Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

Ligand-based design and synthesis of N'-Benzylidene-3,4-dimethoxybenzohydrazide derivatives as potential antimicrobial agents; evaluation by in vitro,in vivo,and in silico approaches with SAR studies

Herein, a series of N''-benzylidene-3,4-dimethoxybenzohydrazide derivatives were designed and synthesised to target the multidrug efflux pump (MATE). The antibacterial activities were screened against S. aureus, Acinetobacter, S. typhi, E. coli, and P. aeruginosa, whereas their antifungal activities were screened against C. albicans. Compounds 4a, 4h, and 4i showed the most promising antibacterial and antifungal activities. Moreover, compounds 4h and 4i being the broader and superior members regarding their antimicrobial effects were selected to be further evaluated via in vivo testing using biochemical analysis and liver/kidney histological examination. Additionally, molecular docking was carried out to attain further deep insights into the synthesised compounds'' binding modes. Also, ADMET studies were performed to investigate the physicochemical/pharmacokinetics features and toxicity parameters of the synthesised derivatives. Finally, a structure-antimicrobial activity relationship study was established to facilitate further structural modifications in the future.

Highlights

• A series of new N''-benzylidene-3,4-dimethoxybenzohydrazide derivatives were designed and synthesised targeting the multidrug efflux pump (MATE) guided by the pharmacophoric features of the co-crystallized native inhibitor of the target protein.
• The newly synthesised compounds were assessed through in vitro, in vivo, and in silico approaches.
• Using the agar well diffusion assay, the antibacterial activities of the synthesised compounds were screened against S. aureus, Acinetobacter, S. typhi, E. coli, and P. aeruginosa, whereas, their antifungal activities were screened against C. albicans.
• The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the synthesised compounds were investigated on variable microbial species.
• Compounds (4h and 4i) -as the broader and superior members regarding their antimicrobial effects- were further evaluated via in vivo testing using bio-chemical analysis and liver/kidney histological examination.
• A molecular docking study and ADMET in silico studies were performed.
• A structure-antimicrobial activity relationship study was established to facilitate further structural modifications in the future.

     

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.     

Conservative initial postoperative anticoagulation strategy after HeartMate 3 left ventricular assist device implantation

IntroductionAlthough anticoagulation therapy is mandated after implantation of a left ventricular assist device (LVAD), postoperative bleedings and reoperations occur relatively frequently and are associated with worse outcomes. We evaluated the use of a conservative postoperative anticoagulation protocol in patients implanted with a HeartMate 3 (HM3) LVAD.MethodsIn a single-centre retrospective analysis of postoperative outcomes after HM3 LVAD implantation, a standard (old) anticoagulation protocol (i.e. early, full-dose anticoagulation with low-molecular weight heparin and overlapping vitamin K antagonist) was compared with a new conservative anticoagulation protocol (i.e. slow initiation of vitamin K antagonists without overlapping heparin). Main outcomes were changes in international normalised ratio (INR), lactate dehydrogenase (LDH), bleeding and/or tamponade events requiring reoperation, length of stay and adverse events.ResultsIn total, 73 patients (48 in old vs 25 in new protocol group) were evaluated. Mean age was 56 years (standard deviation 13) and most patients (78%) were males. Changes in INR and LDH in the first 14 days were similar in both groups (p = 0.50 and p = 0.997 for interaction, respectively). Number of bleeding/tamponade events requiring reoperation was lower in the new than in the old protocol group (4% vs 33%, p = 0.005). Postoperative 30-day mortality was similar, and we observed no thromboembolic events. Median (25th–75th percentiles) total length of postoperative hospital stay (27 (25–41) vs 21 (19–27) days, p < 0.001) and length of intensive care unit stay (5 (2–9) vs 2 (2–5) days, p = 0.022) were significantly shorter in the new protocol group.ConclusionThese retrospective data suggest that conservative slow initiation of anticoagulation therapy after HM3 LVAD implantation is associated with less bleeding/tamponade events requiring reoperation, a similar safety profile and a shorter duration of stay than the currently advised standard anticoagulation protocol.Supplementary InformationThe online version of this article (10.1007/s12471-022-01671-1) contains supplementary material, which is available to authorized users.     

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.     

Solubility enhancement of mefenamic acid by inclusion complex with β-cyclodextrin: in silico modelling,formulation, characterisation,and in vitro studies

The aim of this study was to prepare and characterise inclusion complexes of a low water-soluble drug, mefenamic acid (MA), with β-cyclodextrin (β-CD). First, the phase solubility diagram of MA in β-CD was drawn from 0 to 21 × 10⁻³ M of β-CD concentration. A job’s plot experiment was used to determine the stoichiometry of the MA:β-CD complex (2:1). The stability of this complex was confirmed by molecular modelling simulation. Three methods, namely solvent co-evaporation (CE), kneading (KN), and physical mixture (PM), were used to prepare the (2:1) MA:β-CD complexes. All complexes were fully characterised. The drug dissolution tests were established in simulated liquid gastric and the MA water solubility at pH 1.2 from complexes was significantly improved. The mechanism of MA released from the β-CD complexes was illustrated through a mathematical treatment. Finally, two in vitro experiments confirmed the interest to use a (2:1) MA:β-CD complex.     

In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.     

一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建

双荧光素酶报告基因系统能够提供灵敏的读数,但该系统需要依赖组成型表达的内参对读数进行归一化。然而,大多数内参并不是在所有条件下都组成型表达。为此,文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先,突变BmV gP78启动子上的激素应答相关元件,获得了在家蚕细胞中稳定表达的组成型启动子BmV gP78M;然后,用BmV gP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列,成功构建了pRL-V gP78M内参质粒;最后,通过细胞转染实验证实pRL-V gP78M内参在家蚕细胞系中稳定表达,并且pRL-V gP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终,获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-V gP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时,该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。     

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial–mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.     

Ligand-based design,synthesis, computational insights,and in vitro studies of novel N-(5-Nitrothiazol-2-yl)-carboxamido derivatives as potent inhibitors of SARS-CoV-2 main protease

The global outbreak of the COVID-19 pandemic provokes scientists to make a prompt development of new effective therapeutic interventions for the battle against SARS-CoV-2. A new series of N-(5-nitrothiazol-2-yl)-carboxamido derivatives were designed and synthesised based on the structural optimisation principle of the SARS-CoV Mpro co-crystallized WR1 inhibitor. Notably, compound 3b achieved the most promising anti-SARS-CoV-2 activity with an IC₅₀ value of 174.7 µg/mL. On the other hand, compounds 3a, 3b, and 3c showed very promising SARS-CoV-2 Mpro inhibitory effects with IC₅₀ values of 4.67, 5.12, and 11.90 µg/mL, respectively. Compound 3b docking score was very promising (−6.94 kcal/mol) and its binding mode was nearly similar to that of WR1. Besides, the molecular dynamics (MD) simulations of compound 3b showed its great stability inside the binding pocket until around 40 ns. Finally, a very promising SAR was concluded to help to design more powerful SARS-CoV-2 Mpro inhibitors shortly.     

Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases

We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals.     

Molecular and in vivo phenotyping of missense variants of the human glucagon receptor

Naturally occurring missense variants of G protein–coupled receptors with loss of function have been linked to metabolic disease in case studies and in animal experiments. The glucagon receptor, one such G protein–coupled receptor, is involved in maintaining blood glucose and amino acid homeostasis; however, loss-of-function mutations of this receptor have not been systematically characterized. Here, we observed fewer glucagon receptor missense variants than expected, as well as lower allele diversity and fewer variants with trait associations as compared with other class B1 receptors. We performed molecular pharmacological phenotyping of 38 missense variants located in the receptor extracellular domain, at the glucagon interface, or with previously suggested clinical implications. These variants were characterized in terms of cAMP accumulation to assess glucagon-induced Gα_s coupling, and of recruitment of β-arrestin-1/2. Fifteen variants were impaired in at least one of these downstream functions, with six variants affected in both cAMP accumulation and β-arrestin-1/2 recruitment. For the eight variants with decreased Gα_s signaling (D63^ECDN, P86^ECDS, V96^ECDE, G125^ECDC, R225^3.30H, R308^5.40W, V368^6.59M, and R378^7.35C) binding experiments revealed preserved glucagon affinity, although with significantly reduced binding capacity. Finally, using the UK Biobank, we found that variants with wildtype-like Gα_s signaling did not associate with metabolic phenotypes, whereas carriers of cAMP accumulation-impairing variants displayed a tendency toward increased risk of obesity and increased body mass and blood pressure. These observations are in line with the essential role of the glucagon system in metabolism and support that Gα_s is the main signaling pathway effecting the physiological roles of the glucagon receptor.     

A Cre/LoxP conditional luciferase reporter transgenic mouse for bioluminescence monitoring of tumorigenesis

Genetically engineered, Cre/LoxP‐conditional mouse models of cancer are designed to investigate the genetic contributors of tumorigenesis and are well suited to assess therapeutic treatment responses. The capacity to serially visualize tumor burden in a noninvasive fashion would greatly strengthen their applications. We report the generation of a bioluminescent reporter strain that allows monitoring of tumor development in preexisting conditional mouse tumor models. We demonstrate that, in a Cre‐dependent glioblastoma multiforme model, tumor initiation and progression is readily monitored over time and that luminescent output is related to tumor volume. Our results show that this reporter strain may be combined with various Cre/loxP mouse tumor models to allow for noninvasive longitudinal monitoring of tumor growth and therapeutic response in vivo. genesis 47:659–666, 2009. © 2009 Wiley‐Liss, Inc.     

4‐octyl Itaconate inhibits lipopolysaccharide (LPS)‐induced osteoarthritis via activating Nrf2 signalling pathway

Small molecule drug intervention for chondrocytes is a valuable method for the treatment of osteoarthritis (OA). The 4‐octyl itaconate (OI) is a cellular derivative of itaconate with sound cell permeability and transformation rate. We attempted to confirm the protective role of OI in chondrocytes and its regulatory mechanism. We used lipopolysaccharide (LPS) to induce chondrocyte inflammation injury. After the OI treatment, the secretion and mRNA expression of Il‐6, Il‐10, Mcp‐1 and Tnf‐α were detected by ELISA and qPCR. The protective effect of OI on articular cartilage was further verified in surgical destabilization of the medial meniscus model of OA. Cell death and apoptosis were evaluated based on CCK8, LDH, Typan blue staining, Annexin V and TUNEL analyses. The small interfering RNAs were used to knockout the Nrf2 gene of chondrocytes to verify the OI‐mediated Nrf2 signalling pathway. The results revealed that OI protects cells from LPS‐induced inflammatory injury and attenuates cell death and apoptosis induced by LPS. Similar protective effects were also observed on articular cartilage in mice. The OI activated Nrf2 signalling pathway and promoted the stable expression and translocation of Nrf2 into the nucleus. When the Nrf2 signalling pathway was blocked, the protective effect of OI was significantly counteracted in chondrocytes and a mouse arthritis model. Both itaconate and its derivative (i.e., OI) showed important medical effects in the treatment of OA.     

Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2

Nucleosomes are dynamic entities with wide‐ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo‐EM structures of nucleosome core particles containing human H2A.B (H2A.B‐NCP) at atomic resolution, identifying large‐scale structural rearrangements in the histone octamer in H2A.B‐NCP. H2A.B‐NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left‐handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B‐specific residues, including a ROF (“regulating‐octamer‐folding”) sequence of six consecutive residues, are responsible for loosening of H2A.B‐NCPs. Unlike H2A.B‐NCP, the H2A.Z.2.2‐containing nucleosome (Z.2.2‐NCP) adopts a less‐extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2‐specific ROF in both H2A.Z.2.2‐NCP opening and SWR1‐dependent histone replacement. Taken together, these first high‐resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.     

A polypeptide inhibitor of calcineurin blocks the calcineurin-NFAT signalling pathway in vivo and in vitro

Calcineurin (CN) controls the immune response by regulating nuclear factor of activated T cells (NFAT). Inhibition of CN function is an effective treatment for immune diseases. The PVIVIT peptide is an artificial peptide based on the NFAT-PxIxIT motif, which exhibits stronger binding to CN. A bioactive peptide (named pep4) that inhibits the CN/NFAT interaction was designed. Pep4 contains a segment of A238L as the linker and the LxVP motif and PVIVIT motif as CN binding sites. Pep4 has strong binding capacity to CN and inhibits CN activity competitively. 11-arginine-modified pep4 (11 R-pep4) inhibits the nuclear translocation of NFAT and reduces the expression of IL-2. 11 R-pep4 improves the pathological characteristics of asthmatic mice to a certain extent. The above results indicated that pep4 is a high-affinity CN inhibitor. These findings will contribute to the discovery of new CN inhibitors and promising immunosuppressive drugs.