Internal Labeling Research Articles

Zr-MOF-based substrate with inherent internal standards facilitates reliable SERS analysis of sulfonamide antibiotics

A multifunctional structure comprising gold nanoparticles immobilized within a zirconium-based metal-organic framework (AuNPs/Zr-MOF) was systematically designed. With the capabilities of analyte enrichment, Raman signal enhancement and internal standard calibration, the as-prepared AuNPs/Zr-MOF substrates demonstrate the outstanding SERS sensing performance toward the representative sulfonamide antibiotics (SAs), sulfadiazine, sulfamerazine, sulfamethazine and sulfaquinoxaline. For the first time, these four SAs are detected by SERS in real honey samples at levels of 2.8 ∼ 5.2 ng·g−1 within 10 min. The extensive π-bond conjugated system of Zr-MOF significantly enhances the concentration of SAs through π-π interactions. The entrapped SAs are adsorbed perpendicularly onto the surface of Au via coordinated interactions between Au and two oxygen atoms, or between Au and the terminal amino group. To achieve reliable quantitative analysis, the inherent vibration of the Zr-MOF structure was used as an internal standard label. Combined with a simple clean-up protocol, the established SERE sensing method is superior to conventional methods in terms of time, while achieving the impressive sensitivity. This study presents a robust SERS-based approach for the rapid detection and monitoring of multiple analytes.

The conductor model of consciousness, our neuromorphic twins, and the human-AI deal

AbstractCritics of Artificial Intelligence (AI) posit that artificial agents cannot achieve consciousness even in principle, because they lack certain necessary pre-conditions present in biological agents. Here we highlight arguments from a neuroscientific and neuromorphic engineering perspective as to why such a strict denial of consciousness in artificial agents is not compelling. Based on the construction of a co-evolving neuromorphic twin, we argue that the differences between a developing biological and artificial brain are not fundamental and are vanishing with progress in neuromorphic architecture designs mimicking the human blueprint. To characterise this blueprint, we propose the Conductor Model of Consciousness (CMoC) that builds on neuronal implementations of an external and internal world model, while gating and labelling information flows. An extended turing test lists functional and neuronal correlates of biological consciousness that are captured by the CMoC. These correlates provide the grounding for how biological or artificial agents learn to distinguish between sensory activity generated from outside or inside of the brain, how the perception of these activities can itself be learned, and how the information flow for learning an internal world model is orchestrated by a cortical meta-instance, which we call the conductor. Perception comes with the distinction of sensory and affective components, with the affective component linking to ethical questions that are inherent in our multidimensional model of consciousness. Recognizing the existence of a blueprint for a possible artificial consciousness encompasses functional, neuronal and ethical dimensions, begging the question: How should we behave towards agents that are akin to us in the inner workings of their brains? We sketch a human-AI deal, balancing the growing cognitive abilities of artificial agents, and the possibility to relieve them from suffering of negative affects, with a protection for the rights of humans.

Unraveling new players in helminth pathology: extracellular vesicles from Fasciola hepatica and Dicrocoelium dendriticum exert different effects on hepatic stellate cells and hepatocytes

Fasciola hepatica and Dicrocoelium dendriticum are parasitic trematodes residing in the bile ducts of mammalian hosts, causing, in some cases, impairment of liver function and hepatic fibrosis. Previous studies have shown that extracellular vesicles released by F. hepatica (FhEVs) and D. dendriticum (DdEVs) induce a distinct phenotype in human macrophages, but there is limited information on the effect of parasitic EVs on liver cells, which interact directly with the worms in natural infections. In this study, we isolated FhEVs and DdEVs by size exclusion chromatography and labeled them with a lipophilic fluorescent dye to analyze their uptake by human hepatic stellate cells (HSC) and hepatocytes, important cell types in liver pathology, using synthetic liposomes as internal labeling and uptake control. We analyzed EV uptake and the proteome profiles after the treatment with EVs for both cell types. Our results reveal that EVs establish unique and specific interactions with stellate cells and hepatocytes, suggesting a different role of EVs derived from each parasite, depending on the migration route to reach their final niche. FhEVs have a cytostatic effect on HSCs, but induce the extracellular matrix secretion and elicit anti-inflammatory responses in hepatocytes. DdEVs have a more potent anti-proliferative effect than FhEVs and trigger a global inflammatory response, increasing the levels of NF-κB and other inflammatory mediators in both cell types. These interactions may have a major influence on the progression of the disease, serving to generate conditions that may favor the establishment of the helminths in the host.

Repurposing a plant peptide cyclase for targeted lysine acylation

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine–leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Multiplexed electrochemical liposomes applied to the detection of nucleic acids for Influenza A, Influenza B and SARS-CoV-2

Multiplexing is a relevant strategy for biosensors to improve accuracy and decision-making due to the increased amount of simultaneously obtained information. Liposomes offer unique benefits for label-based multiplexing since a variety of different marker molecules can be encapsulated, leading to intrinsic signal amplification and enabling a variety of detection formats. We successfully developed an electrochemical (EC) liposome-based platform technology for the simultaneous detection of at least three analytes by studying parameters to ensure specific and sensitive bioassay performance. Influenza A and B and SARS-CoV-2 sequences served as model system in a standard sandwich hybridization assay. Studies included encapsulants, probe distribution on liposomes and capture beads, assay setup and interferences between liposomes to also ensure a generalization of the platform. Ruthenium hexamine(III), potassium hexacyanoferrate(II) and m-carboxy luminol, when encapsulated separately into a liposome, provided desirable long-term stability of at least 12 months and no cross-signals between liposomes. Through the optimization process, low limits of detections of 1.6 nmol L−1, 125 pmol L−1 and 130 pmol L−1, respectively, were achieved in a multiplexed assay setup, which were similar to singleplex assays. Non-specific interactions were limited to 25.1%, 7.6% and 7.5%, respectively, through sequential liposome incubations and singleplex capture bead designs. Here, ruthenium hexamine liposomes had only mediocre performance so that low overall signal strength translated into higher LODs and worse specificity. A different marker such as ferroin may be an option in the future. The identification of further electrochemical markers will provide new opportunities for liposomes to function as multiplex, orthogonal or internal standard labels in electrochemical bioassays.Graphical

Effectiveness of Family-Focused Therapy in Bipolar Patients in Zahedan City, Iran: A Randomized Controlled Trial

Background: Family-focused therapy (FFT) has been developed to address the significant impairments in psychosocial functioning among individuals with bipolar disorder (BD). Objectives: This study investigated the effectiveness of FFT combined with drug therapy in patient support in managing this disorder. Methods: In this randomized controlled trial, 50 patients and their caregivers were randomly divided into 2 groups: a drug therapy group that only received medication and a family therapy group that received 15 one-hour sessions of FFT with their caregivers in addition to medication. The Expressed Emotion Questionnaire (EEQ), a modified version of the standard internal label questionnaire for the mentally ill, and the World Health Organization Quality of Life–Brief (WHOQOL-BREF) were used in the present study. Data were analyzed using the t-test. Results: Expressed emotion (EE) significantly decreased in the family therapy group (P = 0.001). Also, mental health and social health were better in the family therapy group than in the drug therapy group (P = 0.001). Nevertheless, no significant change was observed in the dimensions of physical and environmental health in both groups. The results of the study were relatively stable in all measurements in follow-up, except for the stigma. Conclusions: Significant improvements were observed in EE and quality of life (QOL) in the family therapy group. This difference was not statistically significant in the stigma score despite the difference in the means of the 2 groups.

Comparing 13C methyl and deuterated methyl isotopic labeling for the quantification of methyl cellulose patterns using mass spectrometry

The methyl substitution along and among the polymer chains of methyl cellulose (MC) is commonly analyzed by ESI-MS after perdeuteromethylation of the free-OH groups and partial hydrolysis to cello-oligosaccharides (COS). This method requires a correct quantification of the molar ratios of the constituents belonging to a particular degree of polymerization (DP). However, isotopic effects are most pronounced for H/D since their mass difference is 100%. Therefore, we investigated whether more precise and accurate results could be obtained for the methyl distribution of MC by MS of 13CH3 instead of CD3-etherified O-Me-COS. Internal isotope labeling with 13CH3 makes the COS of each DP chemically and physically much more similar, reducing mass fractionation effects, but at the same time requires more complex isotopic correction for evaluation. Results from syringe pump infusion ESI-TOF-MS with 13CH3 and CD3 as isotope label were equal. However, in the case of LC-MS with a gradient system, 13CH3 was superior to CD3. In the case of CD3, the occurrence of a partial separation of the isotopologs of a particular DP resulted in slight distortion of the methyl distribution since the signal response is significantly dependent on the solvent composition. Isocratic LC levels this problem, but one particular eluent-composition is not sufficient for a series of oligosaccharides with increasing DP due to peak broadening. In summary, 13CH3 is more robust to determine the methyl distribution of MCs. Both syringe pump and gradient-LC-MS measurements are possible, and the more complex isotope correction is not a disadvantage.Graphical abstract

Ratiometric response to formaldehyde by 3D silver SERS substrate with polyoxometalate as internal label

For surface-enhanced Raman scattering (SERS) analytical technology, the rational design of efficient substrate and effective internal standard is the key issue to implement the reliable detection. In this paper, a novel three-dimensional (3D) silver(I)-polyoxometalate complex of Ag2[Ag(trz)2][H6CrMo6O24]·2H2O (Ag3CrMo6, trz = 1, 2, 4-triazole) has been designed and synthesized, which can be used for the quantitative detection of formaldehyde (FA). Structural analysis reveals that the [H6CrMo6O24]3- polyoxoanions link Ag+ by diverse Ag−O(Mo−O) bonds to form a stable 3D framework. When Ag3CrMo6 is exposed to FA, the Ag+ can be partially reduced to Ag0 species to become the label-free SERS active substrate and and present the enhanced signals ascribed to the Mo−O vibrations. Due to that the reduced Ag0 species is only close to Mo−O groups, the Raman signals for Cr−O bonds are almost constant and can be used as internal standards. Therefore, the IMo−O/ICr−O values can be used as ratiometric SERS signals to fulfill the elusive SERS quantitative analysis for FA with the detection limit of 0.05 ng/L. This work opens up an innovative way to the rational design of label-free SERS substrate based on polyoxoanions and silver ions, by which the quantitative detection can be realized via the ratiometric Raman signals.

Enzymatic Synthesis of High-Density RNA Microarrays.

Oligonucleotide microarrays are used to investigate the interactome of nucleic acids. DNA microarrays are commercially available, whereas equivalent RNA microarrays are not. This protocol describes a method to convert DNA microarrays of any density and complexity into RNA microarrays using only readily available materials and reagents. This simple conversion protocol will facilitate the accessibility of RNA microarrays to a wide range of researchers. In addition to general considerations for the design of a template DNA microarray, this procedure describes the experimental steps of hybridization of an RNA primer to the immobilized DNA, followed by its covalent attachment via psoralen-mediated photocrosslinking. The subsequent enzymatic processing steps comprise the extension of the primer with T7 RNA polymerase to generate complementary RNA, and finally the removal of the DNA template with TURBO DNase. Beyond the conversion process, we also describe approaches to detect the RNA product either by internal labeling with fluorescently labeled NTPs or via hybridization to the product strand, a step that can then be complemented by an RNase H assay to confirm the nature of the product. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conversion of a DNA microarray to an RNA microarray Alternate Protocol: Detection of RNA via incorporation of Cy3-UTP Support Protocol 1: Detection of RNA via hybridization Support Protocol 2: RNase H assay.

Scale-invariant representation of machine learning.

The success of machine learning has resulted from its structured representation of data. Similar data have close internal representations as compressed codes for classification or emerged labels for clustering. We observe that the frequency of internal codes or labels follows power laws in both supervised and unsupervised learning models. This scale-invariant distribution implies that machine learning largely compresses frequent typical data, and simultaneously, differentiates many atypical data as outliers. In this study, we derive the process by which these power laws can naturally arise in machine learning. In terms of information theory, the scale-invariant representation corresponds to a maximally uncertain data grouping among possible representations that guarantee a given learning accuracy.

Ratiometric Response to Formaldehyde by 3d Silver Sers Substrate with Polyoxometalate As Internal Label

Ratiometric Response to Formaldehyde by 3d Silver Sers Substrate with Polyoxometalate As Internal Label

Testing the agreement of trees with internal labels

BackgroundA semi-labeled tree is a tree where all leaves as well as, possibly, some internal nodes are labeled with taxa. Semi-labeled trees encompass ordinary phylogenetic trees and taxonomies. Suppose we are given a collection {mathcal {P}}= {{mathcal {T}}_1, {mathcal {T}}_2, ldots , {mathcal {T}}_k} of semi-labeled trees, called input trees, over partially overlapping sets of taxa. The agreement problem asks whether there exists a tree {mathcal {T}}, called an agreement tree, whose taxon set is the union of the taxon sets of the input trees such that the restriction of {mathcal {T}} to the taxon set of {mathcal {T}}_i is isomorphic to {mathcal {T}}_i, for each i in {1, 2, ldots , k}. The agreement problems is a special case of the supertree problem, the problem of synthesizing a collection of phylogenetic trees with partially overlapping taxon sets into a single supertree that represents the information in the input trees. An obstacle to building large phylogenetic supertrees is the limited amount of taxonomic overlap among the phylogenetic studies from which the input trees are obtained. Incorporating taxonomies into supertree analyses can alleviate this issue. ResultsWe give a {mathcal {O}}(n k (sum _{i in [k]} d_i + log ^2(nk))) algorithm for the agreement problem, where n is the total number of distinct taxa in {mathcal {P}}, k is the number of trees in {mathcal {P}}, and d_i is the maximum number of children of a node in {mathcal {T}}_i.ConclusionOur algorithm can aid in integrating taxonomies into supertree analyses. Our computational experience with the algorithm suggests that its performance in practice is much better than its worst-case bound indicates.

Site-specific internal protein labeling through trans-splicing

Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.

Strategies for Covalent Labeling of Long RNAs.

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site‐specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.

Effect of trap symmetry and atom-atom interactions on a trapped-atom interferometer with internal state labeling

In this paper, we study the dynamics of a trapped atom interferometer with internal state labelling in the presence of interactions. We consider two situations: an atomic clock in which the internal states remain superposed, and an inertial sensor configuration in which they are separated. From the average spin evolution, we deduce the fringe contrast and the phase-shift. In the clock configuration, we recover the well-known identical spin rotation effect (ISRE) which can significantly increase the spin coherence time. We also find that the magnitude of the effect depends on the trap geometry in a way that is consistent with our recent experimental results in a clock configuration [M. Dupont-Nivet, and al., New J. Phys., 20, 043051 (2018)], where ISRE was not observed. In the case of an inertial sensor, we show that despite the spatial separation it is still possible to increase the coherence time by using mean field interactions to counteract asymmetries of the trapping potential.

Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases.

The mRNA modification N6‐methyladenosine (m6A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3‐METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3‐14 shows remarkable cosubstrate promiscuity, enabling sequence‐specific internal labeling of RNA without additional guide RNAs. The transfer of ortho‐nitrobenzyl and 6‐nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase‐catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo‐enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo‐enzymatic modification of different sites in a single RNA.

Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

AbstractThe mRNA modification N6‐methyladenosine (m6A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3‐METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3‐14 shows remarkable cosubstrate promiscuity, enabling sequence‐specific internal labeling of RNA without additional guide RNAs. The transfer of ortho‐nitrobenzyl and 6‐nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase‐catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo‐enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo‐enzymatic modification of different sites in a single RNA.

Mixed Labeling: Integrating Internal and External Labels.

In this article, we present an algorithm capable of mixed labeling of 2D and 3D objects. In mixed labeling, the given objects are labeled with both internal labels placed (at least partially) over the objects and external labels placed in the space around the objects and connected with the labeled objects with straight-line leaders. The proposed algorithm determines the position and type of each label based on the user-specified ambiguity threshold and eliminates overlaps between the labels, as well as between the internal labels and the straight-line leaders of external labels. The algorithm is a screen-space technique; it operates in an image where the 2D objects or projected 3D objects are encoded. In other words, we can use the algorithm whenever we can render the objects to an image, which makes the algorithm fit for use in many domains. The algorithm operates in real-time, giving the results immediately. Finally, we present results from an expert evaluation, in which a professional illustrator has evaluated the label layouts produced with the proposed algorithm.

Implementation and Evaluation of a Fast Area Feature Labeling Method Using Auxiliary Lines

Appropriate place labels, which provide the name or attribute of a graphical feature, are important in geographical information systems and cartography. Herein, an internal label placement method was proposed for area features, such as cities, prefectures, and lakes, on a map. For internal label placement, placing a large label for an extremely narrow or small area, such that the label does not protrude from the corresponding area is challenging. In such cases, a label can overlap with protruding labels from other areas. Meanwhile, tablet devices have been rapidly employed in recent years. Because tablet devices can easily zoom in on a map, it is possible to eliminate the overlaps by enlarging the map without changing the label size. Therefore, we proposed a method that enables real-time processing, even on tablet devices. The label positions are determined by detecting the intersections of the auxiliary and boundary lines of a given area feature. The proposed method adequately labels the positions of area features, even those with indents and narrow sections. Moreover, it can find tens of thousands of label positions within 100 ms, even on low-performance computers, such as tablet devices.

Hierarchical Representation for Chromatic Processing across Macaque V1, V2, and V4

The perception of color is an internal label for the inferred spectral reflectance of visible surfaces. To study how spectral representation is transformed through modular subsystems of successive cortical areas, we undertook simultaneous optical imaging of intrinsic signals in macaque V1, V2, and V4, supplemented by higher-resolution electrophysiology and two-photon imaging in awake macaques. We find a progressive evolution in the scale and precision of chromotopic maps, expressed by a uniform blob-like architecture of hue responses within each area. Two-photon imaging reveals enhanced hue-specific cell clustering in V2 compared with V1. A phenomenon of endspectral (red and blue) responses that is clear in V1, recedes in V2, and is virtually absent in V4. The increase in mid- and extra-spectral hue representations through V2 and V4 reflects the nature of hierarchical processing as higher areas read out locations in chromatic space from progressive integration of signals relayed by V1.