Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

Polarization-Selective Enhancement of Telecom Wavelength Quantum Dot Transitions in an Elliptical Bullseye Resonator.

Semiconductor quantum dots are promising candidates for the generation of nonclassical light. Coupling a quantum dot to a device capable of providing polarization-selective enhancement of optical transitions is highly beneficial for advanced functionalities, such as efficient resonant driving schemes or applications based on optical cyclicity. Here, we demonstrate broadband polarization-selective enhancement by coupling a quantum dot emitting in the telecom O-band to an elliptical bullseye resonator. We report bright single-photon emission with a degree of linear polarization of 96%, Purcell factor of 3.9 ± 0.6, and count rates up to 3 MHz. Furthermore, we present a measurement of two-photon interference without any external polarization filtering. Finally, we demonstrate compatibility with compact Stirling cryocoolers by operating the device at temperatures up to 40 K. These results represent an important step toward practical integration of optimal quantum dot photon sources in deployment-ready setups.

Design study for an efficient semiconductor quantum light source operating in the telecom C-band based on an electrically-driven circular Bragg grating.

The development of efficient sources of single photons and entangled photon pairs emitting in the low-loss wavelength region around 1550 nm is crucial for long-distance quantum communication. Moreover, direct fiber coupling and electrical carrier injection are highly desirable for deployment in compact and user-friendly systems integrated with the existing fiber infrastructure. Here we present a detailed design study of circular Bragg gratings fabricated in InP slabs and operating in the telecom C-band. These devices enable the simultaneous enhancement of the X and XX spectral lines, with collection efficiency in numerical aperture 0.65 close to 90% for the wavelength range 1520 - 1580 nm and Purcell factor up to 15. We also investigate the coupling into a single mode fiber, which exceeds 70% in UHNA4. Finally, we propose a modified device design directly compatible with electrical carrier injection, reporting Purcell factors up to 20 and collection efficiency in numerical aperture 0.65 close to 70% for the whole telecom C-band.

Development and validation of a QTrap method for sensitive quantification of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid and ligand for five G protein-coupled cell-surface receptors designated S1PR1-5. The determination of low levels of S1P remains a challenge and usually requires sophisticated analytical instrumentation and methodology. This report describes a technique using the linear ion trap mode of a basic QTrap triple-quadrupole mass spectrometer. S1P was extracted from acidified biological samples using a modified Folch extraction procedure. After the addition of C17-sphingosine as an internal standard, a step gradient LC method was used to separate the analytes on a reversed-phase C18 MultoHigh analytical column. After the internal standard C17-sphingosine was detected by multiple reaction monitoring (MRM), the detection mode was switched to enhanced product ion (EPI) mode for the detection of S1P. The mode was switched back to MRM again for the detection of other analytes. Using this QTrap method, we reached a limit of detection of 1 nM and a limit of quantification of 3 nM for S1P, which was up to 30 times more sensitive than the MRM mode with the same instrument. Intra-day precision ranged between -3.8 and 6.3%, and inter-day precision was between -13.8 and 3.3%, depending on the spiked S1P concentration.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

1GHz clocked distribution of electrically generated entangled photon pairs.

Quantum networks are essential for realising distributed quantum computation and quantum communication. Entangled photons are a key resource, with applications such as quantum key distribution, quantum relays, and quantum repeaters. All components integrated in a quantum network must be synchronised and therefore comply with a certain clock frequency. In quantum key distribution, the most mature technology, clock rates have reached and exceeded 1GHz. Here we show the first electrically pulsed sub-Poissonian entangled photon source compatible with existing fiber networks operating at this clock rate. The entangled LED is based on InAs/InP quantum dots emitting in the main telecom window, with a multi-photon probability of less than 10% per emission cycle and a maximum entanglement fidelity of 89%. We use this device to demonstrate GHz clocked distribution of entangled qubits over an installed fiber network between two points 4.6km apart.

Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells.

Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1293) is similar to the protein produced in bacteria (ALKBH1Ec) with regard to its m6A demethylase and AP lyase activities. In addition, we find that ALKBH1293 forms a covalent adduct with the 5' product of the lyase product in a manner analogous to ALKBH1Ec. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding.

The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of ALKBH7, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7's ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.

Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,ß-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

Simple delay-limited sideband locking with heterodyne readout.

We present a robust sideband laser locking technique ideally suited for applications requiring low probe power and heterodyne readout. By feeding back to a high-bandwidth voltage-controlled oscillator, we lock a first-order phase-modulation sideband to a high-finesse Fabry-Perot cavity in ambient conditions, achieving a closed-loop bandwidth of 3.5 MHz (with a single integrator) limited fundamentally by the signal delay. The measured transfer function of the closed loop agrees with a simple model based on ideal system components, and from this we suggest a modified design that should achieve a bandwidth exceeding 6 MHz with a near-causally limited feedback gain as high as 4 × 107 at 1 kHz. The off-resonance optical carrier enables alignment-free heterodyne readout, alleviating the need for additional lasers or optical modulators.

Calorimetric assessment of Fe(2+) binding to α-ketoglutarate/taurine dioxygenase: ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad.

The thermodynamic properties of Fe(2+) binding to the 2-His-1-carboxylate facial triad in α-ketoglutarate/taurine dioxygenase (TauD) were explored using isothermal titration calorimetry. Direct titrations of Fe(2+) into TauD and chelation experiments involving the titration of ethylenediaminetetraacetic acid into Fe(2+)-TauD were performed under an anaerobic environment to yield a binding equilibrium of 2.4 (±0.1) × 10(7) (Kd = 43 nM) and a ΔG° value of -10.1 (±0.03) kcal/mol. Further analysis of the enthalpy/entropy contributions indicates a highly enthalpic binding event, where ΔH = -11.6 (±0.3) kcal/mol. Investigations into the unfavorable entropy term led to the observation of water molecules becoming organized within the Fe(2+)-TauD structure.

Regorafenib (BAY 73-4506): antitumor and antimetastatic activities in preclinical models of colorectal cancer.

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.

Electronic structure of the silicon vacancy color center in diamond.

The negatively charged silicon vacancy (SiV) color center in diamond has recently proven its suitability for bright and stable single photon emission. However, its electronic structure so far has remained elusive. We here explore the electronic structure by exposing single SiV defects to a magnetic field where the Zeeman effect lifts the degeneracy of magnetic sublevels. The similar responses of single centers and a SiV ensemble in a low strain reference sample prove our ability to fabricate almost perfect single SiVs, revealing the true nature of the defect's electronic properties. We model the electronic states using a group-theoretical approach yielding a good agreement with the experimental observations. Furthermore, the model correctly predicts polarization measurements on single SiV centers and explains recently discovered spin selective excitation of SiV defects.

A covalent protein-DNA 5'-product adduct is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1).

ALKBH1 (AlkB homologue 1) is a mammalian AlkB (2-oxoglutarate-dependent dioxygenase) homologue that possesses AP (abasic or apurinic/apyrimidinic) lyase activity. The AP lyase reaction is catalysed by imine formation with an active site lysine residue, and a covalent intermediate can be trapped in the presence of NaBH4. Surprisingly, ALKBH1 also forms a stable protein-DNA adduct in the absence of a reducing agent. Experiments with different substrates demonstrated that the protein covalently binds to the 5' DNA product, i.e. the fragment containing an α,ß-unsaturated aldehyde. The N-terminal domain of ALKBH1 was identified as the main site of linkage with DNA. By contrast, mutagenesis studies suggest that the primary catalytic residue forming the imine linkage is Lys133, with Lys154 and other lysine residues in this region serving in opportunistic roles. These findings confirm the classification of ALKBH1 as an AP lyase, identify the primary and a secondary lysine residues involved in the lyase reaction, and demonstrate that the protein forms a covalent adduct with the 5' DNA product. We propose two plausible chemical mechanisms to account for the covalent attachment.

Cluster-localized sparse logistic regression for SNP data.

The task of analyzing high-dimensional single nucleotide polymorphism (SNP) data in a case-control design using multivariable techniques has only recently been tackled. While many available approaches investigate only main effects in a high-dimensional setting, we propose a more flexible technique, cluster-localized regression (CLR), based on localized logistic regression models, that allows different SNPs to have an effect for different groups of individuals. Separate multivariable regression models are fitted for the different groups of individuals by incorporating weights into componentwise boosting, which provides simultaneous variable selection, hence sparse fits. For model fitting, these groups of individuals are identified using a clustering approach, where each group may be defined via different SNPs. This allows for representing complex interaction patterns, such as compositional epistasis, that might not be detected by a single main effects model. In a simulation study, the CLR approach results in improved prediction performance, compared to the main effects approach, and identification of important SNPs in several scenarios. Improved prediction performance is also obtained for an application example considering urinary bladder cancer. Some of the identified SNPs are predictive for all individuals, while others are only relevant for a specific group. Together with the sets of SNPs that define the groups, potential interaction patterns are uncovered.

Sphingosine-1-phosphate improves outcome of no-reflow acute myocardial infarction via sphingosine-1-phosphate receptor 1.

AIMS: Therapeutic options targeting post-ischaemic cardiac remodelling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischaemia/reperfusion injury. However, its impact on post-ischaemic remodelling independently of its infarct size (IS)-reducing effect is yet unknown and was addressed in this study. METHODS AND RESULTS: Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl6 were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting 7 days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), murine echocardiography, histomorphology, and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+) and Cre- control mice and S1P concentrations measured by LC-MS/MS. IS and cardiac function did not differ between control and DOP-treated groups on day one after LAD-ligation despite fourfold increase in plasma S1P. In contrast, cardiac function was clearly improved and myocardial scar size reduced, respectively, on Day 21 in DOP-treated mice. The latter also exhibited smaller cardiomyocyte size and reduced embryonic gene expression. The benefit of DOP treatment was abolished in S1PR1 Cardio Cre+. CONCLUSIONS: S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size and accomplishes this via the cardiomyocyte S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodelling as adjunctive therapy to revascularization as well as in patients not eligible for standard interventional procedures.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction.

Antiplatelet medication is standard of care in acute myocardial infarction (AMI). However, it may have obscured beneficial properties of the activated platelet secretome. We identify platelets as major source of a sphingosine-1-phosphate (S1P) burst during AMI, and find its magnitude to favorably associate with cardiovascular mortality and infarct size in STEMI patients over 12 months. Experimentally, administration of supernatant from activated platelets reduces infarct size in murine AMI, which is blunted in platelets deficient for S1P export (Mfsd2b) or production (Sphk1) and in mice deficient for cardiomyocyte S1P receptor 1 (S1P1). Our study reveals an exploitable therapeutic window in antiplatelet therapy in AMI as the GPIIb/IIIa antagonist tirofiban preserves S1P release and cardioprotection, whereas the P2Y12 antagonist cangrelor does not. Here, we report that platelet-mediated intrinsic cardioprotection is an exciting therapeutic paradigm reaching beyond AMI, the benefits of which may need to be considered in all antiplatelet therapies.

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

Systematic Metabolic Profiling Identifies De Novo Sphingolipid Synthesis as Hypha Associated and Essential for Candida albicans Filamentation.

The yeast-to-hypha transition is a key virulence attribute of the opportunistic human fungal pathogen Candida albicans, since it is closely tied to infection-associated processes such as tissue invasion and escape from phagocytes. While the nature of hypha-associated gene expression required for fungal virulence has been thoroughly investigated, potential morphotype-dependent activity of metabolic pathways remained unclear. Here, we combined global transcriptome and metabolome analyses for the wild-type SC5314 and the hypha-defective hgc1Δ and cph1Δefg1Δ strains under three hypha-inducing (human serum, N-acetylglucosamine, and alkaline pH) and two yeast-promoting conditions to identify metabolic adaptions that accompany the filamentation process. We identified morphotype-related activities of distinct pathways and a metabolic core signature of 26 metabolites with consistent depletion or enrichment during the yeast-to-hypha transition. Most strikingly, we found a hypha-associated activation of de novo sphingolipid biosynthesis, indicating a connection of this pathway and filamentous growth. Consequently, pharmacological inhibition of this partially fungus-specific pathway resulted in strongly impaired filamentation, verifying the necessity of de novo sphingolipid biosynthesis for proper hypha formation. IMPORTANCE The reversible switch of Candida albicans between unicellular yeast and multicellular hyphal growth is accompanied by a well-studied hypha-associated gene expression, encoding virulence factors like adhesins, toxins, or nutrient scavengers. The investigation of this gene expression consequently led to fundamental insights into the pathogenesis of this fungus. In this study, we applied this concept to hypha-associated metabolic adaptations and identified morphotype-dependent activities of distinct pathways and a stimulus-independent metabolic signature of hyphae. Most strikingly, we found the induction of de novo sphingolipid biosynthesis as hypha associated and essential for the filamentation of C. albicans. These findings verified the presence of morphotype-specific metabolic traits in the fungus, which appear connected to the fungal virulence. Furthermore, the here-provided comprehensive description of the fungal metabolome will help to foster future research and lead to a better understanding of fungal physiology.

Factors determining the success of radiofrequency denervation in lumbar facet joint pain: a prospective study.

BACKGROUND AND AIMS: Radiofrequency denervation (RF) of the lumbar facet joints has been shown to be effective in well-selected patients. However, long-term success varies between studies. We evaluated the influence of selected psychosocial and constitutional factors on the outcome of RF, expressed as the duration of pain relief. METHODS: This prospective observational study included 44 patients who received RF denervations at the University Hospital of Berne. Success was defined as at least 50% pain reduction 7-21 days, 6 months and 1 year after RF therapy. The Cox-regression analysis was performed to evaluate the influence of the following factors on the duration of success: age, sex, depression, work inability and previous surgery. RESULTS: Complete follow-up was available for 41 patients. The success rate 7-21 days after the denervation was 76%. It decreased to 32% at 6 months and to 22% at 1 year. The median success duration was 17 weeks (95% CI 10-26). The Cox-regression analysis showed a significant shorter duration of success for patients with depression (hazard ratio [HR] 2.97, 95% CI 1.32-6.65), previous surgery (HR 2.39, 95% CI 1.10-5.21) and number of treated joints (HR 1.95 for each increase in the number of joints, 95% CI 1.14-3.33). In bivariate analyses, only depression was kept to be significant. CONCLUSIONS: Depression seems to be related with a short duration of success. Based on these findings, a comprehensive study is warranted to evaluate whether psychosocial factors have to be considered when recruiting patients for radiofrequency denervation.