Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7,200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Lactobacillus rhamnosus GG ameliorates hyperuricemia in a novel model.

Hyperuricemia (HUA) is a metabolic syndrome caused by abnormal purine metabolism. Although recent studies have noted a relationship between the gut microbiota and gout, whether the microbiota could ameliorate HUA-associated systemic purine metabolism remains unclear. In this study, we constructed a novel model of HUA in geese and investigated the mechanism by which Lactobacillus rhamnosus GG (LGG) could have beneficial effects on HUA. The administration of antibiotics and fecal microbiota transplantation (FMT) experiments were used in this HUA goose model. The effects of LGG and its metabolites on HUA were evaluated in vivo and in vitro. Heterogeneous expression and gene knockout of LGG revealed the mechanism of LGG. Multi-omics analysis revealed that the Lactobacillus genus is associated with changes in purine metabolism in HUA. This study showed that LGG and its metabolites could alleviate HUA through the gut-liver-kidney axis. Whole-genome analysis, heterogeneous expression, and gene knockout of LGG enzymes ABC-type multidrug transport system (ABCT), inosine-uridine nucleoside N-ribohydrolase (iunH), and xanthine permease (pbuX) demonstrated the function of nucleoside degradation in LGG. Multi-omics and a correlation analysis in HUA patients and this goose model revealed that a serum proline deficiency, as well as changes in Collinsella and Lactobacillus, may be associated with the occurrence of HUA. Our findings demonstrated the potential of a goose model of diet-induced HUA, and LGG and proline could be promising therapies for HUA.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Rational tuning of binding properties of pillar [5] arene-based sensing material by synergistic effect and its application for fluorescent turn-on detection of isoniazid and controlled reversible morphology.

Isoniazid (INH) is crucial in the treatment of tuberculosis; however, its overuse may induce significant gastrointestinal and hepatic side effects. On October 27, 2017, the International Agency for Research on Cancer, under the auspices of the World Health Organization, published a list of carcinogens for preliminary collation and reference. Isoniazid was categorized as a Group 3 carcinogen. The efficient detection of INH poses an important and challenging task. In this study, a "synergistic effect" is incorporated into the pillar (Yamagishi and Ogoshi, 2018) [5] arene-based macrocyclic host (DPA) by strategically attaching bis-p-hydroxybenzoic acid groups to the opposite ends of the pillar (Yamagishi and Ogoshi, 2018) [5] arene. This combination endows DPA with a reversible and selective fluorescence response to isoniazid. Additionally, DPA exhibits excellent analytical capabilities for isoniazid, including speed and selectivity, with a detection limit as low as 4.85 nM. Concurrently, DPA can self-assemble into a microsphere structure, which is convertible into micrometer-sized tubular structures through host-guest interactions with isoniazid. The introduction of a competitive guest, trimethylamine, enables the reversion to its microsphere structure. Consequently, this study presents an innovative and straightforward synthetic approach for smart materials that facilitates the reversible morphological transition between microspheres and microtubes in response to external chemical stimuli. This discovery provides a valuable strategy for designing "synergistic effects" in constructing trace-level isoniazid-responsive interfaces, with potential applications across various fields, such as controlled drug delivery.

Macrocycles and macrocyclization in anticancer drug discovery: Important pieces of the puzzle.

Increasing disease-related proteins have been identified as novel therapeutic targets. Macrocycles are emerging as potential solutions, bridging the gap between conventional small molecules and biomacromolecules in drug discovery. Inspired by successful macrocyclic drugs of natural origins, macrocycles are attracting more attention for enhanced binding affinity and target selectivity. Due to the conformation constraint and structure preorganization, macrocycles can reach bioactive conformations more easily than parent acyclic compounds. Also, rational macrocyclization combined with sequent structural modification will help improve oral bioavailability and combat drug resistance. This review introduces various strategies to enhance membrane permeability in macrocyclization and subsequent modification, such as N-methylation, intramolecular hydrogen bonding modulation, isomerization, and reversible bicyclization. Several case studies highlight macrocyclic inhibitors targeting kinases, HDAC, and protein-protein interactions. Finally, some macrocyclic agents targeting tumor microenvironments are illustrated.

Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9.

The mechanism by which brown adipose tissue (BAT) regulates bone metabolism is unclear. Here, we reveal that BAT secretes S100A8/A9, a previously unidentified BAT adipokine (batokine), to impair bone formation. Brown adipocytes-specific knockout of Rheb (RhebBAD KO), the upstream activator of mTOR, causes BAT malfunction to inhibit osteogenesis. Rheb depletion induces NF-κB dependent S100A8/A9 secretion from brown adipocytes, but not from macrophages. In wild-type mice, age-related Rheb downregulation in BAT is associated with enhanced S100A8/A9 secretion. Either batokines from RhebBAD KO mice, or recombinant S100A8/A9, inhibits osteoblast differentiation of mesenchymal stem cells in vitro by targeting toll-like receptor 4 on their surfaces. Conversely, S100A8/A9 neutralization not only rescues the osteogenesis repressed in the RhebBAD KO mice, but also alleviates age-related osteoporosis in wild-type mice. Collectively, our data revealed an unexpected BAT-bone crosstalk driven by Rheb-S100A8/A9, uncovering S100A8/A9 as a promising target for the treatment, and potentially, prevention of osteoporosis.

An optimized DCO with modified binary-weighted DCTLs based hybrid tuning banks for an E-band DPLL.

An optimized millimeter-wave digital controlled oscillator (DCO) in a 40-nm CMOS process is presented in this work. The coarse-tuning modules and medium-tuning modules of the DCO utilize modified binary-weighted digitally controlled transmission lines (DCTLs) to achieve a better compromise among smaller chip size, higher resonant frequency, better tuning resolution and lower phase noise. The tuning precision and die size of the medium tuning bank are improved without changing the binary coding rules by replacing the lowest-weight bit of the DCTLs with switched capacitors. In comparison with traditional DCTLs, the control bits of the coarse and medium tuning modules have been changed from 30 to 8, resulting in a 34.4% reduction in overall length (from 122[Formula: see text]m to 80[Formula: see text]m). In addition, the DCO's fine-tuning modules are achieved using a binary-weighted switched capacitors array connected to the secondary winding of a low-coupling transformer, which enhances the DCO's fine-tuning bank for better frequency resolution with less circuit complexity. The measured tuning range of the optimized DCO is 76-81GHz with a smaller die size of 0.12mm[Formula: see text]. This results in an outstanding figure of merit ([Formula: see text]) of - 190.52dBc/Hz.

Circ-MBOAT2 Regulates Angiogenesis via the miR-495/NOTCH1 Axis and Associates with Myocardial Perfusion in Patients with Coronary Chronic Total Occlusion.

Revascularization of coronary chronic total occlusion (CTO) still remains controversial. The factors that impact collateral circulation and myocardial perfusion are of interest. Circular RNA (circRNA) has been shown to regulate the process of angiogenesis. However, the effects of circ-membrane-bound O-acyltransferase domain containing 2 (circ-MBOAT2) on angiogenesis in patients with CTO were unclear. In this study, we evaluated circulating circRNAs and miRNAs in patients with CTO and stable coronary artery disease using high-throughput sequencing. Another cohort of patients were selected to verify the expressions of circ-MBOAT2 and miR-495. The role and mechanism of circ-MBOAT2 in the process of angiogenesis were explored through in vitro and vivo studies. Finally, we came back to a clinical perspective and investigated whether circ-MBOAT2 and miR-495 were associated with the improvement of myocardial perfusion evaluated by single-photon emission computed tomography (SPECT). We found that the expression of circ-MBOAT2 was significantly up-regulated while miR-495 was significantly down-regulated in patients with CTO. The expression of circ-MBOAT2 was negatively correlated with miR-495 in patients with CTO. In an in vitro study, we found that circ-MBOAT2 promoted tube formation and cell migration via the miR-495/NOTCH1 axis in endothelial cells. In an in vivo study, we showed that the inhibition of miR-495 caused the increase in collateral formation in mice after hindlimb ischemia. In a human study, we showed the expressions of circ-MBOAT2 and miR-495 were associated with myocardial perfusion improvement after revascularization of CTO. In conclusion, circ-MBOAT2 regulates angiogenesis via the miR-495/NOTCH1 axis and associates with myocardial perfusion in patients with CTO. Our findings suggest that circ-MBOAT2 and miR-495 may be potential therapeutic targets and prognostic factors for patients with CTO.

AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome.

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem-loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.

Narrowband Near-Infrared Multiple-Resonance Thermally Activated Delayed Fluorescence Emitters towards High-Performance and Stable Organic Light-Emitting Diodes.

Multiple-resonance thermally activated delayed fluorescence (MR-TADF) materials are highly coveted for their high efficiency and narrowband emission in organic light-emitting diodes (OLEDs). Nevertheless, the development of near-infrared (NIR) MR-TADF emitters remains a formidable challenge. In this study, we design two new NIR MR-TADF emitters, PXZ-R-BN and BCz-R-BN, by embedding 10H-phenoxazine (PXZ) and 7H-dibenzo[c,g]carbazole (BCz) fragments to increase the electron-donating ability or extending π-conjugation on the framework of para-boron fusing polycyclic aromatic hydrocarbons (PAHs). Both compounds emit in the NIR region, with a full-width at half-maximum (FWHM) of 49 nm (0.13 eV) for PXZ-R-BN and 43 nm (0.11 eV) for BCz-R-BN in toluene. To sensitize the two NIR MR-TADF emitters in OLEDs, a new platinum complex, Pt-1, is designed as a sensitizer. The PXZ-R-BN-based sensitized OLEDs achieve a maximum external quantum efficiency (EQEmax ) of nearly 30 % with an emission band at 693 nm, and exceptional long operational stability with an LT97 (time to 97 % of the initial luminance) value of 39084 h at an initial radiance of 1000 mW sr-1 m-2 . The BCz-R-BN-based OLEDs reach EQEmax values of 24.2 % with an emission band at 713 nm, which sets a record value for NIR OLEDs with emission bands beyond 700 nm.

Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback.

INTRODUCTION: Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. OBJECTIVES: Development of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. METHODS: We designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. RESULTS: When this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. CONCLUSION: Many positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

Influence of two kinds of clearance joints on the dynamics of planar mechanical system based on a modified contact force model.

This study takes the slider-crank mechanism with revolute joint and translational joint as the research object and studies the contact force model of the clearance joint and the influence of the hybrid clearance joints on the nonlinear dynamic behavior of the mechanism. A modified contact force model is established based on the simplified elastic oscillator model, which can be used as a normal force in clearance joint. In the new contact force model, the component n of the indentation depth can be arbitrarily selected and it can support the calculation of contact force for both fully elastic recovery, non-elastic recovery and fully inelastic recovery. Based on the LuGre friction model, the tangential friction model of the clearance joint is given. Thus, the normal force and tangential force during the dynamic contact of the clearance joint are formed. Combining Lagrange's equations of the first kind with the modified normal force and tangential friction force, the dynamic equations of the multi-body system with clearance joints are established. The Baumgarte stabilization method is used to improve the numerical stability. The correctness of the dynamic prediction model in the mechanism with clearance joint is verified by experiment. The dynamic analysis of the slider-crank mechanism with mixed clearance joints shows that the revolute clearance joint has a greater influence on the mechanism than the translational clearance, and the revolute clearance joint plays a leading role in the dynamic response.

Biosynthesis of trans-AT PKS-Derived Shuangdaolides Featuring a trans-acting Enzyme for Online Epoxidation.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) synthesize natural products with intricate structures and potent biological activities. They generally contain various unusual modules or trans-acting enzymes. Herein, we report the trans-AT PKS-derived biosynthetic pathway of the shuangdaolide with a rare internal 2-hydroxycyclopentenone moiety. The multidomain protein SdlR catalyzes the synthesis of 16,17-epoxide during polyketide chain elongation. The SdlR contains a ketoreductase, an acyl carrier protein, a flavoprotein monooxygenase, and a serine hydrolase domain. This online epoxidation occurs at unusual positions away from the thioester. Then, two tailoring enzymes, SdlB and SdlQ, convert a methylene to a carbonyl group and oxidize a hydroxyl group to a carbonyl group, respectively. The following spontaneous opening of 16,17-epoxide induces the formation of a new C-C bond to generate the 2-hydroxycyclopentenone moiety. The characterization of the shuangdaolide pathway extends the understanding of the trans-AT PKSs, facilitating the mining and identification of this class of natural products.

Biosynthesis and engineering of the nonribosomal peptides with a C-terminal putrescine.

The broad bioactivities of nonribosomal peptides rely on increasing structural diversity. Genome mining of the Burkholderiales strain Schlegelella brevitalea DSM 7029 leads to the identification of a class of dodecapeptides, glidonins, that feature diverse N-terminal modifications and a uniform putrescine moiety at the C-terminus. The N-terminal diversity originates from the wide substrate selectivity of the initiation module. The C-terminal putrescine moiety is introduced by the unusual termination module 13, the condensation domain directly catalyzes the assembly of putrescine into the peptidyl backbone, and other domains are essential for stabilizing the protein structure. Swapping of this module to another two nonribosomal peptide synthetases leads to the addition of a putrescine to the C-terminus of related nonribosomal peptides, improving their hydrophilicity and bioactivity. This study elucidates the mechanism for putrescine addition and provides further insights to generate diverse and improved nonribosomal peptides by introducing a C-terminal putrescine.

The Genomic-Driven Discovery of Glutarimide-Containing Derivatives from Burkholderia gladioli.

Glutarimide-containing polyketides exhibiting potent antitumor and antimicrobial activities were encoded via conserved module blocks in various strains that favor the genomic mining of these family compounds. The bioinformatic analysis of the genome of Burkholderia gladioli ATCC 10248 showed a silent trans-AT PKS biosynthetic gene cluster (BGC) on chromosome 2 (Chr2C8), which was predicted to produce new glutarimide-containing derivatives. Then, the silent polyketide synthase gene cluster was successfully activated via in situ promoter insertion and heterologous expression. As a result, seven glutarimide-containing analogs, including five new ones, gladiofungins D-H (3-7), and two known gladiofungin A/gladiostatin (1) and 2 (named gladiofungin C), were isolated from the fermentation of the activated mutant. Their structures were elucidated through the analysis of HR-ESI-MS and NMR spectroscopy. The structural diversities of gladiofungins may be due to the degradation of the butenolide group in gladiofungin A (1) during the fermentation and extraction process. Bioactivity screening showed that 2 and 4 had moderate anti-inflammatory activities. Thus, genome mining combined with promoter engineering and heterologous expression were proved to be effective strategies for the pathway-specific activation of the silent BGCs for the directional discovery of new natural products.

A Two-Step Fluorescence-Resonance Energy Transfer System Constructed by Platinum(II) Metallacycle Based Molecular Recognition.

Considerable progress in the construction of efficient fluorescence-resonance energy transfer (FRET) systems has promoted the development of artificial energy transfer materials. However, despite recent advances, the exploration of efficient and easy strategies to fabricate novel supramolecular systems with FRET activities is still a challenge. Here, we report that a two-step FRET system was successfully achieved, driven by platinum metallacycle based host-guest interactions. The two-step FRET system is used for the preparation of a white-light-emitting diode and serves as a nanoreactor for the photosynthetic process. This work offers a strategy for the fabrication of FRET systems and opens opportunities for functional materials constructed by platinum(II) metallacycle based host-guest interactions.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

The matrix protein of respiratory syncytial virus suppresses interferon signaling via RACK1 association.

IMPORTANCE: Respiratory syncytial virus (RSV) matrix (M) protein is indispensable for virion assembly and release. It is localized to the nucleus during early infection to perturb host transcription. However, the function of RSV M protein in other cellular activities remains poorly understood. In this study, several interferon response-associated host factors, including RACK1, were identified by proteomic analysis as RSV M interactors. Knockdown of RACK1 attenuates RSV-restricted IFN signaling leading to enhanced host defense against RSV infection, unraveling a role of M protein in antagonizing IFN response via association with RACK1. Our study uncovers a previously unrecognized mechanism of immune evasion by RSV M protein and identifies RACK1 as a novel host factor recruited by RSV, highlighting RACK1 as a potential new target for RSV therapeutics development.