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Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non-host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non-ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms ( SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen. [ABSTRACT FROM AUTHOR]

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study. [ABSTRACT FROM AUTHOR]

The main objective of this study is to explore possible synergistic or additive effects of combinations of chemical disinfectants (sodium hypochlorite, peracetic acid, hydrogen peroxide, chlorine dioxide) and UV in their efficacy in inactivating free-living bacteria and removing biofilms. In contrast to most studies, this study examines disinfection of municipal water in a pilot-scale system using a mixed bacterial suspension, which enables a better simulation of the conditions encountered in actual industrial environments. It was shown that the combination of either hypochlorite, hydrogen peroxide, peracetic acid, or chlorine dioxide with UV yielded additive effects on the inactivation of free-living bacteria. Actual synergy was observed for the combination of UV and 5 ppm hydrogen peroxide. Regarding biofilm treatment, additive effects were observed using the combination of hydrogen peroxide and UV. The promising results obtained in this study indicate that the combination of UV and chemical disinfectants can considerably reduce the amount of chemicals required for the effective disinfection and treatment of biofilms. [ABSTRACT FROM AUTHOR]

Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment. [ABSTRACT FROM AUTHOR]

Aims: This study assessed the value of a commercial alkaline solution of hop β-acids (HBA) for prevention of microbial degradation of thick juice, a concentrated intermediate product in the production of beet sugar. Methods and Results: The antimicrobial effect of different concentrations of HBA against microbial degradation of thick juice was tested in a pilot-scale storage experiment. Chemical, biochemical and microbial parameters were monitored during thick juice storage. Thick juice degradation, indicated as a decrease in pH, was generally accompanied by an increase in the count of fastidious bacteria (FB) on Columbia Agar with Sheep Blood (CAwSB), which were mainly identified as Tetragenococcus halophilus. Addition of HBA delayed juice acidification and the development of FB in a concentration-dependent manner. The susceptibility of FB to HBA was determined by plating degraded thick juice (FB > 105 CFU ml−1) on CAwSB plates with different concentrations of HBA (0–160 ppm). None of the HBA concentrations tested reduced the number of FB colonies formed, but increasing HBA concentrations extended the lag time of colony formation. Conclusions: HBA produce no measurable bactericidal effect, but retard the development of FB in thick juice. Moreover, HBA do not prevent the thick juice from deteriorating, but significantly delay its degradation. Significance and Impact of the Study: These results indicate that adding a commercially available HBA formulation can prolong the storage life of thick juice in the sugar industry, although degradation cannot be eliminated. Future research will focus on the detailed characterization of FB consistently isolated from degraded thick juice and on determining their role in thick juice degradation. [ABSTRACT FROM AUTHOR]

Twenty-five Aeromonas hydrophila strains, isolated from freshwater fish and from freshwater samples were identified, using API 20NE and CH50. They were screened for the presence of plasmids. A group of 10 strains was found without plasmids, 11 strains harbored one plasmid (20kb), and a third group (four strains) contained two or three plasmids. A 20kb plasmid was common to all plasmid-positive strains. Although their plasmid profile differs, all groups arc very similar with regard to their biochemical and physiological characterization. However, the differences observed using clustering can be explained by characteristics, some of which have been reported as virulence associated. No relationship between these factors and the presence of plasmids is found. The possibility of differentiating A. hydrophila strains on the basis of biochemical characteristics using a cluster analysis is considered. [ABSTRACT FROM AUTHOR]

The purpose of the study was to provide an example of integrated monitoring and control of foodborne viruses using the assessment of one vertical production enterprise and the recommendations given to it. A risk assessment for virological hazards in lettuce was carried out based on the Codex Alimentarius framework, modified to consider viral hazards associated with fresh (whole) lettuce. The fit-for-purpose model was constructed by a complex process of: 1) identification of premises-selection of sampling sites, through the analysis of background information questionnaires based on HACCP audit principles and food safety fact-finding visits, 2) development of sampling guidance documents, 3) a half-year sampling campaign, 4) molecular analysis of the presence of index, human adenoviruses (hAdV) and human pathogenic viruses, noroviruses (NoVGI, NoVGII), and hepatitis A virus (HAV), 5) fit-for-purpose risk assessment, 6) development of a fit-for-purpose guidance sheet for the enterprise food safety manager for the prevention of contamination of leafy greens by viruses. Molecular virological analysis resulted in the detection of hAdV (55.65%, 123/221), and NoVGII (16.66%, 4/24), while NoVGI (0/55) and HAV (0/60) were not detected in the analyzed samples. HAdVs were detected in samples of all three phases (production, processing, and point of sale). The elevated prevalence of hAdVs supports the existence of routes of produce viral contamination. NoVs GI were detected at the point of sale in fresh lettuce heads, supporting the previous finding that pathogenic viruses can follow the routes of index viruses. NoVs GII were detected in irrigation water, harvesters' hands and manure samples, indicating sewage contamination of water, and unsatisfactory levels of hygiene concerning hands hygiene and use of toilet facilities. As a result of the study a fit-for-purpose guidance sheet was finally produced for the prevention of contamination of leafy green vegetables by viruses. The integrated monitoring and control process of the study can be applied to all leafy green vegetables production sites. [ABSTRACT FROM AUTHOR]

Abstract: Characterization of the microflora during malting is an essential step towards process management and optimization. Up till now, however, microbial characterization in the malting process has mostly been done using culture-dependent methods, probably leading to biased estimates of microbial diversity. The aim of this study was to characterize the bacterial communities using two culture-independent methods, including Terminal Restriction Fragment Length Polymorphism (T-RFLP) and 454 pyrosequencing, targeting the 16S rRNA gene. Studied samples originated from two harvest years and two malting houses malting the same batch of barley. Besides targeting the entire bacterial community (T-RFLP), emphasis was put on lactic acid bacteria (LAB) (T-RFLP and 454 pyrosequencing). The overall bacterial community richness was limited, but the community structure changed during the process. Zooming in on the LAB community using 454 pyrosequencing revealed a total of 47 species-level operational taxonomic units (OTUs). LAB diversity appeared relatively limited since 88% of the sequences were covered by the same five OTUs (representing members of Weissella, Lactobacillus and Leuconostoc) present in all samples investigated. Fluctuations in the relative abundances of the dominant LAB were observed with the process conditions. In addition, both the year of harvest and malting house influenced the LAB community structure. [Copyright &y& Elsevier]

Applications such as nucleic acid synthesis or next-generation sequencing involve repeated fluidic cycles with the same set of reagents. The large dead volumes present in external valves and pumps with relatively long supply lines mandate the inclusion of extensive rinsing steps in current protocols, resulting in the consumption of significant quantities of reagents. To allow for fast rinsing, to reduce reagent consumption, and to ensure high reagent purity, we propose a fluidic concept based on a hierarchical branching structure. The working principle comprises a 3D fluidic network of supply lines - one line per reagent - that ensures reagents to be provided up to the entrance of every single reaction cavity, called supercells. Because all reagents are always present inside or at the inlet of a supercell, the principle allows for very rapid reagent switching, while a continuous flow avoids cross contamination. Selection of a specific reagent to enter the supercells is controlled by adjusting the pressure over different supply lines. As the pressure is regulated by a single, external controller per reagent, no integrated valves are needed. The very small distances to the reaction cavities also results in the use of minimal reagent volumes and, hence, largely reduces operational costs. We demonstrated the working principle of this concept and show an average switching time of 0.23 ± 0.09 s for the current design at a flow rate of 10 nL s -1 . We used a 10 × 10 matrix of supercells to validate the fluidic concept to be scalable towards a large number of reaction sites. In summary, we believe the presented fluidic 3D hierarchical concept allows designing flow cells that enable highly parallel, more cost-efficient, and faster work flows for applications requiring many reagent cycles.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s -1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s -1 , with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Single-molecule nanopore electrophysiology is an emerging technique for the detection of analytes in aqueous solutions with high sensitivity. These detectors have proven applicable for the enzyme-assisted sequencing of oligonucleotides. There has recently been an increased interest in the use of nanopores for the fingerprinting of peptides and proteins, referred to as single-molecule nanopore spectrometry. However, the analysis of the resulting electrophysiology traces remains complicated due to the fast unassisted translocation of such analytes, usually in the order of micro- to milliseconds, and the small ion current signal produced (in the picoampere range). Here, we present the application of a generalized normal distribution function (gNDF) for the characterization of short-lived ion current signals (blockades). We show that the gNDF can be used to determine if the observed blockades have adequate time to reach their maximum current plateau while also providing a description of each blockade based on the open pore current ( I O ), the difference caused by the pore blockade (Δ I B ), the position in time (μ), the standard deviation (σ), and a shape parameter (β), leaving only the noise component. In addition, this method allows the estimation of an ideal range of low-pass filter frequencies that contains maximum information with minimal noise. In summary, we show a parameter-free and generalized method for the analysis of short-lived ion current blockades, which facilitates single-molecule nanopore spectrometry with minimal user bias., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Despite the power of VR in immersing viewers in an experience, it generally only targets viewers via visual and auditory cues. Human beings use more senses to gather information, so expectedly, the full potential of this medium is currently not yet tapped. This study contributes in answering two research questions: (1) How can conventional VR ads be enriched by also addressing the forgotten sense of smell ?; and (2) Does doing so indeed instill more engaging experiences? A 2 × 3 between-subjects study ( n = 235) is conducted, whereby an existing branded VR commercial (Boursin Sensorium Experience) is augmented with "sound" (on/off) and (congruent/incongruent/no) "scents." The power of these sensory augmentations is evaluated by inspecting emotional, cognitive and conative dimensions of customer engagement . The results identify product-scent congruence (with sound) as a deal-maker, albeit product-scent incongruence is not necessarily a deal-breaker. The article concludes with further research avenues and a translation into managerial implications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brengman, Willems and De Gauquier.)

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin., (© 2022. The Author(s).)

We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters.

Despite the broad success of biological nanopores as powerful instruments for the analysis of proteins and nucleic acids at the single-molecule level, a fast simulation methodology to accurately model their nanofluidic properties is currently unavailable. This limits the rational engineering of nanopore traits and makes the unambiguous interpretation of experimental results challenging. Here, we present a continuum approach that can faithfully reproduce the experimentally measured ionic conductance of the biological nanopore Cytolysin A (ClyA) over a wide range of ionic strengths and bias potentials. Our model consists of the extended Poisson-Nernst-Planck and Navier-Stokes (ePNP-NS) equations and a computationally efficient 2D-axisymmetric representation for the geometry and charge distribution of the nanopore. Importantly, the ePNP-NS equations achieve this accuracy by self-consistently considering the finite size of the ions and the influence of both the ionic strength and the nanoscopic scale of the pore on the local properties of the electrolyte. These comprise the mobility and diffusivity of the ions, and the density, viscosity and relative permittivity of the solvent. Crucially, by applying our methodology to ClyA, a biological nanopore used for single-molecule enzymology studies, we could directly quantify several nanofluidic characteristics difficult to determine experimentally. These include the ion selectivity, the ion concentration distributions, the electrostatic potential landscape, the magnitude of the electro-osmotic flow field, and the internal pressure distribution. Hence, this work provides a means to obtain fundamental new insights into the nanofluidic properties of biological nanopores and paves the way towards their rational engineering.

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76-81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

An anxiety disorder can manifest in multiple ways. We saw a 21-year-old woman with an anxiety disorder in combination with superior mesenteric artery syndrome. For we know, this is the first description of this combination. There was also hypersensitivity to certain foods and rigid thinking patterns, and an autism spectrum disorder was discovered later. The recognition of the influence of autism on comorbid disorders is important in order to create an individual adapted treatment protocol.

Background: Polyetheretherketone (PEEK) is a popular material for posterior lumbar interbody fusion (PLIF) cages, although osseointegration remains limited. To optimize PEEK cage characteristics, titanium (Ti) and calcium phosphate (CaP) nanocoatings have been developed with proven mechanical safety. This multicenter randomized controlled trial compared the clinical and radiological outcome parameters of nanocoated and uncoated PEEK cages, up to 1 year after surgery., Methods: Standard open PLIF surgery was performed on 127 patients, randomized in 3 groups: Ti-nanocoated (n = 44), CaP-nanocoated (n = 46), and uncoated PEEK cages (n = 37). Clinical assessments up to 1 year after surgery included visual analogue scales (VASs), Oswestry Disability Index (ODI), and 36-Item Short Form Survey (SF-36). Primary radiological outcome parameters were implant stability and fusion status, assessed by x-ray and computed tomography (CT) scans. Patients, surgeons, and postsurgery analysts were blinded., Results: PLIF surgery with all cage types resulted in significant improvements of clinical outcome parameters, exceeding the minimum clinically important differences. No significant differences in VAS, ODI, or SF-36 scores were found among the 3 groups. One year after the surgery, 65.6% of patients with uncoated PEEK cages achieved definite fusion. Significantly more patients with nanocoated PEEK cages achieved definite fusion: 93.9% for Ti nanocoating ( P  = .0034) and 88.0% for CaP nanocoating ( P  = .032). No significant differences in fusion were found between the nanocoated cage types ( P  = .4318)., Conclusions: The similar clinical outcome improvements after 1 year suggest that nanocoated PEEK cages have the same safety and efficacy as the clinically accepted uncoated PEEK cages. Furthermore, nanocoated PEEK cages achieved a better fusion rate than uncoated PEEK cages at the 1-year follow-up. A 5-year follow-up study is warranted to revisit the findings., Clinical Relevance: The safety, efficacy, and enhanced osseointegration of nanocoated PEEK cages were demonstrated. Osseointegration is a significant predictor of positive long-term clinical outcomes and improved implant longevity, implying a clinical added value of nanocoatings. Enhanced osseointegration becomes even more important in minimally invasive spine surgery and in patients at risk for incomplete fusion., Competing Interests: Disclosures and COI: This work was funded by Orthobion GmbH (Konstanz, Germany), which developed the technology of the fine osseointegrative nanocoated implants. The funder had no role in the study design, surgical procedures, data collection, data analysis, decision to publish, nor in the preparation of the manuscript., (©International Society for the Advancement of Spine Surgery 2019.)

The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here, we describe a system where steric, electrostatic, electrophoretic, and electro-osmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electro-osmotic flow on the protein (≅9 pN at -50 mV) to be retrieved. The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies.

In order to improve osseointegration, polyetheretherketone (PEEK) interbody fusion cages are frequently surface coated. The bonding strength of the coatings is mostly investigated under unrealistic loading conditions. To close this gap, in this study, uncoated and coated cages were loaded in a clinical setup in order to investigate their real resistance against abrasion. Six uncoated, six calcium phosphate (CaP) nanocoated, and six titanium (Ti) nanocoated PEEK cages were tested in this study. The experimental setup was designed to mimic cage impaction into the intervertebral disc space using polyurethane (PU) foam blocks as vertebral body substitutes. The cage surface was inspected before and after impaction, and their weight was measured. Impaction resulted in abrasion at the tip of the ridges on the implant surface. The mean weight loss was 0.39 mg for the uncoated cages, 0.57 mg for the CaP nanocoated cages, and 0.75 mg for the Ti nanocoated cages. These differences were statistically significant. In conclusion, differences between the three cage types were found concerning the amount of abrasion. However, all three cages lost less weight than a comparative Ti plasma spray coated cage, which showed a mean weight loss of 2.02 mg. This may be because the plasma spray coating is significantly thicker than the two nanocoatings. If compared with the permitted amount of weight loss derived from an FDA guidance document, which is approximately 1.7 mg, the wear of the Ti plasma spray coated cage is above this limit, whereas the wear for all other cage types is below.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Background: Neck pain (NP) and low back pain (LBP) are highly prevalent. Exercise therapy helps, but effect sizes and therapy compliance remain low. Client-centred therapy and technology use may play a role to improve therapy outcomes. To offer technology supported rehabilitation matching patient's goals, training preferences for rehabilitation and technology familiarity need to be known., Purpose: This study aims to (1) inventory training preferences and motives, (2) evaluate whether these change during rehabilitation, and (3) evaluate familiarity with using technologies, in persons with NP/LBP., Method: Semi-structured interviews were conducted with regard to training preferences and usage of mainstream technological devices., Results: Persons with NP (n = 40) preferred to train on "lifting", "prolonged sitting" and "driving a car". Persons with LBP (n = 40) preferred to train on "household activities", "lifting" and "prolonged walking". Motives were predominantly "ability to work" and "ability to do free time occupations". Preferences shifted in ranking but remained the same during rehabilitation. Participants were familiar with the surveyed technologies., Conclusion: Persons with NP or LBP prefer to train on exercises supporting the improvement of everyday life skills. They use technologies in their professional and personal life, which may lower the threshold for the adoption of rehabilitation technologies. Implications for rehabilitation Persons with neck pain (NP) and persons with low back pain (LBP) prefer to train on specific activities that limit their functional ability during daily tasks. The underlying motives linked to preferred training activities are predominantly "being able to work" and "being able to perform free time occupations". Persons with NP and persons with LBP are accustomed to the use of mainstream technologies and the integration of these technologies in rehabilitation settings seems feasible. In order to enable technology supported rehabilitation that is client-centred, technologies need to offer an extensive number of exercises that support (components of) patient training preferences.

Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.3 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.Biological nanopore-based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Here the authors show that engineered FraC nanopores can overcome these problems and recognize biomarkers in the form of oligopeptides, polypeptides and folded proteins.

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'., (© 2017 The Author(s).)

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.