DNA surface exploration and operator bypassing during target search.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 µs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

Spatiotemporal kinetics of the SRP pathway in live E. coli cells.

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Uncatalyzed Diboron Activation by a Strained Hydrocarbon: Experimental and Theoretical Study of [1.1.1]Propellane Diborylation.

The synthesis of strained carbocyclic building blocks is relevant for Medicinal Chemistry, and methylenecyclobutanes are particularly challenging with current synthetic technology. Careful inspection of the reactivity of [1.1.1]propellane and diboron reagents has revealed that bis(catecholato)diboron (B2cat2) can produce a bis(borylated) methylenecyclobutane in a few minutes at room temperature. This reaction constitutes the first example of B-B bond activation by a special apolar hydrocarbon and also the first time that propellane is electrophilically activated by boron. Mechanistic studies including in situ NMR kinetics and DFT calculations demonstrate that the diboron moiety can be directly activated through coordination with the inverted sigma bond of propellane, and reveal that DMF is involved in the stabilization of diboronate ylide intermediates rather than the activation of the B-B bond. These results enable new possibilities for both diboron and propellane chemistry, and for further developments in the synthesis of methylenecyclobutanes based on propellane strain release.

HSQC Spectra Simulation and Matching for Molecular Identification.

In the pursuit of improved compound identification and database search tasks, this study explores heteronuclear single quantum coherence (HSQC) spectra simulation and matching methodologies. HSQC spectra serve as unique molecular fingerprints, enabling a valuable balance of data collection time and information richness. We conducted a comprehensive evaluation of the following four HSQC simulation techniques: ACD/Labs (ACD), MestReNova (MNova), Gaussian NMR calculations (DFT), and a graph-based neural network (ML). For the latter two techniques, we developed a reconstruction logic to combine proton and carbon 1D spectra into HSQC spectra. The methodology involved the implementation of three peak-matching strategies (minimum-sum, Euclidean-distance, and Hungarian distance) combined with three padding strategies (zero-padding, peak-truncated, and nearest-neighbor double assignment). We found that coupling these strategies with a robust simulation technique facilitates the accurate identification of correct molecules from similar analogues (regio- and stereoisomers) and allows for fast and accurate large database searches. Furthermore, we demonstrated the efficacy of the best-performing methodology by rectifying the structures of a set of previously misidentified molecules. This research indicates that effective HSQC spectral simulation and matching methodologies significantly facilitate molecular structure elucidation. Furthermore, we offer a Google Colab notebook for researchers to use our methods on their own data (https://github.com/AstraZeneca/hsqc_structure_elucidation.git).

A mixed method study exploring similarities and differences in general and social services-specific barriers to treatment-seeking among individuals with a problematic use of alcohol, cannabis, or gambling.

INTRODUCTION: The treatment gap for addictive disorders is one of the largest in health care. Several studies have investigated barriers to treatment for different addictive disorders, but very few studies conducted have explored whether the barriers differ depending on substance or behavior or if they are common among all addictive disorders. In Sweden, addiction care is provided both by the healthcare and social services, where the latter is common, but also less popular. To our knowledge, there are no studies exploring whether the barriers are different depending on where the treatment is given. AIM: The aim was to thoroughly explore both which general and social services-specific barriers to treatment that are common, which barriers that differs, and how the barriers are described among individuals with a problematic use of alcohol, cannabis and/or gambling. METHOD: A mixed method convergent parallel design was conducted. For the quantitative measures, surveys including the validated Barriers to Treatment Inventory as well as questions regarding barriers in the Swedish multi-provider landscape, were collected from individuals with a problematic use of alcohol (n = 207), cannabis (n = 51), and gambling (n = 37). In parallel, 17 semi-structured interviews from the same population were conducted and analyzed with thematic analysis. Thereafter, the quantitative and qualitative data was compared, contrasted, and at last, interpreted. RESULTS: The quantitative data showed that the largest general barriers in all groups were privacy concern and poor availability, and the largest barriers for seeking help from the social services was stigma, unawareness of what is offered, and fear of consequences for all groups. The qualitative data resulted in five general barriers: stigma, ambivalence, accessibility, fear of consequences, and lack of knowledge about addiction and its' treatments, and three barriers specifically towards social services: social services reputation, fear of meeting acquaintances, and lack of knowledge. The themes were developed from data from all groups, but different aspects of the themes were mentioned by different groups. CONCLUSION: There are details and aspects that differentiates both the general and social service-specific barriers to treatment between individuals with a problematic use of alcohol, cannabis, and gambling, but in large they perceive similar barriers.

Real-time measurements of aminoglycoside effects on protein synthesis in live cells.

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections.

OBJECTIVE: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo. DESIGN: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer. RESULTS: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection. CONCLUSION: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection.

Hybrid Machine Learning Approach to Predict the Site Selectivity of Iridium-Catalyzed Arene Borylation.

The borylation of aryl and heteroaryl C-H bonds is valuable for the site-selective functionalization of C-H bonds in complex molecules. Iridium catalysts ligated by bipyridine ligands catalyze the borylation of the C-H bond that is most acidic and least sterically hindered in an arene, but predicting the site of borylation in molecules containing multiple arenes is difficult. To address this challenge, we report a hybrid computational model that predicts the Site of Borylation (SoBo) in complex molecules. The SoBo model combines density functional theory, semiempirical quantum mechanics, cheminformatics, linear regression, and machine learning to predict site selectivity and to extrapolate these predictions to new chemical space. Experimental validation of SoBo showed that the model predicts the major site of borylation of pharmaceutical intermediates with higher accuracy than prior machine-learning models or human experts, demonstrating that SoBo will be useful to guide experiments for the borylation of specific C(sp2)-H bonds during pharmaceutical development.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Ruthenium-Catalyzed Aminocarbonylation with Isocyanates Through Weak Coordinating Groups.

Introducing amide functional groups under mild conditions has growing importance owing to the prevalence of such moiety in biologically active molecules. Herein, we disclose a mild protocol for the directed ruthenium-catalyzed C-H aminocarbonylation with isocyanates as the amidating agents developed through high-throughput experimentation (HTE). The redox-neutral and base-free reaction is guided by weakly Lewis basic functional groups, including anilides, lactams and carbamates to access anthranilamide derivatives. The synthetic utility of this transformation is reflected by large-scale synthesis and late-stage functionalization.

A psychometric evaluation of the Swedish translation of the Perceived Stress Scale: a Rasch analysis.

BACKGROUND: Stress reflects physical and psychological reactions to imposing demands and is often measured using self-reports. A widely-used instrument is the Perceived Stress Scale (PSS), intended to capture more general aspects of stress. A Swedish translation of the PSS is available but has not previously been examined using modern test theory approaches. The aim of the current study is to apply Rasch analysis to further the understanding of the PSS' measurement properties, and, in turn, improve its utility in different settings. METHODS: Data from 793 university students was used to investigate the dimensionality of different version of the PSS (14, 10, and 4 items) as well as potential response patterns among the participants. RESULTS: The current study demonstrates that the PSS-14 has two separate factors, divided between negatively worded items (perceived stress) and positively worded items (perceived [lack of] control), although with only the negative subscale exhibiting good reliability. Response patterns were analyzed using Differential Item Functioning, which did not find an influence of gender on any of the items, but for age regarding the positive subscale (items 6 and 9). The PSS-10 also demonstrated adequate reliability for the negative subscale, but the PSS-4 was not deemed suitable as a unidimensional scale. CONCLUSIONS: Based on the results, none of the versions of the PSS should be used by sum-scoring all of the items. Only the negative items from the PSS-14 or PSS-10 can be used as unidimensional scales to measure general aspects of stress. As for different response patterns, gender may nevertheless be important to consider, as prior research has found differences on several items. Meanwhile, content validity is discussed, questioning the relevance of anger and being upset when measuring more general aspects of stress. Finally, a table to convert the PSS-7 (i.e., negative items) ordinal sum scores to interval level scores is provided.

Comment on "A new class of out-gap discrete solitons in binary waveguide arrays" [Chaos 32, 073113 (2022)].

Recent results [M. C. Tran and T. X. Tran, Chaos 32, 073113 (2022)] are put into the context of earlier work [A. V. Gorbach and M. Johansson, Eur. Phys. J. D 29, 77-93 (2004); M. Johansson and A. V. Gorbach, Phys. Rev. E 70, 057604 (2004)]. The two newly found families of "beyond-band discrete solitons" [M. C. Tran and T. X. Tran, Chaos 32, 073113 (2022)] are found to be smooth continuations of "on-top breathers" briefly mentioned by Gorbach and Johansson [Eur. Phys. J. D 29, 77-93 (2004)] and Johansson and Gorbach [Phys. Rev. E 70, 057604 (2004)] and the relevant bifurcation scenarios are described. One family is shown to be linearly unstable with respect to symmetry-breaking oscillations.

Heterogeneous Copper-Catalyzed Cross-Coupling for Sustainable Synthesis of Chiral Allenes: Application to the Synthesis of Allenic Natural Products.

Classical Crabbé type SN 2' substitutions of propargylic substrates has served as one of the standard methods for the synthesis of allenes. However, the stereospecific version of this transformation often requires either stoichiometric amounts of organocopper reagents or special functional groups on the substrates, and the chirality transfer efficiency is also capricious. Herein, we report a sustainable methodology for the synthesis of diverse 1,3-di and tri-substituted allenes by using a simple and cheap cellulose supported heterogeneous nanocopper catalyst (MCC-Amp-Cu(I/II)). This approach represents the first example of heterogeneous catalysis for the synthesis of chiral allenes. High yields and excellent enantiospecificity (up to 97 % yield, 99 % ee) were achieved for a wide range of di- and tri-substituted allenes bearing various functional groups. It is worth noting that the applied heterogeneous catalyst could be recycled at least 5 times without any reduced reactivity. To demonstrate the synthetic utility of the developed protocol, we have applied it to the total synthesis of several chiral allenic natural products.

Echocardiographic diagnosis of heart failure with preserved ejection fraction in elderly patients with hypertension.

Objectives. The aim of this study is to evaluate the diagnostic performance of echocardiography for the diagnosis of heart failure with preserved ejection fraction (HFpEF) in the elderly and to validate the Heart Failure Association diagnostic algorithm (HFA-PEFF). Design. A case-control study was conducted in patients with hypertension with or without HFpEF who were matched for age (n = 33; 78.4 ± 5.3 years) and sex. Participants underwent echocardiography including assessment of left atrial (LA) volume index (LAVI), early mitral filling to early diastolic mitral annulus velocity ratio (E/e'), LA reservoir strain (LASr), tissue Doppler LA contraction (a'), right ventricular isovolumic relaxation time (RVIVRT), and a 6-minute walk test (6-MWT). The filling pressure algorithm from the European association of cardiovascular imaging (EACVI) 2021 was applied. The HFA-PEFF score was also applied, using echocardiography parameters and the value of NT pro-BNP, without considering symptomatic status. Results. Echocardiographic parameters identified patients with HFpEF with an area under the curve (AUC) >0.9 for E/e', RVIVRT, LASr, a', and the ratio of LAVI/a'. LASr correlated with 6-MWT (r = 0.59, p = .0003). The EACVI algorithm classified all controls with normal filling pressure and 94% of patients with HFpEF with increased filling pressure. When the HFA-PEFF diagnostic algorithm was validated, a high score (≥5 points) had 100% sensitivity for HFpEF, while 88% of controls had intermediate scores (2-4 points). Conclusion. The EACVI filling pressure algorithm, RVIVRT, LASr, and the ratio LAVI/a' were accurate for diagnosing HFpEF in elderly patients with hypertension. The HFA-PEFF score had high sensitivity but limited ability to exclude HFpEF.

Blocking Entry of Hepatitis B and D Viruses to Hepatocytes as a Novel Immunotherapy for Treating Chronic Infections.

BACKGROUND: Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T-cell response. We therefore designed an immunotherapy driven by naive healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry. METHODS: Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV-specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes. RESULTS: The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice. CONCLUSIONS: We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation.

Oxalate Oxidase for In Situ H2 O2 -Generation in Unspecific Peroxygenase-Catalysed Drug Oxyfunctionalisations.

H2 O2 -driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2 O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by-product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C-H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high-throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 µL-scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one-pot approach ensures adequate H2 O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.

Development of a method for heat shock stress assessment in yeast based on transcription of specific genes.

All living cells, including yeast cells, are challenged by different types of stresses in their environments and must cope with challenges such as heat, chemical stress, or oxidative damage. By reversibly adjusting the physiology while maintaining structural and genetic integrity, cells can achieve a competitive advantage and adapt environmental fluctuations. The yeast Saccharomyces cerevisiae has been extensively used as a model for study of stress responses due to the strong conservation of many essential cellular processes between yeast and human cells. We focused here on developing a tool to detect and quantify early responses using specific transcriptional responses. We analyzed the published transcriptional data on S. cerevisiae DBY strain responses to 10 different stresses in different time points. The principal component analysis (PCA) and the Pearson analysis were used to assess the stress response genes that are highly expressed in each individual stress condition. Except for these stress response genes, we also identified the reference genes in each stress condition, which would not be induced under stress condition and show stable transcriptional expression over time. We then tested our candidates experimentally in the CEN.PK strain. After data analysis, we identified two stress response genes (UBI4 and RRP) and two reference genes (MEX67 and SSY1) under heat shock (HS) condition. These genes were further verified by real-time PCR at mild (42°C), severe (46°C), to lethal temperature (50°C), respectively.

Late-Stage Amination of Drug-Like Benzoic Acids: Access to Anilines and Drug Conjugates through Directed Iridium-Catalyzed C-H Activation.

The functionalization of C-H bonds, ubiquitous in drugs and drug-like molecules, represents an important synthetic strategy with the potential to streamline the drug-discovery process. Late-stage aromatic C-N bond-forming reactions are highly desirable, but despite their significance, accessing aminated analogues through direct and selective amination of C-H bonds remains a challenging goal. The method presented herein enables the amination of a wide array of benzoic acids with high selectivity. The robustness of the system is manifested by the large number of functional groups tolerated, which allowed the amination of a diverse array of marketed drugs and drug-like molecules. Furthermore, the introduction of a synthetic handle enabled expeditious access to targeted drug-delivery conjugates, PROTACs, and probes for chemical biology. This rapid access to valuable analogues, combined with operational simplicity and applicability to high-throughput experimentation has the potential to aid and considerably accelerate drug discovery.

Author Correction: tRNA tracking for direct measurements of protein synthesis kinetics in live cells.

In the version of this article originally published, the values on the y axis of Fig. 6d were incorrect. They should be 0.00, 0.02, 0.04, 0.06 and 0.08 instead of the previous 0.00, 0.04, 0.08 and 0.12. The error has been corrected in the HTML and PDF versions of this paper.