared to existing approaches. In addition, ChiRAViz can be used to visualize and filter large chimeric datasets intuitively., Conclusion: ChiRA tool suite provides a complete analysis and visualization framework along with ready-to-use Galaxy workflows and tutorials for RNA-RNA interactome and structurome datasets. Common read loci built by ChiRA can rescue multi-mapped reads on paralogous genes without requiring any information on gene relations. We showed that ChiRA is sensitive in detecting new RNA-RNA interactions from published RNA-RNA interactome datasets., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

For soft robotics and programmable metamaterials, novel approaches are required enabling the design of highly integrated thermoresponsive actuating systems. In the concept presented here, the necessary functional component was obtained by polymer syntheses. First, poly(1,10-decylene adipate) diol (PDA) with a number average molecular weight M n of 3290 g·mol -1 was synthesized from 1,10-decanediol and adipic acid. Afterward, the PDA was brought to reaction with 4,4'-diphenylmethane diisocyanate and 1,4-butanediol. The resulting polyester urethane (PEU) was processed to the filament, and samples were additively manufactured by fused-filament fabrication. After thermomechanical treatment, the PEU reliably actuated under stress-free conditions by expanding on cooling and shrinking on heating with a maximum thermoreversible strain of 16.1%. Actuation stabilized at 12.2%, as verified in a measurement comprising 100 heating-cooling cycles. By adding an actuator element to a gripper system, a hen's egg could be picked up, safely transported and deposited. Finally, one actuator element each was built into two types of unit cells for programmable materials, thus enabling the design of temperature-dependent behavior. The approaches are expected to open up new opportunities, e.g., in the fields of soft robotics and shape morphing.

Background: Galaxy is a web-based and open-source scientific data-processing platform. Researchers compose pipelines in Galaxy to analyse scientific data. These pipelines, also known as workflows, can be complex and difficult to create from thousands of tools, especially for researchers new to Galaxy. To help researchers with creating workflows, a system is developed to recommend tools that can facilitate further data analysis., Findings: A model is developed to recommend tools using a deep learning approach by analysing workflows composed by researchers on the European Galaxy server. The higher-order dependencies in workflows, represented as directed acyclic graphs, are learned by training a gated recurrent units neural network, a variant of a recurrent neural network. In the neural network training, the weights of tools used are derived from their usage frequencies over time and the sequences of tools are uniformly sampled from training data. Hyperparameters of the neural network are optimized using Bayesian optimization. Mean accuracy of 98% in recommending tools is achieved for the top-1 metric., Conclusions: The model is accessed by a Galaxy API to provide researchers with recommended tools in an interactive manner using multiple user interface integrations on the European Galaxy server. High-quality and highly used tools are shown at the top of the recommendations. The scripts and data to create the recommendation system are available under MIT license at https://github.com/anuprulez/galaxy&#95;tool&#95;recommendation., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)