Advancement of microfluidic modulation spectroscopy as a highly sensitive protein characterization technique for the detection of small structural changes.

The higher order structure (HOS) of a protein is vital to its function and activity, making it a critical component in the development of protein-based therapeutics. Characterization of HOS can be performed using various biophysical techniques, but many of these methods have limitations such as low throughput, complicated workflow, narrow concentration range, and low sensitivity. Microfluidic Modulation Spectroscopy (MMS) is an emerging technology that addresses these limitations, offering high sensitivity and automated analysis for protein secondary structure. This study evaluates and compares the different well plate formats and scan modes of two MMS instruments. The newer Apollo system features a high throughput 96-well plate format and sweep scan mode that allows a 50% reduction in sample volume consumption and measurement time compared to the previous system. By measuring two proteins with drastically different secondary structures, the results demonstrated that the measurements were highly repeatable (>99% repeatability by area of overlap) regardless of the well plate formats or the scan modes. The limit of quantitation (LOQ) for determining structural impurity using the sweep scan mode was 3.2% and significantly better than that of FTIR at 23% from previous studies. This work highlighted the advancement of MMS as a highly sensitive technique to detect small changes of protein structures due to aggregation or misfolding.

Structure, Mechanics, and Instability of Fibrin Clot Infected with Staphylococcus epidermidis.

Health care-associated infection, over half of which can be attributed to indwelling medical devices, is a strong risk factor for thromboembolism. Although most experimental models of medical device infection draw upon isolated bacterial biofilms, in fact there is no infection without host protein contribution. Here we study, to our knowledge, a new model for medical device infection-that of an infected fibrin clot-and show that the common blood-borne pathogen Staphylococcus epidermidis influences this in vitro model of a blood clot mechanically and structurally on both microscopic and macroscopic scales. Bacteria present during clot formation produce a visibly disorganized microstructure that increases clot stiffness and triggers mechanical instability over time. Our results provide insight into the observed correlation between medical device infection and thromboembolism; the increase in model clot heterogeneity shows that S. epidermidis can rupture a fibrin clot. The resultant embolization of the infected clot can contribute to the systemic dissemination of the pathogen.

Effect of Antimicrobial and Physical Treatments on Growth of Multispecies Staphylococcal Biofilms.

The prevalence and structure of Staphylococcus aureus and Staphylococcus epidermidis within multispecies biofilms were found to depend sensitively on physical environment and antibiotic dosage. Although these species commonly infect similar sites, such as orthopedic implants, little is known about their behavior in multispecies communities, particularly in response to treatment. This research establishes that S. aureus is much more prevalent than S. epidermidis when simultaneously seeded and grown under unstressed conditions (pH 7, 37°C) in both laboratory and clinical strains. In multispecies communities, S. epidermidis is capable of growing a more confluent biofilm when the addition of S. aureus is delayed 4 to 6 h during 18 h of growth. Different vancomycin dosages generate various behaviors: S. epidermidis is more prevalent at a dose of 1.0 µg/ml vancomycin, but reduced growth of both species occurs at 1.9 µg/ml vancomycin. This variability is consistent with the different MICs of S. aureus and S. epidermidis Growth at higher temperature (45°C) results in an environment where S. aureus forms porous biofilms. This porosity allows S. epidermidis to colonize more of the surface, resulting in detectable S. epidermidis biomass. Variations in pH result in increased prevalence of S. epidermidis at low pH (pH 5 and 6), while S. aureus remains dominant at high pH (pH 8 and 9). This work establishes the structural variability of multispecies staphylococcal biofilms as they undergo physical and antimicrobial treatments. It provides a basis for understanding the structure of these communities at infection sites and how treatments disrupt their multispecies behaviors.IMPORTANCEStaphylococcus aureus and Staphylococcus epidermidis are two species of bacteria that are commonly responsible for biofilm infections on medical devices. Biofilms are structured communities of bacteria surrounded by polysaccharides, proteins, and DNA; bacteria are more resistant to antimicrobials as part of a biofilm than as individual cells. This work investigates the structure and prevalence of these two organisms when grown together in multispecies biofilms and shows shifts in the behavior of the polymicrobial community when grown in various concentrations of vancomycin (an antibiotic commonly used to treat staphylococcal infections), in a high-temperature environment (a condition previously shown to lead to cell disruption and death), and at low and high pH (a change that has been previously shown to soften the mechanical properties of staphylococcal biofilms). These shifts in community structure demonstrate the effect such treatments may have on multispecies staphylococcal infections.

Displacement back analysis for a high slope of the Dagangshan Hydroelectric Power Station based on BP neural network and particle swarm optimization.

The right bank high slope of the Dagangshan Hydroelectric Power Station is located in complicated geological conditions with deep fractures and unloading cracks. How to obtain the mechanical parameters and then evaluate the safety of the slope are the key problems. This paper presented a displacement back analysis for the slope using an artificial neural network model (ANN) and particle swarm optimization model (PSO). A numerical model was established to simulate the displacement increment results, acquiring training data for the artificial neural network model. The backpropagation ANN model was used to establish a mapping function between the mechanical parameters and the monitoring displacements. The PSO model was applied to initialize the weights and thresholds of the backpropagation (BP) network model and determine suitable values of the mechanical parameters. Then the elastic moduli of the rock masses were obtained according to the monitoring displacement data at different excavation stages, and the BP neural network model was proved to be valid by comparing the measured displacements, the displacements predicted by the BP neural network model, and the numerical simulation using the back-analyzed parameters. The proposed model is useful for rock mechanical parameters determination and instability investigation of rock slopes.

Mercury distribution, mobilization and bioavailability in polluted sediments of Scheldt Estuary and Belgian Coastal Zone.

In this study, the vertical distribution of mercury (Hg) in estuarine and marine sediment porewaters and solid phases was assessed by conventional and passive sampling techniques in the historically polluted Scheldt Estuary and Belgian Coastal Zone (BCZ). The Diffusive Gradients in Thin-films (DGT) measured labile Hg concentrations (HgLA) were mostly lower than the porewater Hg concentrations (HgPW), and they also presented different vertical distribution patterns. Still high Hg concentrations in the sediment solid phases, comparable to the historical ones, were observed. Even though pH, redox potential and dissolved sulfide concentration could influence the Hg biogeochemical behaviour, organic matter (OM) played a key role in governing Hg mobilization from sediment solid phase to porewater and in its partitioning between porewater and solid phase over depth. In the marine sediments, where OM had a marine signature, higher labile Hg concentrations in the porewater and faster resupply from the solid phase were observed. The DGT technique showed significant potential not only for the measurement of bioavailable Hg fractions in porewater, but also for the assessment of kinetic parameters governing the release of labile Hg species from the solid phase with the assistance of the DGT Induced Fluxes in Sediments (DIFS) model.

Origin and partitioning of mercury in the polluted Scheldt Estuary and adjacent coastal zone.

Estuaries and coastal zones are areas with complex biogeochemical and hydrological cycles and are generally facing intense pollution due to anthropogenic activities. An emblematic example is the Scheldt Estuary which ends up in the North Sea and has been historically heavily contaminated by multiple pollutants, including mercury (Hg). We report here Hg species and their levels in surface waters of the Scheldt Estuary and the Belgian Part of North Sea (BPNS) from different sampling campaigns in February-April 2020 and 2021. Along the estuary, Hg concentration on suspended particles ([HgSPM]) progressively decreased with increasing salinity and was strongly correlated with organic matter content (%Corg) and origin (identified with δ13Corg). While [HgSPM] drives total Hg concentration in the estuary (total dissolved Hg, HgTD is only 7 ± 6 %), annual and daily variations of total Hg levels were mostly attributed to changes in SPM loads depending on river discharge and tidal regime. In the BPNS, a significant fraction of total Hg occurs as HgTD (40 ± 21 %) and the majority of this HgTD was reducible (i.e. labile Hg), meaning potentially available for microorganisms. Compared to the '90s, a significant decrease of [HgSPM] was observed in the estuary, but this was not the case for [HgTD], which can be due to (1) still significant discrete discharges from Antwerp industrial area, and (2) higher Hg partitioning towards the dissolved phase in the water column relative to the '90s. Our results highlight the important contribution of the Scheldt estuary for the Hg budget in North Sea coastal waters, as well as the need for seasonal monitoring of all Hg species.

Investigation on metal geochemical cycling in an anthropogenically impacted tidal river in Belgium.

The geochemical behavior of metals in water and sediment was investigated in the tidal section of the Zenne River in Belgium. Twelve-hour sampling campaigns were performed in October 2013 and March 2021 at the mouth of the Zenne River, under dry and rainy weather conditions respectively. Water samples were collected every hour while the passive samplers of Diffusive Gradients in Thin-films (DGT) were deployed continuously during a tidal cycle. In addition, bottom sediments were sampled at the tidal station and water samples were taken upstream and downstream of that station to identify the metal sources. The highest concentrations of Fe, Mn, Pb, Cr, Ni and Zn appear at low tide, indicating the Zenner River as a main source. However, for Co, Cd and Cu, other sources including upstream transport may explain their behavior during a tidal cycle. Fe, Pb and Cr are essentially transported in the particulate phase (<10 % dissolved) while the other metals in the dissolved phase (20 to 90 %). Rainfall and wind gust events also play an important role in trace metal distribution, increasing sediment resuspension and metal desorption. A good agreement was found between the time-averaged dissolved and DGT-labile metal concentrations with the exception of Cu and Fe, which form strong organic Cu complexes and Fe colloids respectively. The sediments of the tidal Zenne are contaminated by trace metals, thus acting as a secondary pollution source to the river. The reductive dissolution of Mn and Fe oxyhydroxides and the release of associated trace metals are the main mobilization mechanisms. Knowledge of the upstream and downstream levels in the water column, the benthic fluxes, which are based on turbulent diffusion, and the partitioning between dissolved and particulate phases allow to explain the metal concentration variations during the tidal cycle.

Anthropogenic activities influence the mobilization of trace metals and oxyanions in coastal sediment porewaters.

The biogeochemical cycle of trace metals and oxyanions in marine sediments is mainly controlled by early diagenesis of organic material, but anthropogenic activities such as dumping of dredged sludge or shipyard activities can strongly perturb these natural processes. Therefore, the impact of dumping of dredged sludge, shipyard activities such as blasting of boat hulls and the mixing of polluted Scheldt estuary water with less polluted coastal water on the mobilization of trace metals and oxyanions in the sediments was studied in the pre- and post-bloom period (March and October) in Belgian Coastal Zone (BCZ). Vertical profiles of total and labile dissolved trace element concentrations, dissolved sulfide, pH and redox potential were assessed in sediment porewaters. Generally, total and labile dissolved element profiles in the porewaters are well correlated, showing higher concentrations in March when the redox potential is lower and sulfide concentrations higher. Low pH levels were documented together with high amounts of Fe and associated elements in March at the dumping station. At the coastal station in the vicinity of the shipyards, sediment porewaters are enriched in dissolved Zn while at the mouth of the Scheldt estuary the highest dissolved Cu levels were found. Theoretical calculations with Visual MINTEQ confirm the increased Fe and associated element concentrations in March (lower pH) and the lower metal concentrations in October (lower Eh). R values, ratio of labile dissolved to total dissolved concentrations, reflect the potential of the solid sediment phase to supply labile elements to the porewater. They were lowest for Cu and highest for V. This study showed that anthropogenic activities in the BCZ such as dumping of dredged sludge and blasting of boat hulls have a serious impact on the levels, distribution and bioavailability of contaminants in the sediments, which may form a real threat to the benthic ecosystem.

A multimethodological evaluation of arsenic in the Zenne River, Belgium: Sources, distribution, geochemistry, and bioavailability.

The distribution and geochemistry of arsenic (As) in water and sediments of the Zenne River, a small urban river flowing through Brussels (Belgium), were assessed based on the results of 18 sampling campaigns performed between 2010 and 2021. In general, concentrations of As sharply increase between Vilvoorde and Eppegem and are up to 6-8 times higher in the section downstream of Eppegem in comparison to the upstream part of the Zenne. The monitoring surveys in which the grab water samples were taken at a 1-hour sampling frequency revealed that the large temporal variability in As concentrations found in the downstream part of the river is driven by the tidal cycle. The diffusive gradients in thin films (DGT) technique was used to assess the DGT labile As species in surface water and sediment porewater. Three DGT sorbents (Metsorb, Lewatit FO 36, and ZrO2) for the determination of total As were applied to compare their performance, and the 3-mercaptopropyl-functionalized silica (3-MFS) was used for the speciation of As(III) in porewater. Arsenic species are fully labile in surface waters as the DGT time-integrated concentrations of As were in good agreement with the average concentrations calculated from the grab samplings. In sediment porewaters, As is predominantly present as non-DGT labile species (66-93 %), and the DGT labile As fraction is dominated by As(III). Flux calculations evaluating the relative importance of different As sources to the Zenne River revealed the presence of a point source on the tributary Tangebeek, which contributes to 87 % of the As load carried by the Zenne River.

Tensor Completion via Complementary Global, Local, and Nonlocal Priors.

Completing missing entries in multidimensional visual data is a typical ill-posed problem that requires appropriate exploitation of prior information of the underlying data. Commonly used priors can be roughly categorized into three classes: global tensor low-rankness, local properties, and nonlocal self-similarity (NSS); most existing works utilize one or two of them to implement completion. Naturally, there arises an interesting question: can one concurrently make use of multiple priors in a unified way, such that they can collaborate with each other to achieve better performance? This work gives a positive answer by formulating a novel tensor completion framework which can simultaneously take advantage of the global-local-nonlocal priors. In the proposed framework, the tensor train (TT) rank is adopted to characterize the global correlation; meanwhile, two Plug-and-Play (PnP) denoisers, including a convolutional neural network (CNN) denoiser and the color block-matching and 3 D filtering (CBM3D) denoiser, are incorporated to preserve local details and exploit NSS, respectively. Then, we design a proximal alternating minimization algorithm to efficiently solve this model under the PnP framework. Under mild conditions, we establish the convergence guarantee of the proposed algorithm. Extensive experiments show that these priors organically benefit from each other to achieve state-of-the-art performance both quantitatively and qualitatively.

Cysteine-modified silica resin in DGT samplers for mercury and trace metals assessment.

Diffusive gradients in thin-films (DGT) is an in situ passive sampling technique to assess labile trace metal concentrations in different environmental matrix. The technique is consisting of a diffusive domain backed up by a resin gel that binds free metals and metal complexes that dissociate in the diffusive domain. This technique requires specific resin for special metals, for example mercury (Hg), since the classic resin (Chelex-100) gel is not applicable for Hg measurement. A simultaneous determination of Hg with other metals by the DGT was not yet reported. Two biomolecule-based resins were prepared by glutaraldehyde immobilisation of cysteine onto 3-amino-functionalised silica and 3-aminopropyl-functionalised silica, respectively. The load of functional groups on modified resins was qualitatively and quantitatively characterised. The modified resins were applied in the DGT technique and the uptake efficiency, elution efficiency, and linear accumulation of analytes of the DGT were tested. This novel DGT technique, using two cysteine-modified resins, can accumulate Hg and other metals in a broad range of pH and ionic strength in solutions. In the Belgian coastal zone (BCZ), the concentrations of Hg and other trace metals sampled by cysteine-modified resin-DGTs were similar as those by the other two DGT assemblies for Hg and other trace metals, respectively. The cysteine-modified silica resin combined the features of Chelex-100 resin and 3-mercaptopropyl silica resin and allowed simultaneous determination of labile Hg and other trace metals. The resin with a higher load of functional groups also showed higher performance in the further application in the DGT technique.

Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity.

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm2s) versus exponential phase (2,212 ± 264, cells/cm2 s). Furthermore, the expression of sdrG-a cell surface adhesion protein with specificity for fibrinogen-was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G' = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G' = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

Investigation on trace metal speciation and distribution in the Scheldt estuary.

The biogeochemical behavior of Cd, Co, Cr, Cu, Ni and Pb along the historically polluted Scheldt estuary (Belgium - The Netherlands) was investigated in this study. As never studied before in this area, labile trace metals were measured using the passive sampling technique of Diffusive Gradients in Thin-films (DGT), while total dissolved and particulate trace metal concentrations were assessed using classic active sampling techniques. This dual approach allowed us to highlight the variations of trace metal speciation and distribution in the estuarine surface waters, considering environmental and physicochemical gradients along the transect. The large data set obtained was then compared with literature data of historical measurements along the Scheldt (from 1980 until now), but also from other estuaries. As emphasized by our results, trace metal mobility and partitioning along the Scheldt estuary was mainly driven by biogeochemical reactions which were strongly influenced by gradients of specific estuarine physico-chemical parameters, such as salinity, turbidity, temperature and so on. Hence, all species of trace metals displayed a non-conservative behavior. More precisely, dissolved labile fractions of trace metals showed higher levels in the middle estuary, where many solubilization and remobilization processes occurred due to turbulent mixing mechanisms and an increasing salinity. Our study confirmed the decreasing trend historically observed for particulate metals along the Scheldt, as well as the rising concentrations recorded for dissolved trace metals which might also lead to an increase of their labile fraction measured by the DGT. Finally, these preliminary results suggested that a more regular monitoring of labile metal along the Scheldt estuary is essential to have an in-depth understanding of trace metal speciation and to review bioavailability of trace metals within estuarine ecosystems.

Patch-Based Principal Component Analysis for Face Recognition.

We have proposed a patch-based principal component analysis (PCA) method to deal with face recognition. Many PCA-based methods for face recognition utilize the correlation between pixels, columns, or rows. But the local spatial information is not utilized or not fully utilized in these methods. We believe that patches are more meaningful basic units for face recognition than pixels, columns, or rows, since faces are discerned by patches containing eyes and noses. To calculate the correlation between patches, face images are divided into patches and then these patches are converted to column vectors which would be combined into a new "image matrix." By replacing the images with the new "image matrix" in the two-dimensional PCA framework, we directly calculate the correlation of the divided patches by computing the total scatter. By optimizing the total scatter of the projected samples, we obtain the projection matrix for feature extraction. Finally, we use the nearest neighbor classifier. Extensive experiments on the ORL and FERET face database are reported to illustrate the performance of the patch-based PCA. Our method promotes the accuracy compared to one-dimensional PCA, two-dimensional PCA, and two-directional two-dimensional PCA.

Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel.

Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic X-linked recessive disorders that affect approximately 1 in 3500 and 1 in 20,000 male individuals, respectively. Approximately 65% of patients with DMD have deletions, 7% to 10% have duplications, and 25% to 30% have point mutations in one or more of the 79 exons of the dystrophin gene. Most clinical genetics laboratories test for deletions, and some use technologies that can detect smaller mutations and duplications. Reference and quality control materials for DMD/BMD diagnostic and carrier genetic testing are not commercially available. To help address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing and the DMD/BMD patient communities and the Coriell Cell Repositories, have characterized new and existing cell lines to create a comprehensive DMD/BMD reference material panel. Samples from 31 Coriell DMD cell lines from male probands and female carriers were analyzed using the Affymetrix SNP Array 6.0 and Multiplex Ligation-Dependent Probe Amplification (MRC-Holland BV, Amsterdam, the Netherlands), a multiplex PCR assay, and DNA sequence analysis. Identified were 16 cell lines with deletions, 9 with duplications, and 4 with point mutations distributed throughout the dystrophin gene. There were no discordant results within assay limitations. These samples are publicly available from Coriell Institute for Medical Research (Camden, NJ) and can be used for quality assurance, proficiency testing, test development, and research, and should help improve the accuracy of DMD testing.