Search

Exploration of new organometallic systems based on polyhedral boron clusters has the potential to solve challenging chemical problems such as the stabilization of reactive intermediates and transition-state-like species postulated for E-H (E = H, B, C, Si) bond activation reactions. We report on facile and clean B-H activation of a hydroborane by a new iridium boron cluster complex. The product of this reaction is an unprecedented and fully characterized transition metal-stabilized boron cation or borenium. Moreover, this intermediate bears an unusual intramolecular B···H interaction between the hydrogen originating from the activated hydroborane and the cyclometallated metal-bonded boron atom of the boron cluster. This B···H interaction is proposed to be an arrested insertion of hydrogen into the B

Here, we report six novel, easily accessible BODIPY-based agents for cancer treatment. In contrast to established photodynamic therapy (PDT) agents, these BODIPY-based compounds show additional photothermal activity and their cytotoxicity is not dependent on the generation of reactive oxygen species (ROS). The agents show high photocytotoxicity upon irradiation with light and low dark toxicity in different cancer cell lines in 2D culture as well as in 3D multicellular tumor spheroids (MCTSs). The ratio of dark to light toxicity (phototoxic index, PI) of these agents reaches striking values exceeding 830,000 after irradiation with energetically low doses of light at 630 nm. The oxygen-dependent mechanism of action (MOA) of established photosensitizers (PSs) hampers effective clinical deployment of these agents. Under hypoxic conditions (0.2% O2), which are known to limit the efficiency of conventional PSs in solid tumors, photocytotoxicity was induced at the same concentration levels, indicating an oxygen-independent photothermal MOA. With a PI exceeding 360,000 under hypoxic conditions, both PI values are the highest reported to date. We anticipate that small molecule agents with a photothermal MOA, such as the BODIPY-based compounds reported in this work, may overcome this barrier and provide a new avenue to cancer therapy.

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16–22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.

Inverse electron demand [4+2] Diels–Alder (iEDDA) reactions as well as unprecedented nucleophilic (azaphilic) additions of R-substituted silyl-enol ethers (where R is Phenyl, Methyl, and Hydrogen) to 1,2,4,5-tetrazine (s-tetrazine) catalyzed by $$\hbox {BF}_{3}$$ BF 3 have recently been discovered (Simon et al. in Org Lett 23(7):2426–2430, 2021), where static calculations were employed for calculation of activation energies. In order to have a more realistic dynamic description of these reactions in explicit solution at ambient conditions, in this work we use a semiempirical tight-binding method combined with enhanced sampling techniques to calculate free energy surfaces (FESs) of the iEDDA and azaphilic addition reactions. Relevant products of not only s-tetrazine but also its derivatives such as $$\hbox {BF}_{3}$$ BF 3 -mediated s-tetrazine adducts are investigated. We reconstruct the FESs of the iEDDA and azaphilic addition reactions using metadynamics and blue moon ensemble, and compare the ability of different collective variables (CVs) including bond distances, Social PeRmutation INvarianT (SPRINT) coordinates, and path-CV to describe the reaction pathway. We find that when a bulky Phenyl is used as a substituent at the dienophile the azaphilic addition is preferred over the iEDDA reaction. In addition, we also investigate the effect of $$\hbox {BF}_{3}$$ BF 3 in the diene and steric hindrance in the dienophile on the competition between the iEDDA and azaphilic addition reactions, providing chemical insight for reaction design.

The nuclear receptor farnesoid X receptor (FXR, NR1H4) is a bile acid (BA) sensor that links the enterohepatic circuit that regulates BA metabolism and elimination to systemic lipid homeostasis. Furthermore, FXR represents a real guardian of the hepatic function, preserving, in a multifactorial fashion, the integrity and function of hepatocytes from chronic and acute insults. This review summarizes how FXR modulates the expression of pathway-specific as well as polyspecific transporters and enzymes, thereby acting at the interface of BA, lipid and drug metabolism, and influencing the onset and progression of hepatotoxicity of varying etiopathogeneses. Furthermore, this review article provides an overview of the advances and the clinical development of FXR agonists in the treatment of liver diseases.

In nature, thiolate-based systems are the primary targets of divalent mercury (Hg-II) toxicity. The formation of Hg(Cys)(x) cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct Hg-II-thiolate coordination preferences, among which linear Hg-II complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral Hg-II complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating Zn-II. Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with Hg-II. Using UV-Vis, CD, PAC, and Hg-199 NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)(2) species with a distorted tetrahedral HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher Hg-II content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)(4) core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)(2) complex is approximately three orders of magnitude higher than those of the strongest Hg-II complexes known to date.

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.

The direct pathophysiological effects of obstructive sleep apnea (OSA) have been well described. However, the systemic and metabolic consequences of OSA are less well understood. The aim of this secondary analysis was to translate recent findings in healthy subjects on vigilance-state-dependent metabolism into the context of OSA patients and answer the question of how symptomatic OSA influences metabolism and whether these changes might explain metabolic and cardiovascular consequences of OSA. Patients with suspected OSA were assigned according to their oxygen desaturation index (ODI) and Epworth Sleepiness Scale (ESS) score into symptomatic OSA and controls. Vigilance-state-dependent breath metabolites assessed by high-resolution mass spectrometry were used to test for a difference in both groups. In total, 44 patients were eligible, of whom 18 (40.9%) were assigned to the symptomatic OSA group. Symptomatic OSA patients with a median [25%, 75% quartiles] ODI of 40.5 [35.0, 58.8] events/h and an ESS of 14.0 [11.2, 15.8] showed moderate to strong evidence for differences in 18 vigilance-state-dependent breath compounds compared to controls. These identified metabolites are part of major metabolic pathways in carbohydrate, amino acid, and lipid metabolism. Thus, beyond hypoxia per se, we hypothesize that disturbed sleep in OSA patients persists as disturbed sleep-dependent metabolite levels during daytime., International Journal of Molecular Sciences, 23 (22), ISSN:1422-0067

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols., Angewandte Chemie. International Edition, 61 (48), ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.

Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.

High-throughput engineering has the potential to revolutionize the customization of biosynthetic assembly lines for the sustainable production of pharmaceutically relevant natural product analogs. Here, we show that the substrate specificity of gatekeeper adenylation domains of nonribosomal peptide synthetases can be switched from an α-amino acid to an α-hydroxy acid in a single round of combinatorial mutagenesis and selection using yeast cell surface display. In addition to shedding light on how such proteins discriminate between amino and hydroxy groups, the remodeled domains function in a pathway context to produce α-hydroxy acid-containing linear peptides and cyclic depsipeptides with high efficiency. Site-specific replacement of backbone amines with oxygens by an engineered synthetase provides the means to probe and tune the activities of diverse peptide metabolites in a simple and predictable fashion.

Flattening helices while keeping the handedness: On-surface cyclodehydrogenation of bishelicene enantiomers leads stereospecifically to (M,M) and (P,P) chiral planar polyaromatic hydrocarbons. This is followed by their homochiral aggregation into a 2D conglomerate. Thermally induced cyclodehydrogenation proceeds stereospecifically to chiral, planar coronocoronene. Such a reaction is a special example of topochemistry in which enantiospecific conversion is supported by the alignment of the reactant by the surface.

Catalytic systems for direct C-H activation of arenes commonly show preference for electronically activated and sterically exposed C-H sites. Here we show that a range of functionally rich and pharmaceutically relevant arene classes can undergo site-selective C-H arylation ortho to small alkyl substituents, preferably endocyclic methylene groups. The C-H activation is experimentally supported as being the selectivity-determining step, while computational studies of the transition state models indicate the relevance of non-covalent interactions between the catalyst and the methylene group of the substrate. Our results suggest that preference for C(sp

Aims: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. Objectives: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. Methods and results: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (ICa-L) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. Conclusions: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant.

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL., International Journal of Molecular Sciences, 23 (16), ISSN:1422-0067

Here we report on carbon monoxide-photoreleasable compounds (photoCORMs) that combine heptamethine cyanine and flavonol chromophores and are activated upon irradiation with near-infrared light. Excellent CO-release yields and uncaging cross sections in aqueous solutions, enhanced water solubilities thanks to polar substituents or a host-guest approach using cucurbit[7]uril are demonstrated. The hybrids display outstanding biocompatibility and diverse, structure-dependent cell penetrability and internalization.

Thoracic spinal cord injury (SCI) results in urinary dysfunction, which majorly affects the quality of life of SCI patients. Abnormal sprouting of lumbosacral bladder afferents plays a crucial role in this condition. Underlying mechanisms may include changes in expression of regulators of axonal growth, including chondroitin sulphate proteoglycans (CSPGs), myelin-associated inhibitors (MAIs) and repulsive guidance molecules, known to be upregulated at the injury site post SCI. Here, we confirmed lumbosacral upregulation of the growth-associated protein GAP43 in SCI animals with bladder dysfunction, indicating the occurrence of axonal sprouting. Neurocan and Phosphacan (CSPGs), as well as Nogo-A (MAI), at the same spinal segments were upregulated 7 days post injury (dpi) but returned to baseline values 28 dpi. In turn, qPCR analysis of the mRNA levels for receptors of those repulsive molecules in dorsal root ganglia (DRG) neurons showed a time-dependent decrease in receptor expression. In vitro assays with DRG neurons from SCI rats demonstrated that exposure to high levels of NGF downregulated the expression of some, but not all, receptors for those regulators of axonal growth. The present results, therefore, show significant molecular changes at the lumbosacral cord and DRGs after thoracic lesion, likely critically involved in neuroplastic events leading to urinary impairment.

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models.

The first example of a neutral spin‐delocalized carbon‐nanoring radical was achieved by integration of the open‐shell phenalenyl unit into cycloparaphenylene (CPP). Spin distribution in this hydrocarbon is localized primarily on the phenalenyl segment and partially on the CPP segment as a consequence of steric and electronic effects. The resulting geometry is reminiscent of a diamond ring, with pseudo‐perpendicular arrangement of the radial and the planar π‐surface. The phenylene rings attached directly to the phenalenyl unit give rise to a steric effect that governs a highly selective dimerization pathway, yielding a giant double nanohoop. Its π‐framework made of 158 sp2‐carbon atoms was elucidated by single‐crystal X‐ray diffraction, which revealed a three‐segment CPP‐peropyrene‐CPP structure. This nanocarbon shows a fluorescence profile characteristic of peropyrene, regardless of which segment gets excited. These results in conjunction with DFT suggest that adjusting the size of the CPP segments in this double nanohoop could deliver donor–acceptor systems., A neutral open‐shell carbon nanoring was achieved by integration of the phenalenyl unit into the cycloparaphenylene framework. It is shown that the spin distribution and reactivity of this hydrocarbon in the shape of a diamond ring are a result of steric and electronic effects and that selective dimerization yields a double nanohoop with a radial‐planar‐radial arrangement of its three π‐conjugated segments comprising a total of 158 sp2‐carbon atoms.

We report a method of glycosylated enzymes' surface immobilisation and stabilisation. The enzyme is immobilised at the surface of silica nanoparticles through the reversible covalent binding of vicinal diols of the enzyme glycans with a surface-attached boronate derivative. A soft organosilica layer of controlled thickness is grown at the silica surface, entrapping the enzyme and thus avoiding enzyme leaching. We demonstrate that this approach results not only in high and durable activity retention but also enzyme stabilisation.

Burkholderia cenocepacia is an opportunistic pathogen that can lead to severe infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Being an obligate aerobe, B. cenocepacia is unable to grow in the absence of oxygen. In this study, we show that the CF isolate B. cenocepacia H111 can survive in the absence of oxygen. Using a transposon sequencing (Tn-seq) approach, we identified 71 fitness determinants involved in anoxic survival, including a Crp-Fnr family transcriptional regulatory gene (anr2), genes coding for the sensor kinase RoxS and its response regulator RoxR, the sigma factor for flagella biosynthesis (FliA) and subunits of a cytochrome bd oxidase (CydA, CydB and the potentially novel subunit CydP). Individual knockouts of these fitness determinants significantly reduced anoxic survival, and inactivation of both anr copies is shown to be lethal under anoxic conditions. We also show that the two-component system RoxS/RoxR and FliA are important for virulence and swarming/swimming, respectively.

In this study, we explored expression of microRNA (miR), miR-target genes and matrix remodelling molecules in temporal artery biopsies (TABs) from treatment-naïve patients with giant cell arteritis (GCA, n = 41) and integrated these analyses with clinical, laboratory, ultrasound and histological manifestations of GCA. NonGCA patients (n = 4) served as controls. GCA TABs exhibited deregulated expression of several miRs (miR-21-5p, -145-5p, -146a-5p, -146b-5p, -155-5p, 424-3p, -424-5p, -503-5p), putative miR-target genes (YAP1, PELI1, FGF2, VEGFA, KLF4) and matrix remodelling factors (MMP2, MMP9, TIMP1, TIPM2) with key roles in Toll-like receptor signaling, mechanotransduction and extracellular matrix biology. MiR-424-3p, -503-5p, KLF4, PELI1 and YAP1 were identified as new deregulated molecular factors in GCA TABs. Quantities of miR-146a-5p, YAP1, PELI1, FGF2, TIMP2 and MMP9 were particularly high in histologically positive GCA TABs with occluded temporal artery lumen. MiR-424-5p expression in TABs and the presence of facial or carotid arteritis on ultrasound were associated with vision disturbances in GCA patients. Correlative analysis of miR-mRNA quantities demonstrated a highly interrelated expression network of deregulated miRs and mRNAs in temporal arteries and identified KLF4 as a candidate target gene of deregulated miR-21-5p, -146a-5p and -155-5p network in GCA TABs. Meanwhile, arterial miR and mRNA expression did not correlate with constitutive symptoms and signs of GCA, elevated markers of systemic inflammation nor sonographic characteristics of GCA. Our study provides new insights into GCA pathophysiology and uncovers new candidate biomarkers of vision impairment in GCA.

Contains fulltext : 252024.pdf (Publisher’s version ) (Open Access) Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.

New strategies to efficiently treat bacterial infections are crucial to circumvent the increase of resistant strains and to mitigate side effects during treatment. Skin and soft tissue infections represent one of the areas suffering the most from these resistant strains. We developed a new drug delivery system composed of the green algae, Chlamydomonas reinhardtii, which is generally recognized as safe, to target specifically skin diseases. A two‐step functionalization strategy was used to chemically modify the algae with the antibiotic vancomycin. Chlamydomonas reinhardtii was found to mask vancomycin and the insertion of a photocleavable linker was used for the release of the antibiotic. This living drug carrier was evaluated in presence of Bacillus subtilis and, only upon UVA1‐mediated release, growth inhibition of bacteria was observed. These results represent one of the first examples of a living organism used as a drug delivery system for the release of an antibiotic by UVA1‐irradiation., Algae control the release: A new approach for antibiotic caging and controlled release was developed using Chlamydomonas reinhardtii. The algae were covalently modified with vancomycin, and using a photocleavable linker, light‐mediated release of an antibacterial agent from the cell surface and inhibited the growth of Bacillus subtilis (see figure).

Ni/CoOx sites were supported on an around 2 nm-TiOx modified hematite photoanode for water oxidation. TiOx demonstrates insignificant hole accumulation and a catalytically inactive surface that serves as an ideal platform. We reveal that the NiOx favors the extraction of holes from the TiOx surface, which are efficiently transferred to active CoOx for water oxidation.

Low back pain (LBP) has been among the leading causes of disability for the past 30 years. This highlights the need for improvement in LBP management. Many clinical trials focus on developing treatments against degenerative disc disease (DDD). The multifactorial etiology of DDD and associated risk factors lead to a heterogeneous patient population. It comes as no surprise that the outcomes of clinical trials on intradiscal mesenchymal stem cell (MSC) injections for patients with DDD are inconsistent. Intradiscal MSC injections have demonstrated substantial pain relief and significant disability-related improvements, yet they have failed to regenerate the intervertebral disc (IVD). Increasing evidence suggests that the positive outcomes in clinical trials might be attributed to the immunomodulatory potential of MSCs rather than to their regenerative properties. Therefore, patient stratification for inflammatory DDD phenotypes may (i) better serve the mechanisms of action of MSCs and (ii) increase the treatment effect. Modic type 1 changes—pathologic inflammatory, fibrotic changes in the vertebral bone marrow—are frequently observed adjacent to degenerated IVDs in chronic LBP patients and represent a clinically distinct subpopulation of patients with DDD. This review discusses whether degenerated IVDs of patients with Modic type 1 changes should be treated with an intradiscal MSC injection.

A variety of different nanomaterials (NMs) such as microbubbles (MBs), nanobubbles (NBs), nanodroplets (NDs), and silica hollow meso-structures have been tested as ultrasound contrast agents for the detection of heart diseases. The inner part of these NMs is made gaseous to yield an ultrasound contrast, which arises from the difference in acoustic impedance between the interior and exterior of such a structure. Furthermore, to specifically achieve a contrast in the diseased heart region (DHR), NMs can be designed to target this region in essentially three different ways (i.e., passively when NMs are small enough to diffuse through the holes of the vessels supplying the DHR, actively by being associated with a ligand that recognizes a receptor of the DHR, or magnetically by applying a magnetic field orientated in the direction of the DHR on a NM responding to such stimulus). The localization and resolution of ultrasound imaging can be further improved by applying ultrasounds in the DHR, by increasing the ultrasound frequency, or by using harmonic, sub-harmonic, or super-resolution imaging. Local imaging can be achieved with other non-gaseous NMs of metallic composition (i.e., essentially made of Au) by using photoacoustic imaging, thus widening the range of NMs usable for cardiac applications. These contrast agents may also have a therapeutic efficacy by carrying/activating/releasing a heart disease drug, by triggering ultrasound targeted microbubble destruction or enhanced cavitation in the DHR, for example, resulting in thrombolysis or helping to prevent heart transplant rejection.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing–remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS–RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies., International Journal of Molecular Sciences, 23 (3), ISSN:1422-0067

The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established microRNA Next-Generation-Sequencing Discovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.

Roux-en-Y gastric bypass (RYGB) surgery has been proven successful in weight loss and improvement of co-morbidities associated with obesity. Chronic complications such as malabsorption of micronutrients in up to 50% of patients underline the need for additional therapeutic approaches. We investigated systemic RYGB surgery effects in a liquid sucrose diet-induced rat obesity model. After consuming a diet supplemented with high liquid sucrose for eight weeks, rats underwent RYGB or control sham surgery. RYGB, sham pair-fed, and sham ad libitum-fed groups further continued on the diet after recovery. Notable alterations were revealed in microbiota composition, inflammatory markers, feces, liver, and plasma metabolites, as well as in brain neuronal activity post-surgery. Higher fecal 4-aminobutyrate (GABA) correlated with higher Bacteroidota and Enterococcus abundances in RYGB animals, pointing towards the altered enteric nervous system (ENS) and gut signaling. Favorable C-reactive protein (CRP), serine, glycine, and 3-hydroxybutyrate plasma profiles in RYGB rats were suggestive of reverted obesity risk. The impact of liquid sucrose diet and caloric restriction mainly manifested in fatty acid changes in the liver. Our multi-modal approach reveals complex systemic changes after RYGB surgery and points towards potential therapeutic targets in the gut-brain system to mimic the surgery mode of action.

International audience; Single-molecule Förster resonance energy transfer (FRET) is a versatile technique for probing the structure and dynamics of biomolecules even in heterogeneous ensembles. However, due to the limited fluorescence brightness per molecule and the relatively long fluorescence lifetimes, probing ultrafast structural dynamics in the nanosecond timescale has thus far been very challenging. Here we demonstrate that nanophotonic fluorescence enhancement in zero-mode waveguides enables measurements of previously inaccessible low-nanosecond dynamics by dramatically improving time resolution and reduces data acquisition times by more than an order of magnitude. As a prototypical example, we use this approach to probe the dynamics of a short intrinsically disordered peptide that were previously inaccessible with single-molecule FRET measurements. We show that we are now able to detect the low-nanosecond correlations in this peptide, and we obtain a detailed interpretation of the underlying distance distributions and dynamics in conjunction with all-atom molecular dynamics simulations, which agree remarkably well with the experiments. We expect this combined approach to be widely applicable to the investigation of very rapid biomolecular dynamics.

Little is known regarding the interplay between microbiota and pancreas functions in humans as investigations are usually limited to distal sites, namely the analyses of fecal samples. The aim of this study was to investigate both ileal and fecal microbiota in response to pancreatic enzyme replacement therapy (PERT) in a porcine model of exocrine pancreatic insufficiency (EPI). PERT was stopped for ten days in ileo-cecal fistulated minipigs with experimentally induced EPI (n = 8) and ileal digesta as well as fecal samples were obtained before withdrawal, during withdrawal and after the reintroduction of PERT. Profound community changes occurred three days after enzyme omission and were maintained throughout the withdrawal phase. A reduction in α-diversity together with relative abundance changes in several taxa, in particular increases in Bifidobacteria (at both sites) and Lactobacilli (only feces) were observed. Overall, dysbiosis events from the ileum had accumulating effects in distal parts of the gastrointestinal tract with additional alterations occurring only in the colon. Changes were reversible after continuing PERT, and one week later, bacterial communities resembled those at baseline. Our study demonstrates the rapid and profound impacts of enzyme withdrawal in bacterial communities, contributing to our understanding of the interplay between pancreas function and microbiota.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant Mrps5G315R/G315R mice did not show a phenotype distinct from wild-type animals., International Journal of Molecular Sciences, 23 (8), ISSN:1422-0067

Triangulene is the smallest non-Kekulé graphene fragment known as Clar's hydrocarbon. Due to its open-shell electronic structure, triangulene is a promising molecular building block of carbon-based organic materials for spintronics and quantum molecular science. It comprises six benzenoid rings arranged in a triangular shape with two unpaired electrons delocalized over the entire conjugated core, making this molecule highly reactive. A triplet ground state is predicted for this hydrocarbon by Ovchinnikov's rule, or Lieb's theorem, in accord with Hund's rule. The pioneering work on triangulene was performed almost 70 years ago by Erich Clar, who attempted to prepare the pristine compound. Since then, several synthetic approaches to prepare this molecule have been exploited. The extreme reactivity of triangulene can be circumvented using on-surface techniques or by installation of sterically demanding substituents, which kinetically stabilize the diradical core against oligomerization in solution. The first two examples of a persistent derivative of triangulene were simultaneously and independently developed last year. This article presents a historical development in the synthesis of triangulene and its derivatives and outlines possible future applications in ferromagnetic materials, electrically conductive polymers or quantum computing.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up-or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms., International Journal of Molecular Sciences, 23 (3), ISSN:1422-0067

The spectroscopy and structural dynamics of a deep eutectic mixture (KSCN/acetamide) with varying water content is investigated from 2D IR (with the C-N stretch vibration of the SCN$^-$ anions as the reporter) and THz spectroscopy. Molecular dynamics simulations correctly describe the non-trivial dependence of both spectroscopic signatures depending on water content. For the 2D IR spectra, the MD simulations relate the steep increase in the cross relaxation rate at high water content to parallel alignment of packed SCN$^-$ anions. Conversely, the non-linear increase of the THz absorption with increasing water content is mainly attributed to the formation of larger water clusters. The results demonstrate that a combination of structure sensitive spectroscopies and molecular dynamics simulations provides molecular-level insights into emergence of heterogeneity of such mixtures by modulating their composition.

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-β signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC.