Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues.

Glycopeptide antibiotics (GPAs) are peptide natural products used as last resort treatments for antibiotic resistant bacterial infections. They are produced by the sequential activities of a linear nonribosomal peptide synthetase (NRPS), which assembles the heptapeptide core of GPAs, and cytochrome P450 (Oxy) enzymes, which perform a cascade of cyclisation reactions. The GPAs contain proteinogenic and nonproteinogenic amino acids, including phenylglycine residues such as 4-hydroxyphenylglycine (Hpg). The ability to incorporate non-proteinogenic amino acids in such peptides is a distinctive feature of the modular architecture of NRPSs, with each module selecting and incorporating a desired amino acid. Here, we have exploited this ability to produce and characterise GPA derivatives containing fluorinated phenylglycine (F-Phg) residues through a combination of mutasynthesis, biochemical, structural and bioactivity assays. Our data indicate that the incorporation of F-Phg residues is limited by poor acceptance by the NRPS machinery, and that the phenol moiety normally present on Hpg residues is essential to ensure both acceptance by the NRPS and the sequential cyclisation activity of Oxy enzymes. The principles learnt here may prove useful for the future production of GPA derivatives with more favourable properties through mixed feeding mutasynthesis approaches.

The neural signature of an erroneous thought.

The human brain detects errors in overt behavior fast and efficiently. However, little is known about how errors are monitored that emerge on a mental level. We investigate whether neural correlates of error monitoring can be found during inner speech and whether the involved neural processes differ between these non-motor responses and behavioral motor responses. Therefore, electroencephalographic data were collected while participants performed two versions of a decision task that only differed between these response modalities. Erroneous responses were identified based on participants' metacognitive judgments. Correlates of error monitoring in event-related potentials were analyzed by applying residue iteration decomposition on stimulus-locked activity. Non-motor responses elicited the same cascade of early error-related negativity and late error positivity as motor responses. An analysis of oscillatory brain activity showed a similar theta response for both error types. A multivariate pattern classifier trained on theta from the motor condition could decode theta from the non-motor condition, demonstrating the similarity of both neural responses. These results show that errors in inner speech are monitored and detected utilizing the same neural processes as behavioral errors, suggesting that goal-directed cognition and behavior are supported by a generic error-monitoring system.

Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Approach to the Core Structure of Signermycin B.

Among the natural tetramic acids with a decalinoyl part, signermycin B is unique because it contains a cis-decalin. In this paper, we demonstrate that the cis-decalin section of signermycin B can be accessed by an anionic oxy-Cope rearrangement. The substrate, a tricyclic dienol was prepared by an intramolecular Diels-Alder reaction of a masked ortho-benzoquinone, generated by oxidation of an α-methoxyphenol in presence of cis-2-hexenol. After a superfluous bromine on the cycloadduct was removed, reaction of the tricyclic ketone with isopropenylmagnesium bromide led to the tricyclic trienol that underwent the oxy-Cope rearrangement to a cis-decalinone. While we could show, that introduction of the 4-ethyl substituent (signermycin B numbering) is possible by enolate alkylation, the 4-epi-isomer was formed.

Lightweight Potential of Anisotropic Plate Lattice Metamaterials.

Additive manufacturing enables the production of lattice structures, which have been proven to be a superior class of lightweight mechanical metamaterials whose specific stiffness can reach the theoretical limit of the upper Hashin-Shtrikman bound for isotropic cellular materials. To achieve isotropy, complex structures are required, which can be challenging in powder bed additive manufacturing, especially with regard to subsequent powder removal. The present study focuses on the Finite Element Method simulation of 2.5D anisotropic plate lattice metamaterials and the investigation of their lightweight potential. The intentional use of anisotropic structures allows the production of a cell architecture that is easily manufacturable via Laser Powder Bed Fusion (LPBF) while also enabling straightforward optimization for specific load cases. The work demonstrates that the considered anisotropic plate lattices exhibit high weight-specific stiffnesses, superior to those of honeycomb structures, and, simultaneously, a good de-powdering capability. A significant increase in stiffness and the associated surpassing of the upper Hashin-Shtrikman bound due to anisotropy is achievable by optimizing wall thicknesses depending on specific load cases. A stability analysis reveals that, in all lattice structures, plastic deformation is initiated before linear buckling occurs. An analysis of stress concentrations indicates that the introduction of radii at the plate intersections reduces stress peaks and simultaneously increases the weight-specific stiffnesses and thus the lightweight potential. Exemplary samples illustrate the feasibility of manufacturing the analyzed metamaterials within the LPBF process.

Constructing cropland ecological stability assessment method based on disturbance-resistance-response processes and classifying cropland ecological types.

The cropland ecosystem stability (CES) has received increasing attention, especially in ecologically fragile areas, because of its impact on cropland quality, agricultural production and its ability to resist external disturbances. In this study, we first introduced the concepts of resilience and resistance, proposed the ecosystem disturbance-resistance-response process, and established a framework for evaluating the spatial and temporal dynamics of the CES based on RS data, and innovatively combined the RS assessment results of CES with soil field samples data to further classify cropland ecological types (CET) in a key agricultural areas of the Qinghai-Tibetan Plateau, which can effectively identify those croplands in need of priority ecological protection. Results indicate that the combined interactions of disturbance, resistance and response systems affect CES, forming a complex process with significant fluctuations and spatial variations. We also conclude that the disturbance system is positively influenced by topography and precipitation, while slope negatively affects resistance system. Hydrothermal conditions positively influence resistance system, while the response system is influenced by environmental factors at a lower intensity in six periods. It was interesting to note that soil α-biodiversity indicators are significantly and positively correlated with CES at the end of the study period. Therefore, based on the CES assessment results, we further combined the soil α-biodiversity indicators to classify the type of spatial pattern of CET and found that the eastern and northern areas have better quality, which implied an increase in the CES and a higher level of soil biodiversity, which was ideal for cropland expansion. On the contrary, we concluded that the ecosystem maintenance of the Huangshui headwaters and the northern mountainous areas needs to be strengthened in order to reverse the ecological fragility here and safeguard the cropland productive capacity.

RNA interference in the era of nucleic acid therapeutics.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.

Error-related cardiac deceleration: Functional interplay between error-related brain activity and autonomic nervous system in performance monitoring.

Coordinated interactions between the central and autonomic nervous systems are crucial for survival due to the inherent propensity for human behavior to make errors. In our ever-changing environment, when individuals make mistakes, these errors can have life-threatening consequences. In response to errors, specific reactions occur in both brain activity and heart rate to detect and correct errors. Specifically, there are two brain-related indicators of error detection and awareness known as error-related negativity and error positivity. Conversely, error-related cardiac deceleration denotes a momentary slowing of heart rate following an error, signaling an autonomic response. However, what is the connection between the brain and the heart during error processing? In this review, we discuss the functional and neuroanatomical connections between the brain and heart markers of error processing, exploring the experimental conditions in which they covary. Given the current limitations of available data, future research will continue to investigate the neurobiological factors governing the brain-heart interaction, aiming to utilize them as combined markers for assessing cognitive control in healthy and pathological conditions.

Deepfake smiles matter less-the psychological and neural impact of presumed AI-generated faces.

High-quality AI-generated portraits ("deepfakes") are becoming increasingly prevalent. Understanding the responses they evoke in perceivers is crucial in assessing their societal implications. Here we investigate the impact of the belief that depicted persons are real or deepfakes on psychological and neural measures of human face perception. Using EEG, we tracked participants' (N = 30) brain responses to real faces showing positive, neutral, and negative expressions, after being informed that they are either real or fake. Smiling faces marked as fake appeared less positive, as reflected in expression ratings, and induced slower evaluations. Whereas presumed real smiles elicited canonical emotion effects with differences relative to neutral faces in the P1 and N170 components (markers of early visual perception) and in the EPN component (indicative of reflexive emotional processing), presumed deepfake smiles showed none of these effects. Additionally, only smiles presumed as fake showed enhanced LPP activity compared to neutral faces, suggesting more effortful evaluation. Negative expressions induced typical emotion effects, whether considered real or fake. Our findings demonstrate a dampening effect on perceptual, emotional, and evaluative processing of presumed deepfake smiles, but not angry expressions, adding new specificity to the debate on the societal impact of AI-generated content.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

Effects of the number of competing responses on neural signatures of pre- and post-response conflict.

The optimization of human performance requires the continuous monitoring of behavioral conflicts. According to conflict monitoring theory, the dorsal anterior cingulate cortex registers response conflict which is reflected by two electrophysiological signatures, the N2 and the Ne/ERN. The theory assumes that, if a stimulus activates an incorrect response that competes with the correct response, pre-response conflict on correct trials (reflected by the N2) is enhanced but post-response conflict on error trials (reflected by the Ne/ERN) is reduced. Here, we asked whether response conflict depends on the number of competing incorrect responses activated by a stimulus, that is, whether the N2 is further enhanced and the Ne/ERN is further reduced if two incorrect responses are activated as compared to one. To this end, we used a modified flanker paradigm, in which the two flankers were associated either with the same incorrect response or with different incorrect responses. Our results indicate an increased N2 on correct trials and a reduced Ne/ERN on error trials in the latter as compared to the former condition. These results confirm central predictions of conflict monitoring theory and demonstrate that response conflict is directly related to the number of competing incorrect responses.

Cyclic Dichalcogenides Extend the Reach of Bioreductive Prodrugs to Harness Thiol/Disulfide Oxidoreductases: Applications to seco-Duocarmycins Targeting the Thioredoxin System.

Small-molecule prodrug approaches that can activate cancer therapeutics selectively in tumors are urgently needed. Here, we developed the first antitumor prodrugs designed for activation by thiol-manifold oxidoreductases, targeting the thioredoxin (Trx) system. The Trx system is a critical cellular redox axis that is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidized nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of 10 "off-to-on" reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular reductase-dependent activity in 177 cell lines, establishing broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well tolerated in vivo in mice, indicating low systemic release of their duocarmycin cargo, and in vivo anti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising indications of effective tumoral drug release, presumably by in situ bioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase chemotype, validates its ability to access in vivo-compatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes.

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

To Isomerize or not to Isomerize? E/Z Isomers of Cyclic Azobenzene Derivatives and Their Reactivity Upon One-Electron Reduction.

Azo compounds are efficient electron acceptors. Upon one-electron reduction they generally isomerize forming the thermodynamically most stable radical anion. Herein we show that the size of the central ring in 1,2-diazocines and diazonines has a ruling influence on the configuration of the one-electron reduced species. Markedly, diazonines, which bear a central nine membered heterocycle, show light-induced E/Z isomerization, but retain the configuration of the diazene N=N moiety upon one-electron reduction. Accordingly, E/Z isomerization is not induced by reduction.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

The role of action inhibition for behavioral control in joint action.

When two individuals share a task with a common goal, coordinating one's own and the other's actions is pivotal. Inhibition of one's own actions when it is the other's turn to act is assumed to play a crucial role in this process. For instance, in the joint Simon task, two individuals share a two-choice task such that one of them responds to one stimulus type and ignores the stimulus type to which the other responds. Because stimuli can either appear on one's own or on the other's side, stimulus location can conflict with stimulus identity, thus slowing response time. It has previously been shown that such conflict leads to a reduction of the detrimental effects of conflict on immediately upcoming trials both following own responses and even more so following the other's responses. This amplified trial-to-trial adjustment following the other's responses has been assumed to reflect the inhibition of own responses on the other's trials. The present study tested this hypothesis by comparing sequential trial-to-trial adjustments following correct responses and commission errors on which the inhibition of own responses has failed. As expected, adjustments were stronger following the other's correct responses than following own correct responses. Crucially, such amplification of sequential adjustment was not observed following own commission errors on the other's trials. This shows that amplification of sequential adjustments following the other's trials depend on successful inhibition of own responses on these trials and points to a crucial role of response inhibition for behavioral control in joint action.

Gene Silencing Therapeutics in Cardiology: A Review Article

Abstract Therapeutics that inhibit enzymes, receptors, ion channels, and cotransporters have long been the mainstay of cardiovascular medicine. Now, oligonucleotide therapeutics offer a modern variation on this paradigm of protein inhibition. Rather than target a protein, however, small interfering ribonucleic acids and antisense oligonucleotides target the messenger RNA (mRNA) from which a protein is translated. Endogenous, cellular mechanisms enable the oligonucleotides to bind a selected sequence on a target mRNA, leading to its degradation. The catalytic nature of the process confers an advantage over the stoichiometric binding of traditional small molecule therapeutics to their respective protein targets. Advances in nucleic acid chemistry and delivery have enabled development of oligonucleotide therapeutics against a wide range of diseases, including hyperlipidemias and hereditary transthyretin-mediated amyloidosis with polyneuropathy. While most of these therapeutics were initially designed for rare diseases, recent clinical trials highlight the potential impact of oligonucleotides on more common forms of cardiovascular disease.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe.

Quantifying the activity of key cellular redox players is crucial for understanding physiological homeostasis, and for targeting their perturbed states in pathologies including cancer and inflammatory diseases. However, cellularly-selective probes for oxidoreductase turnover are sorely lacking. We rationally developed the first probes that selectively target the mammalian selenoprotein thioredoxin reductase (TrxR), using a cyclic selenenylsulfide oriented to harness TrxR's unique selenolthiol chemistry while resisting the cellular monothiol background. Lead probe RX1 had excellent TrxR1-selective performance in cells, cross-validated by knockout, selenium starvation, knock-in, and chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors, which indicated that tempered SNAr electrophiles may be more selective TrxR drugs than the classical electrophiles used hitherto. The RX1 design thus sets the stage for in vivo imaging of the activity of this key oxidoreductase in health and disease, and can also drive TrxR1-inhibitor drug design.

Assembling the carbon skeleton of A-74528.

The 2'-phosphodiesterase inhibitor A-74528, which combines an intriguing biosynthesis with unusual biological activity, is one of the most complex type II polyketides. As a synthetic target, it represents a significant challenge due to its size but also due to a unique carbon skeleton that features a hexacarbocyclic core with an appended pyrone. Here we report our efforts toward the synthesis of A-74528, which culminated in the construction of the full carbon skeleton and the correct installation of all but one stereocenter. Our strategy employs a molybdenum-catalyzed branched allylation to establish the central quaternary carbon and relies on establishing the remaining stereocenters in a substrate-controlled manner. Carbocycles were established using a spiro epoxide annulation, a 1,3-dipolar cycloaddition, followed by an aldol condensation, and a gold-catalyzed hydroarylation. The pyrone was appended to an aldehyde branching off the quaternary stereocenter by a one-carbon homologation and Mukaiyama aldol addition.