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Follicular cell suspension (FCS) transplantation is a novel surgical method for treating resistant stable vitiligo, whereas mini punch grafting is an established effective method for treating stable vitiligo. The combination of FCS and mini punch grafting is a better strategy for the treatment of resistant stable vitiligo. The aim of the study was to evaluate the efficacy of follicular cell suspension, mini punch grafting, and a combination of both techniques in the treatment of stable vitiligo. This prospective comparative study was conducted on 48 patients with stable vitiligo. They were divided into three equal groups, including group A (treated with follicular cell suspension), group B (treated with mini punch grafting), and group C (treated with the combination of both techniques). All patients were followed-up for six months for the assessment of their therapeutic response regarding clinical outcomes. By comparing the data of the three studied groups, we found that the difference in the degree of re-pigmentation after one and three months of treatment was not significant. However, the progress of re-pigmentation was significantly different after six months of treatment among the three studied groups (P = 0.027). Specifically, re-pigmentation was significantly better in group C than in groups A and B (P = 0.037 and 0.017, respectively), but it was not significantly different between groups A and B. [ABSTRACT FROM AUTHOR]

This work investigates the significant effects of SrO on the structural, physical, and UV–visible (UV–Vis) characteristics and the γ-ray and neutron shielding ability of samarium sodium borotellurite glasses with the composition (46.5-X)B2O3 + 0.5Sm2O3 + 15TeO2 + 18Bi2O3 + 20Na2O + XSrO, where X = 0 (SrO0)–12 (SrO12)% with steps of 3 mol.%, designated as SrOX. The glass samples were prepared via the well-known melt quenching technique. The amorphous nature of the fabricated glasses was confirmed by XRD. The density (ρ) of the SrOX samples increased gradually from 3.73 ± 0.01 g/cm3 to 4.04 ± 0.01 g/cm3. The direct optical gap increased from 2.94 ± 0.01 eV to 2.98 ± 0.01 eV, and the indirect gap increased from 2.74 ± 0.01 eV to 2.95 ± 0.01 eV. Several absorption peaks were found for 4f5–4f5 electronic transitions from ground manifold 6H5/2 to excited samarium ion manifold states. The free volume (Vf), packing density (Pd), ion concentration (N), polaron radius (rp), internuclear distance (ri), field strength (F), molar volume of the boron atoms (Vb), and average boron–boron distance (dBB) changed significantly as the SrO concentration varied in the glass network. The linear attenuation coefficient (µ) followed the order: SrO0 < SrO3 < SrO6 < SrO9 < SrO12. The synthesized SrO12 glass sample had the lowest half-value layer (HVL). The fast neutron removal cross-section (FCS) values of the SrOX glass samples were 0.085 cm−1, 0.085 cm−1, 0.085 cm−1, 0.086 cm−1, and 0.086 cm−1 for samples SrO0, SrO3, SrO6, SrO9, and SrO12, respectively. The obtained results showed that the proposed compositions can be used in optical applications as well as γ-ray and neutron shielding materials. [ABSTRACT FROM AUTHOR]

Recent studies show that biofilm substances in contact with nanoplastics play an important role in the aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in the transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics–biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as, e.g., the acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with an ionic strength being realistic for fresh-water conditions. Biomolecule concentrations ranging from 0.5 g/L up to at maximum 21 g/L are considered. We use non-functionalized PS NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA-mediated bridging. In BSA–alginate mixtures, the alginate hampers this interaction, most likely due to alginate–BSA complex formation. In most BSA–alginate mixtures as in alginate alone, NP aggregation is predominantly driven by weaker, pH-independent depletion forces. The stabilizing effect of alginate is only weakened at high BSA contents, when the electrostatic BSA–BSA attraction is not sufficiently screened by the alginate. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single-component biofilm model systems based on comparing the total organic carbon (TOC) content of the extracellular biofilm substances, as usually considered, would have led to a misjudgment of the stability towards aggregation. [ABSTRACT FROM AUTHOR]

When reviewing the safety of food packaging, safety assessors must determine the migration levels of a food contact substance (FCS) from the packaging into the contained food products. Migration values can be expressed relative to levels in food using a single 'food mass-to-surface area (mF/A) ratio', representative of the general food supply. Because no published literature has examined mF/A ratios for the current U.S. food supply, this study aimed to use food databases to develop a standard mF/A ratio representative of packaged foods in the current U.S. food supply. First, we obtained information on the food-contact surface area (A) and food mass (mF) for a selection of packaged foods sold in U.S. retail stores. To adequately represent the variation of food products on the market, we identified a representative product per food category, using a 95-category list that was derived from the USDA WWEIA Food Categories. The values for A and mF per package were determined for each representative product using product size information from the Syndigo Nutrition Database. These data were used to calculate the A/mF ratio for each of the 95 representative products, and the average A/mF ratio was determined. The average A/mF ratio was weighted using consumption data from the WWEIA component of NHANES, so that more weight was given to foods that are consumed in greater quantities. The final step was to then take the reciprocal of the averaged A/mF ratio to obtain the final mF/A ratio. The resulting mF/A ratio was 4.8 g/in², which rounds to 5 g/in². In summary, we determined that 5 g/in² is representative of the current U.S. food supply and could be used as a standard mF/A ratio when estimating migration levels of an FCS from packaging into food. [ABSTRACT FROM AUTHOR]

Introduction: The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in cells is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood. Methods: Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the open cytoplasm, the stress fibers in the open cytoplasm, and those below the nucleus using three kinds of biologically inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients. Results: We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggest that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers. Conclusion: The data presented here would provide a basis for better understanding of the complex protein diffusion in the nonuniform cytoplasm, shedding light on cellular responses to mechanical stress, their local mechanical properties, and reduced turnover in senescent cells. [ABSTRACT FROM AUTHOR]

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment. [ABSTRACT FROM AUTHOR]

T Michelle Brown,1 Montserrat Vera-Llonch,2 Chisom Kanu,1 Asia Sikora Kessler,2 Aaron Yarlas,2 Sheri E Fehnel1 1Patient-Centered Outcomes Assessment, RTI Health Solutions, Research Triangle Park, NC, USA; 2Health Economics & Outcomes Research, Ionis Pharmaceuticals, Carlsbad, CA, USACorrespondence: Asia Sikora Kessler, Ionis Pharmaceuticals, Carlsbad, CA, USA, Email ASikoraKessler@ionisph.comBackground: Familial chylomicronemia syndrome (FCS) is a rare metabolic disorder that impacts physical, emotional, social, and cognitive functioning. The FCS-Symptom and Impact Scale (FCS-SIS) patient-reported outcome (PRO) measure assesses common symptoms and impacts of FCS. This study was conducted to evaluate cross-sectional psychometric properties of the FCS-SIS and its scoring method.Methods: This multisite, cross-sectional, observational study of individuals with FCS was conducted in the United States and Canada. Participants completed a survey composed of 7 PRO measures, including the FCS-SIS, and questions about clinical characteristics and demographics. The structure of the FCS-SIS was evaluated using inter-item and item-scale correlations and internal consistency reliability. Construct, known-groups, and criterion validity were evaluated by examining associations between FCS-SIS item and composite scores and other measures included within the survey.Results: Most of the 33 participants were female (63.6%) and White (78.1%). On average, participants reported first noticing FCS symptoms at ~16 years, with abdominal pain the most frequently reported initial symptom (n=20). Participants reported 2.5 acute pancreatitis attacks on average over the past year. Average FCS-SIS symptom item scores ranged from 1.8 to 3.9 (on a 0-to-10 scale [none-to-worst-possible]) within the 24-hour recall period, with an average Symptom composite score of 2.7. The average impact item scores on the FCS-SIS ranged from 1.6 to 3.0 (on a 0-to-4 scale), with an average Impact composite score of 2.1. Inter-item correlations between the FCS-SIS Symptom items ranged from 0.32 to 0.78. Corrected item-total correlations were highly satisfactory for Impact items, ranging from 0.62 to 0.85. All a priori validity hypotheses were supported by observed correlations and score differences between known groups.Conclusion: The results of this study support the structure, reliability, and validity of the FCS-SIS, laying the psychometric groundwork for longitudinal evaluation of its utility in assessing treatment benefit in FCS clinical studies.Keywords: psychometric evaluation, FCS, validity, reliability, rare disease, patient reported outcome measure

In order to tackle the global increase in overweight and obesity prevalence, several nutrient profiling systems have been developed; among others, Food Compass Score (FCS) has been designed to encompass multiple domains of food healthfulness. However, environmental sustainability of healthy diets is another crucial dimension which should not be overlooked in the context of human health. The aim of the present study is to assess the association between healthiness and environmental sustainability of food items, using the FCS and Agribalyse databases, respectively. A total of 806 matching food items were identified, grouped in 12 food categories; within each category, differences in median Z-scores between FCS and Single Environmental Footprint (EF) Score were assessed. While Fruits, Legumes and Nuts, Mixed foods, Meat Poultry and Eggs (MPE), Savory and Sweets, and Vegetables showed statistically significant differences (all p < 0.001), Beverages (p = 0.361), Dairy (p = 0.092), Fats and Oils (p = 0.594), Grains (p = 0.436), Sauce and Condiments (p = 0.093), and Seafood (p = 0.241) had similar Food Compass and Single EF Z-scores distributions. These findings underscore a relevant lack of difference between healthfulness and environmental impact of some prominent food categories, such as Grains and Seafood. Therefore, we suggest matching nutrient profiling systems with adequate environmental sustainability indices. [ABSTRACT FROM AUTHOR]

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring–DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms. [ABSTRACT FROM AUTHOR]

The effect of the changes in buffer concentrations or any additives like surfactants in the protein samples during the analysis on the single-biomolecule diffusion is one of the hidden points in the single-molecule time-resolved measurements. In the current work, phosphoglycerate kinase (PGK) labeled with Atto-647 has been investigated on the single molecule level while it diffuses in Na2CO3 buffer at concentrations that vary from 10 mg/l up to 50 mg/l. The fluorescence lifetime of PGK labeled with Atto-647 in 50 mg/ml Na2CO3 has been measured, and it was found to be 2.7 ns. The fluorescence cross correlation of the diffused protein has also been measured, which confirms that the used samples are at a single molecule level. Time decay fluorescence anisotropy has been performed for PGK labeled with Atto-647 in different concentrations of Na2CO3, and the results confirmed that there is a clear impact on the molecular translational and rotational diffusion even with slight changes in the buffer concentration. [ABSTRACT FROM AUTHOR]

When reviewing the safety of food packaging, safety assessors must determine the migration levels of a food contact substance (FCS) from the packaging into the contained food products. Migration values can be expressed relative to levels in food using a single ‘food mass-to-surface area (mF/A) ratio’, representative of the general food supply. Because no published literature has examined mF/A ratios for the current U.S. food supply, this study aimed to use food databases to develop a standard mF/A ratio representative of packaged foods in the current U.S. food supply. First, we obtained information on the food-contact surface area (A) and food mass (mF) for a selection of packaged foods sold in U.S. retail stores. To adequately represent the variation of food products on the market, we identified a representative product per food category, using a 95-category list that was derived from the USDA WWEIA Food Categories. The values for A and mF per package were determined for each representative product using product size information from the Syndigo Nutrition Database. These data were used to calculate the A/mF ratio for each of the 95 representative products, and the average A/mF ratio was determined. The average A/mF ratio was weighted using consumption data from the WWEIA component of NHANES, so that more weight was given to foods that are consumed in greater quantities. The final step was to then take the reciprocal of the averaged A/mF ratio to obtain the final mF/A ratio. The resulting mF/A ratio was 4.8 g/in2, which rounds to 5 g/in2. In summary, we determined that 5 g/in2 is representative of the current U.S. food supply and could be used as a standard mF/A ratio when estimating migration levels of an FCS from packaging into food.

Abstract Background Familial chylomicronemia syndrome (FCS) is a rare, hereditary, metabolic disorder. FCS causes high levels of triglycerides in the blood, which can lead to abdominal pain, xanthomas, and acute pancreatitis (AP). Volanesorsen, along with adherence to a very low-fat diet is used to reduce triglyceride levels in individuals with FCS. We aimed to understand the symptoms of FCS and their impact on health-related quality of life (HRQoL). Methods Interviews were conducted with individuals with genetically confirmed FCS in the UK and Spain, some of whom had been treated with volanesorsen. Interview guides were developed with input from a patient advocacy group to explore the symptoms, impacts and management of FCS. Interviews were conducted by telephone and were recorded and transcribed. Data were analyzed using thematic analysis and saturation was recorded. Results Seventeen interviews were conducted with individuals with FCS (aged 27–68 years), thirteen of whom were currently/previously treated with volanesorsen. Episodes of AP were the most impactful reported symptom, resulting in severe abdominal pain, nausea, vomiting, fever, bloating and appetite loss. Other symptoms and functional issues included abdominal pain, gastrointestinal symptoms, impaired cognitive function and fatigue. These had an impact on work, social activities, relationships and psychological wellbeing. These symptoms and impacts were illustrated in a conceptual model, including management strategies. The challenges of managing a low-fat diet and experience with volanesorsen were discussed. Conclusion Individuals with FCS experience a range of interrelated symptoms and functional limitations which impact their broader HRQoL. Treatments which alleviate symptoms and reduce the incidence of AP episodes have the potential to improve the HRQoL of these individuals.

Recent studies show that biofilm substances in contact with nanoplastics play an important role in the aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in the transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics–biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as, e.g., the acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with an ionic strength being realistic for fresh-water conditions. Biomolecule concentrations ranging from 0.5 g/L up to at maximum 21 g/L are considered. We use non-functionalized PS NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA-mediated bridging. In BSA–alginate mixtures, the alginate hampers this interaction, most likely due to alginate–BSA complex formation. In most BSA–alginate mixtures as in alginate alone, NP aggregation is predominantly driven by weaker, pH-independent depletion forces. The stabilizing effect of alginate is only weakened at high BSA contents, when the electrostatic BSA–BSA attraction is not sufficiently screened by the alginate. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single-component biofilm model systems based on comparing the total organic carbon (TOC) content of the extracellular biofilm substances, as usually considered, would have led to a misjudgment of the stability towards aggregation.

Background: Familial chylomicronemia syndrome (FCS) is a rare, hereditary, metabolic disorder. FCS causes high levels of triglycerides in the blood, which can lead to abdominal pain, xanthomas, and acute pancreatitis (AP). Volanesorsen, along with adherence to a very low-fat diet is used to reduce triglyceride levels in individuals with FCS. We aimed to understand the symptoms of FCS and their impact on health-related quality of life (HRQoL). Methods: Interviews were conducted with individuals with genetically confirmed FCS in the UK and Spain, some of whom had been treated with volanesorsen. Interview guides were developed with input from a patient advocacy group to explore the symptoms, impacts and management of FCS. Interviews were conducted by telephone and were recorded and transcribed. Data were analyzed using thematic analysis and saturation was recorded. Results: Seventeen interviews were conducted with individuals with FCS (aged 27–68 years), thirteen of whom were currently/previously treated with volanesorsen. Episodes of AP were the most impactful reported symptom, resulting in severe abdominal pain, nausea, vomiting, fever, bloating and appetite loss. Other symptoms and functional issues included abdominal pain, gastrointestinal symptoms, impaired cognitive function and fatigue. These had an impact on work, social activities, relationships and psychological wellbeing. These symptoms and impacts were illustrated in a conceptual model, including management strategies. The challenges of managing a low-fat diet and experience with volanesorsen were discussed. Conclusion: Individuals with FCS experience a range of interrelated symptoms and functional limitations which impact their broader HRQoL. Treatments which alleviate symptoms and reduce the incidence of AP episodes have the potential to improve the HRQoL of these individuals. [ABSTRACT FROM AUTHOR]

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Background: The fibrinogen to albumin ratio (FAR) is increasingly regarded as a potential biomarker for predicting prognosis in variety of malignant tumors, but not in gastric signet ring cell carcinoma (GSRC). This study seeks to examine the prognostic value of the FAR and explore a novel FAR-CA125 score (FCS) in resectable GSRC patients. Methods: A retrospective cohort was conducted including 330 GSRC patients who underwent curative resection. Kaplan–Meier (K–M) and Cox regression were used to analysis the prognostic value of FAR and FCS. And a predictive nomogram model was developed. Results: The optimal cut-off values for CA125 and FAR were 9.88 and 0.0697, respectively, according to the receiver operating characteristic curve (ROC). Th area under the ROC curve of FCS is higher than CA125 and FAR. 330 patients were grouped into three groups according to the FCS. High FCS was related to males, anemia, tumor size, TNM stage, lymph node metastasis, tumor invasion depth, SII, and pathological subtypes. K–M analysis showed that high FCS and FAR were associated with poor survival. In the multivariate analysis, FCS, TNM stage, and SII were independent prognostic factors for poor OS in resectable GSRC patients. And the predictive accuracy of clinical nomogram contained FCS was better than TNM stage. Conclusion: This study indicated that the FCS is a prognostic, and effective biomarker for patients with surgically resectable GSRC. Such developed FCS-based nomogram could be effective tools to assist the clinicians to determine the treatment strategy. [ABSTRACT FROM AUTHOR]

[Display omitted] Modification of polyallylamine hydrochloride (PAH) with heterobifunctional low molecular weight polyethylene glycol (PEG) (600 and 1395 Da), and subsequent attachment of mannose, glucose, or lactose sugars to PEG, can lead to formation of polyamine phosphate nanoparticles (PANs) with lectin binding affinity and narrow size distribution. Size, polydispersity, and internal structure of glycosylated PEGylated PANs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). Fluorescence correlation spectroscopy (FCS) was used to study the association of labelled glycol-PEGylated PANs. The number of polymer chains forming the nanoparticles was determined from the changes in amplitude of the cross-correlation function of the polymers after formation of the nanoparticles. SAXS and fluorescence cross-correlation spectroscopy were used to investigate the interaction of PANs with lectins: concanavalin A with mannose modified PANs, and jacalin with lactose modified ones. Glyco-PEGylated PANs are highly monodispersed, with diameters of a few tens of nanometers and low charge, and a structure corresponding to spheres with Gaussian chains. FCS shows that the PANs are single chain nanoparticles or formed by two polymer chains. Concanavalin A and jacalin show specific interactions for the glyco-PEGylated PANs with higher affinity than bovine serum albumin. [ABSTRACT FROM AUTHOR]

Plants have evolved signaling mechanisms such as the multi-step phosphorelay (MSP) to respond to different internal and external stimuli. MSP responses often result in gene transcription regulation that is modulated through transcription factors such as B-type Arabidopsis response regulator (ARR) proteins. Among these proteins, ARR2 is a key component that is expressed ubiquitously and is involved in many aspects of plant development. Although it has been noted that B-type ARRs bind to their cognate genes through a DNA-binding domain termed the GARP domain, little is known about the structure and function of this type of DNA-binding domain; thus, how ARRs bind to DNA at a structural level is still poorly understood. In order to understand how the MSP functions in planta, it is crucial to unravel both the kinetics as well as the structural identity of the components involved in such interactions. For this reason, this work focusses on resolving how the GARP domain of ARR2 (GARP2) binds to the promoter region of ARR5, one of its native target genes in cytokinin signaling. We have established that GARP2 specifically binds to the ARR5 promoter with three different bi-molecular interaction systems—qDPI-ELISA, FCS, and MST—and we also determined the KD of this interaction. In addition, structural modeling of the GARP2 domain confirms that GARP2 entails a HTH motif, and that protein–DNA interaction most likely occurs via the α3-helix and the N-terminal arm of this domain since mutations in this region hinder ARR2's ability to activate transcription. [ABSTRACT FROM AUTHOR]

Fin type Field Effect Transistor (FinFET) technology is a promising technology for single-digit nanometric dimensions. FinFETs are used to increase the performance by reducing the power consumption as the two gate voltages are controlled dependent and/or independently. This work explores the design of a Floating Current Source (FCS) using Double Gate (DG) FinFET and its application of a universal filter. The designed FinFET based FCS operates at 0.15 V power supply, the power consumption of the circuit is 51nW and bandwidth is approximately 100 MHz. The proposed FCS based filter circuit is simulated in LTspice with the FinFET 45 nm PTM-MG model. The filter topology behavior has been tested with an experimental study using the LM13700 integrated circuit and the experimental results are demonstrated. The proposed FinFET based circuit and its application can be used for the design of systems that require low power consumption and low chip area. [ABSTRACT FROM AUTHOR]

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer’s and Parkinson’s disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques.

Cell-free DNA (cfDNA) is of growing clinical and research significance. In vitro cfDNA models are a useful tool in cfDNA research; however, artifacts in these models may have implications for the interpretation of new and published data. This report aimed to establish how endogenous cfDNA in fetal bovine serum (FBS) may influence in vitro cfDNA measurements. Three commercial cell culture media, supplemented with 10% FBS, were analyzed for the presence of cfDNA, with and without culture with ovarian cancer cell lines. cfDNA from FBS was identified with all three commercial media and contributed a major portion of 167-bp cfDNA. Future studies should account for bovine cfDNA in FBS-supplemented media when conducting in vitro cfDNA research.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation–modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments. [ABSTRACT FROM AUTHOR]

Zn (II) complexes of Schiff bases have potential applications in biomedical sciences as imaging agents, cancer therapeutics and diagnostics. Thus, it is important to understand their interaction with carrier proteins, like serum albumins. The present paper focuses on the binding interactions between Human serum albumin (HSA) and Znsalampy, making use of fluorescence spectroscopic techniques at ensemble as well as at single molecular level. An idea about the binding constant is obtained from the quenching of the single Trp (Tryptophan) residue of HSA by Znsalampy. Fluorescence correlation spectroscopy (FCS) has also been used to monitor the protein-ligand binding. The location of Znsalampy in its complex with HSA is determined by competitive binding experiments and molecular docking calculations. The binding constant obtained from the Znsalampy-HSA interaction falls in the ideal range for biological applications and the location is found to be in the proximity of Sudlow's site I. The esterase activity of HSA is retained in the presence of the Znsalampy. Hence, it is concluded that this Znsalampy may be a potential probe and biomarker in biomedical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Outer membrane proteins (OMPs) maintain the viability of Gram‐negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi‐protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single‐molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis. [ABSTRACT FROM AUTHOR]

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer's and Parkinson's disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques. [ABSTRACT FROM AUTHOR]

The presence of Ca-ferrite and silico-ferrite-of-calcium-and-aluminium (SFCA) bonding phases is thought to be crucial to maintain sinter quality due to their high reducibility and strength levels. However, new evidence suggests that porosity might be an equally important factor controlling reducibility, in addition to mineralogy. This work aims to fundamentally understand the development of porosity in simple sinter analogues from the Fe2O3-(Fe3O4)-CaO-SiO2 (FCS) ternary system (with no SFCA), and to connect results back to overall sinter mineralogy, strength, and reducibility properties. Laboratory-scale experiments were conducted to simulate the sintering process by firing tablets of magnetite, hematite, lime and silica mixtures under tightly controlled temperature, holding time and atmosphere conditions. Mineralogy of the fired samples was observed using microscopy techniques, porosity was measured by Mercury Intrusion Porosimetry (MIP), strength was determined using laboratory-scale tumble index equipment and reducibility was measured by the weight loss obtained after reaction of the tablets in a reducing atmosphere of CO/N2. The results confirmed that reducibility is strongly influenced by porosity, and highly reducible sinters can be produced without forming SFCA-like phases. Magnetite-containing samples had similar reducibility to hematite-containing samples, suggesting that magnetite-based sinters could potentially be used in the blast furnace. [ABSTRACT FROM AUTHOR]

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Missing covariate values are a common problem in survival studies, and the method of choice when handling such incomplete data is often multiple imputation. However, it is not obvious how this can be used most effectively when an incomplete covariate is a function of other covariates. For example, body mass index (BMI) is the ratio of weight and height-squared. In this situation, the following question arises: Should a composite covariate such as BMI be imputed directly, or is it advantageous to impute its constituents, weight and height, first and to construct BMI afterwards? We address this question through a carefully designed simulation study that compares various approaches to multiple imputation of composite covariates in a survival context. We discuss advantages and limitations of these approaches for various types of missingness and imputation models. Our results are a first step towards providing much needed guidance to practitioners for analysing their incomplete survival data effectively.

Plants have evolved signaling mechanisms such as the multi-step phosphorelay (MSP) to respond to different internal and external stimuli. MSP responses often result in gene transcription regulation that is modulated through transcription factors such as B-type Arabidopsis response regulator (ARR) proteins. Among these proteins, ARR2 is a key component that is expressed ubiquitously and is involved in many aspects of plant development. Although it has been noted that B-type ARRs bind to their cognate genes through a DNA-binding domain termed the GARP domain, little is known about the structure and function of this type of DNA-binding domain; thus, how ARRs bind to DNA at a structural level is still poorly understood. In order to understand how the MSP functions in planta, it is crucial to unravel both the kinetics as well as the structural identity of the components involved in such interactions. For this reason, this work focusses on resolving how the GARP domain of ARR2 (GARP2) binds to the promoter region of ARR5, one of its native target genes in cytokinin signaling. We have established that GARP2 specifically binds to the ARR5 promoter with three different bi-molecular interaction systems—qDPI-ELISA, FCS, and MST—and we also determined the KD of this interaction. In addition, structural modeling of the GARP2 domain confirms that GARP2 entails a HTH motif, and that protein–DNA interaction most likely occurs via the α3-helix and the N-terminal arm of this domain since mutations in this region hinder ARR2’s ability to activate transcription.

The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β1- and β2-adrenergic receptors (β1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs. [ABSTRACT FROM AUTHOR]

The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae, the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression. [ABSTRACT FROM AUTHOR]

Fluorescent Correlation Spectroscopy (FCS) allows us to determine interactions of labeled proteins or changes in the oligomeric state. The FCS method needs a low amount of fluorescent dye, near nanomolar concentrations. To control the amount of fluorescent dye, we used new photoconvertible FP SAASoti. This work is devoted to the proof of principle of using photoconvertible proteins to measure caspase enzymatic activity in a single live cell. The advantage of this approach is that partial photoconversion of the FP makes FCS measurements possible when studying enzymatic reactions. To investigate the process, in vivo we used HeLa cell line expressing the engineered FRET sensor, SAASoti-23-KFP. This FRET sensor has a cleavable (DEVD) sequence in the linker between two FPs for the detection of one of the key enzymes of apoptosis, caspase-3. Caspase-3 activity was detected by registering the increase in the fluorescent lifetimes of the sensor, whereas the diffusion coefficient of SAASoti decreased. This can be explained by an increase in the total cell viscosity during apoptosis. We can suppose that in the moment of detectible caspase-3 activity, cell structure already has crucial changes in viscosity. [ABSTRACT FROM AUTHOR]

Protein crystallography has become a popular method for biochemists, but obtaining high-quality protein crystals for precise structural analysis and larger ones for neutron analysis requires further technical progress. Many studies have noted the importance of solvent viscosity for the probability of crystal nucleation and for mass transportation; therefore, in this paper, we have reported on experimental results and simulation studies regarding the use of viscous polyethylene glycol (PEG) solvents for protein crystals. We investigated the diffusion rates of proteins, peptides, and small molecules in viscous PEG solvents using fluorescence correlation spectroscopy. In high-molecular-weight PEG solutions (molecular weights: 10,000 and 20,000), solute diffusion showed deviations, with a faster diffusion than that estimated by the Stokes–Einstein equation. We showed that the extent of the deviation depends on the difference between the molecular sizes of the solute and PEG solvent, and succeeded in creating equations to predict diffusion coefficients in viscous PEG solutions. Using these equations, we have developed a new numerical model of 1D diffusion processes of proteins and precipitants in a counter-diffusion chamber during crystallization processes. Examples of the application of anomalous diffusion in counter-diffusion crystallization are shown by the growth of lysozyme crystals. [ABSTRACT FROM AUTHOR]

Missing covariate values are a common problem in survival studies, and the method of choice when handling such incomplete data is often multiple imputation. However, it is not obvious how this can be used most effectively when an incomplete covariate is a function of other covariates. For example, body mass index (BMI) is the ratio of weight and height-squared. In this situation, the following question arises: Should a composite covariate such as BMI be imputed directly, or is it advantageous to impute its constituents, weight and height, first and to construct BMI afterwards? We address this question through a carefully designed simulation study that compares various approaches to multiple imputation of composite covariates in a survival context. We discuss advantages and limitations of these approaches for various types of missingness and imputation models. Our results are a first step towards providing much needed guidance to practitioners for analysing their incomplete survival data effectively. [ABSTRACT FROM AUTHOR]

Copyright of Railway Signalling & Communication Engineering is the property of Railway Signalling & Communication Engineering and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)