Characterization of the dehydrogenase-reductase DHRS2 and its involvement in histone deacetylase inhibition in urological malignancies.

BACKGROUND: Being implicated during tumor migration, invasion, clonogenicity, and proliferation, the nicotinamide adenine dinucleotide (NAD)/-phosphate (NADP)-dependent dehydrogenase/reductase member 2 (DHRS2) has been considered to be induced upon inhibition of histone deacetylases (HDACi). In this study, we evaluated the current knowledge on the underlying mechanisms of the (epi)genetic regulation of DHRS2, as well as its function during tumor progression. METHODS: DHRS2 expression was evaluated on mRNA- and protein-level upon treatment with HDACi by means of qRT-PCR and western blot analyses, respectively. Re-analysis of RNA-sequencing data gained insight into expression of specific DHRS2 isoforms, while re-analysis of ATAC-sequencing data shed light on the chromatin accessibility at the DHRS2 locus. Further examination of the energy and lipid metabolism of HDACi-treated urologic tumor cells was performed using liquid chromatography-mass spectrometry. RESULTS: Enhanced DHRS2 expression levels upon HDACi treatment were directly linked to an enhanced chromatin accessibility at the DHRS2 locus. Particularly the DHRS2 ENST00000250383.11 protein-coding isoform was increased upon HDACi treatment. Application of the HDACi quisinostat only mildly influenced the energy metabolism of urologic tumor cells, though, the analysis of the lipid metabolism showed diminished sphingosine levels, as well as decreased S1P levels. Also the ratios of S1P/sphingosine and S1P/ceramides were reduced in all four quisinostat-treated urologic tumor cells. CONCLUSIONS: With the emphasis on urologic malignancies (testicular germ cell tumors, urothelial, prostate, and renal cell carcinoma), this study concluded that elevated DHRS2 levels are indicative of a successful HDACi treatment and, thereby offering a novel putative predictive biomarker.

Average power scaling of THz spintronic emitters efficiently cooled in reflection geometry.

Metallic spintronic terahertz (THz) emitters have become well-established for offering ultra-broadband, gapless THz emission in a variety of excitation regimes, in combination with reliable fabrication and excellent scalability. However, so far, their potential for high-average-power excitation to reach strong THz fields at high repetition rates has not been thoroughly investigated. In this article, we explore the power scaling behavior of tri-layer spintronic emitters using an Yb-fiber excitation source, delivering an average power of 18.5 W (7 W incident on the emitter after chopping) at 400 kHz repetition rate, temporally compressed to a pulse duration of 27 fs. We confirm that a reflection geometry with back-side cooling is ideally suited for these emitters in the high-average-power excitation regime. In order to understand limiting mechanisms, we disentangle the effects on THz power generation by average power and pulse energy by varying the repetition rate of the laser. Our results show that the conversion efficiency is predominantly determined by the incident fluence in this high-average-power, high-repetition-rate excitation regime if the emitters are efficiently cooled. Using these findings, we optimize the conversion efficiency and reach highest excitation powers in the back-cooled reflection geometry. Our findings provide guidelines for scaling the power of THz radiation emitted by spintronic emitters to the milliwatt-level by using state-of-the-art femtosecond sources with multi-hundred-Watt average power to reach ultra-broadband, strong-field THz sources with high repetition rate.

Nanoscale coherent phonon spectroscopy.

Coherent phonon spectroscopy can provide microscopic insight into ultrafast lattice dynamics and its coupling to other degrees of freedom under nonequilibrium conditions. Ultrafast optical spectroscopy is a well-established method to study coherent phonons, but the diffraction limit has hampered observing their local dynamics directly. Here, we demonstrate nanoscale coherent phonon spectroscopy using ultrafast laser-induced scanning tunneling microscopy in a plasmonic junction. Coherent phonons are locally excited in ultrathin zinc oxide films by the tightly confined plasmonic field and are probed via the photoinduced tunneling current through an electronic resonance of the zinc oxide film. Concurrently performed tip-enhanced Raman spectroscopy allows us to identify the involved phonon modes. In contrast to the Raman spectra, the phonon dynamics observed in coherent phonon spectroscopy exhibit strong nanoscale spatial variations that are correlated with the distribution of the electronic local density of states resolved by scanning tunneling spectroscopy.

Femtosecond Thermal and Nonthermal Hot Electron Tunneling Inside a Photoexcited Tunnel Junction.

Efficient operation of electronic nanodevices at ultrafast speeds requires understanding and control of the currents generated by femtosecond bursts of light. Ultrafast laser-induced currents in metallic nanojunctions can originate from photoassisted hot electron tunneling or lightwave-induced tunneling. Both processes can drive localized photocurrents inside a scanning tunneling microscope (STM) on femto- to attosecond time scales, enabling ultrafast STM with atomic spatial resolution. Femtosecond laser excitation of a metallic nanojunction, however, also leads to the formation of a transient thermalized electron distribution, but the tunneling of thermalized hot electrons on time scales faster than electron-lattice equilibration is not well understood. Here, we investigate ultrafast electronic heating and transient thermionic tunneling inside a metallic photoexcited tunnel junction and its role in the generation of ultrafast photocurrents in STM. Phase-resolved sampling of broadband terahertz (THz) pulses via the THz-field-induced modulation of ultrafast photocurrents allows us to probe the electronic temperature evolution inside the STM tip and to observe the competition between instantaneous and delayed tunneling due to nonthermal and thermal hot electron distributions in real time. Our results reveal the pronounced nonthermal character of photoinduced hot electron tunneling and provide a detailed microscopic understanding of hot electron dynamics inside a laser-excited tunnel junction.

Establishment and Evaluation of Dual HDAC/BET Inhibitors as Therapeutic Options for Germ Cell Tumors and Other Urological Malignancies.

Urological malignancies represent major challenges for clinicians, with annually rising incidences. In addition, cisplatin treatment induced long-term toxicities and the development of therapy resistance emphasize the need for novel therapeutics. In this study, we analyzed the effects of novel histone deacetylase (HDAC) and bromodomain and extraterminal domain-containing (BET) inhibitors to combine them into a potent HDAC-BET-fusion molecule and to understand their molecular mode-of-action. Treatment of (cisplatin-resistant) germ cell tumors (GCT), urothelial, renal, and prostate carcinoma cells with the HDAC, BET, and dual inhibitors decreased cell viability, induced apoptosis, and affected the cell cycle. Furthermore, a dual inhibitor considerably decreased tumor burden in GCT xenograft models. On a molecular level, correlating RNA- to ATAC-sequencing data indicated a considerable induction of gene expression, accompanied by site-specific changes of chromatin accessibility after HDAC inhibitor application. Upregulated genes could be linked to intra- and extra-cellular trafficking, cellular organization, and neuronal processes, including neuroendocrine differentiation. Regarding chromatin accessibility on a global level, an equal distribution of active or repressed DNA accessibility has been detected after HDAC inhibitor treatment, questioning the current understanding of HDAC inhibitor function. In summary, our HDAC, BET, and dual inhibitors represent a new treatment alternative for urological malignancies. Furthermore, we shed light on new molecular and epigenetic mechanisms of the tested epi-drugs, allowing for a better understanding of the underlying modes-of-action and risk assessment for the patient.

Procalcitonin mediates vascular dysfunction in obesity.

AIMS: Obesity is accompanied by a chronic low-grade inflammation associated with endothelial dysfunction and vascular complications. Procalcitonin is a marker of inflammation, secreted by adipose tissue and elevated in obese subjects. We here investigated whether visceral or perivascular fat-derived procalcitonin is a target to improve obesity-induced endothelial dysfunction. MATERIALS AND METHODS: Procalcitonin expression was identified by Western blot. Murine endothelial cells were isolated using CD31-antibody-coated magnetic beads and reactive oxygen species and nitric oxide (NO) determined by H2DCF- or DAF-FM diacetate loading. Endothelium-dependent vasorelaxation was analyzed using pressure myography of murine arterioles. Calcitonin gene-related peptide (CGRP) was used to activate the calcitonin receptor-like receptor (CRLR)/RAMP1 complex and olcegepant or the dipeptidyl-peptidase 4 (DPP4) inhibitor sitagliptin to block procalcitonin signaling or activation. KEY FINDINGS: In addition to visceral adipose tissue, procalcitonin was present in perivascular and epicardial tissue. In concentrations typical for obesity, procalcitonin doubled reactive oxygen species formation and decreased endothelial nitric oxide production in murine endothelial cells. Intravenous delivery of procalcitonin to mice in obesity-associated concentrations impaired endothelium-dependent vasorelaxation in a CRLR/RAMP1-dependent manner and antagonized CGRP-induced endothelial NO release in vitro. Use of CRLR/RAMP1-receptor antagonist olcegepant counteracted procalcitonin effects on vasodilation, nitric oxide production and reactive oxygen species formation. Similarly, blocking procalcitonin activation by the DPP4 inhibitor sitagliptin antagonized endothelial procalcitonin effects. SIGNIFICANCE: Procalcitonin, liberated either from visceral or perivascular adipose tissue, contributes to endothelial dysfunction by antagonizing CGRP signaling in obesity. Targeting hyperprocalcitonemia may be a means to preserve endothelial function and reduce comorbidity burden in obese subjects.

Molecular View into Preferential Binding of the Factor VII Gla Domain to Phosphatidic Acid.

Factor VII (FVII) is a serine protease with a key role in initiating the coagulation cascade. It is part of a family of vitamin K-dependent clotting proteins, which require vitamin K for formation of their specialized membrane-binding domains (Gla domains). Membrane binding of the FVII Gla domain is critical to the activity of FVII, mediating the formation of its complex with other clotting factors. While Gla domains among coagulation factors are highly conserved in terms of amino acid sequence and structure, they demonstrate differential binding specificity toward anionic lipids. Although most Gla domain-containing clotting proteins display a strong preference for phosphatidylserine (PS), it has been demonstrated that FVII and protein C instead bind preferentially to phosphatidic acid (PA). We have developed the first model of the FVII Gla domain bound to PA lipids in membranes containing PA, the highly mobile membrane mimetic model, which accelerates slow diffusion of lipids in molecular dynamics simulations and therefore facilitates the membrane binding process and enhances sampling of lipid interactions. Simulations were performed using atomic level molecular dynamics, requiring a fixed charge to all atoms. The overall charge assigned to each PA lipid for this study was -1. We also developed an additional model of the FVII Gla domain bound to a 1:1 PS/PC membrane and compared the modes of binding of PS and PA lipids to FVII, allowing us to identify potential PA-specific binding sites.

Targeting Procalcitonin Protects Vascular Barrier Integrity.

Rationale: Capillary leakage frequently occurs during sepsis and after major surgery and is associated with microvascular dysfunction and adverse outcome. Procalcitonin is a well-established biomarker in inflammation without known impact on vascular integrity. Objectives: We determined how procalcitonin induces endothelial hyperpermeability and how targeting procalcitonin protects vascular barrier integrity. Methods: In a prospective observational clinical study, procalcitonin levels were assessed in 50 patients who underwent cardiac surgery and correlated to postoperative fluid and vasopressor requirements along with sublingual microvascular functionality. Effects of the procalcitonin signaling pathway on endothelial barrier and adherens junctional integrity were characterized in vitro and verified in mice. Inhibition of procalcitonin activation by dipeptidyl-peptidase 4 (DPP4) was evaluated in murine polymicrobial sepsis and clinically verified in cardiac surgery patients chronically taking the DPP4 inhibitor sitagliptin. Measurements and Main Results: Elevated postoperative procalcitonin levels identified patients with 2-fold increased fluid requirements (P < 0.01), 1.8-fold higher vasopressor demand (P < 0.05), and compromised microcirculation (reduction to 63.5 ± 2.8% of perfused vessels, P < 0.05). Procalcitonin induced 1.4-fold endothelial and 2.3-fold pulmonary capillary permeability (both Ps < 0.001) by destabilizing VE-cadherin. Procalcitonin effects were dependent on activation by DPP4, and targeting the procalcitonin receptor or DPP4 during sepsis-induced hyperprocalcitonemia reduced capillary leakage by 54 ± 10.1% and 60.4 ± 6.9% (both Ps < 0.01), respectively. Sitagliptin before cardiac surgery was associated with augmented microcirculation (74.1 ± 1.7% vs. 68.6 ± 1.9% perfused vessels in non-sitagliptin-medicated patients, P < 0.05) and with 2.3-fold decreased fluid (P < 0.05) and 1.8-fold reduced vasopressor demand postoperatively (P < 0.05). Conclusions: Targeting procalcitonin's action on the endothelium is a feasible means to preserve vascular integrity during systemic inflammation associated with hyperprocalcitonemia.

Orf Virus-Based Vectors Preferentially Target Professional Antigen-Presenting Cells, Activate the STING Pathway and Induce Strong Antigen-Specific T Cell Responses.

Background: Orf virus (ORFV)-based vectors are attractive for vaccine development as they enable the induction of potent immune responses against specific transgenes. Nevertheless, the precise mechanisms of immune activation remain unknown. This study therefore aimed to characterize underlying mechanisms in human immune cells. Methods: Peripheral blood mononuclear cells were infected with attenuated ORFV strain D1701-VrV and analyzed for ORFV infection and activation markers. ORFV entry in susceptible cells was examined using established pharmacological inhibitors. Using the THP1-Dual™ reporter cell line, activation of nuclear factor-κB and interferon regulatory factor pathways were simultaneously evaluated. Infection with an ORFV recombinant encoding immunogenic peptides (PepTrio-ORFV) was used to assess the induction of antigen-specific CD8+ T cells. Results: ORFV was found to preferentially target professional antigen-presenting cells (APCs) in vitro, with ORFV uptake mediated primarily by macropinocytosis. ORFV-infected APCs exhibited an activated phenotype, required for subsequent lymphocyte activation. Reporter cells revealed that the stimulator of interferon genes pathway is a prerequisite for ORFV-mediated cellular activation. PepTrio-ORFV efficiently induced antigen-specific CD8+ T cell recall responses in a dose-dependent manner. Further, activation and expansion of naïve antigen-specific CD8+ T cells was observed in response. Discussion: Our findings confirm that ORFV induces a strong antigen-specific immune response dependent on APC uptake and activation. These data support the notion that ORFV D1701-VrV is a promising vector for vaccine development and the design of innovative immunotherapeutic applications.

Therapeutical interference with the epigenetic landscape of germ cell tumors: a comparative drug study and new mechanistical insights.

BACKGROUND: Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs. RESULTS: We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers (MZ-1, LP99) and polycomb-repressive complexes (PRT4165, GSK343). Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT. CONCLUSION: Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

A Novel Orf Virus D1701-VrV-Based Dengue Virus (DENV) Vaccine Candidate Expressing HLA-Specific T Cell Epitopes: A Proof-of-Concept Study.

Although dengue virus (DENV) affects almost half of the world's population there are neither preventive treatments nor any long-lasting and protective vaccines available at this time. The complexity of the protective immune response to DENV is still not fully understood. The most advanced vaccine candidates focus specifically on humoral immune responses and the production of virus-neutralizing antibodies. However, results from several recent studies have revealed the protective role of T cells in the immune response to DENV. Hence, in this study, we generated a novel and potent DENV vaccine candidate based on an Orf virus (ORFV, genus Parapoxvirus) vector platform engineered to encode five highly conserved or cross-reactive DENV human leukocyte antigen (HLA)-A*02- or HLA-B*07-restricted epitopes as minigenes (ORFV-DENV). We showed that ORFV-DENV facilitates the in vitro priming of CD8+ T cells from healthy blood donors based on responses to each of the encoded immunogenic peptides. Moreover, we demonstrated that peripheral blood mononuclear cells isolated from clinically confirmed DENV-positive donors stimulated with ORFV-DENV generate cytotoxic T cell responses to at least three of the expressed DENV peptides. Finally, we showed that ORFV-DENV could activate CD8+ T cells isolated from donors who had recovered from Zika virus (ZIKV) infection. ZIKV belongs to the same virus family (Flaviviridae) and has epitope sequences that are homologous to those of DENV. We found that highly conserved HLA-B*07-restricted ZIKV and DENV epitopes induced functional CD8+ T cell responses in PBMCs isolated from confirmed ZIKV-positive donors. In summary, this proof-of-concept study characterizes a promising new ORFV D1701-VrV-based DENV vaccine candidate that induces broad and functional epitope-specific CD8+ T cell responses.

Development of heart failure with preserved ejection fraction in type 2 diabetic mice is ameliorated by preserving vascular function.

AIMS: Heart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction and is frequent in people with type 2 diabetes mellitus. In diabetic patients, increased levels of the eicosanoid 12-hydroxyeicosatetraenoic acid (12-HETE) are linked to vascular dysfunction. Here, we aimed to identify the importance of 12-HETE in type 2 diabetic patients exhibiting diastolic dysfunction, and mice exhibiting HFpEF and whether targeting 12-HETE is a means to ameliorate HFpEF progression by improving vascular function in diabetes. MATERIAL AND METHODS: Subjects with diagnosed type 2 diabetes mellitus and reported diastolic dysfunction or healthy controls were recruited and 12(S)-HETE levels determined by ELISA. 12(S)-HETE levels were determined in type 2 diabetic, leptin receptor deficient mice (LepRdb/db) and HFpEF verified by echocardiography. Mitochondrial function, endothelial function and capillary density were assessed using Seahorse technique, pressure myography and immunohistochemistry in LepRdb/db or non-diabetic littermate controls. 12/15Lo generation was inhibited using ML351 and 12(S)-HETE action by using the V1-cal peptide. KEY FINDINGS: Endothelium-dependent vasodilation and mitochondrial functional capacity both improved in response to either application of ML351 or the V1-cal peptide. Correlating to improved vascular function, mice treated with either pharmacological agent exhibited improved diastolic filling and left ventricular relaxation that correlated with increased myocardial capillary density. SIGNIFICANCE: Our results suggest that 12-HETE may serve as a biomarker indicating endothelial dysfunction and the resulting cardiovascular consequences such as HFpEF in type 2 diabetic patients. Antagonizing 12-HETE is a potent means to causally control HFpEF development and progression in type 2 diabetes by preserving vascular function.

Molecular and epigenetic pathogenesis of germ cell tumors.

The development of germ cell tumors (GCTs) is a unique pathogenesis occurring at an early developmental stage during specification, migration or colonization of primordial germ cells (PGCs) in the genital ridge. Since driver mutations could not be identified so far, the involvement of the epigenetic machinery during the pathogenesis seems to play a crucial role. Currently, it is investigated whether epigenetic modifications occurring between the omnipotent two-cell stage and the pluripotent implanting PGCs might result in disturbances eventually leading to GCTs. Although progress in understanding epigenetic mechanisms during PGC development is ongoing, little is known about the complete picture of its involvement during GCT development and eventual classification into clinical subtypes. This review will shed light into the current knowledge of the complex epigenetic and molecular contribution during pathogenesis of GCTs by emphasizing on early developmental stages until arrival of late PGCs in the gonads. We questioned how misguided migrating and/or colonizing PGCs develop to either type I or type II GCTs. Additionally, we asked how pluripotency can be regulated during PGC development and which epigenetic changes contribute to GCT pathogenesis. We propose that SOX2 and SOX17 determine either embryonic stem cell-like (embryonal carcinoma) or PGC-like cell fate (seminoma). Finally, we suggest that factors secreted by the microenvironment, i.e. BMPs and BMP inhibiting molecules, dictate the fate decision of germ cell neoplasia in situ (into seminoma and embryonal carcinoma) and seminomas (into embryonal carcinoma or extraembryonic lineage), indicating an important role of the microenvironment on GCT plasticity.

Dietary spermidine improves cognitive function.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Phase-Resolved Detection of Ultrabroadband THz Pulses inside a Scanning Tunneling Microscope Junction.

Coupling phase-stable single-cycle terahertz (THz) pulses to scanning tunneling microscope (STM) junctions enables spatiotemporal imaging with femtosecond temporal and Ångstrom spatial resolution. The time resolution achieved in such THz-gated STM is ultimately limited by the subcycle temporal variation of the tip-enhanced THz field acting as an ultrafast voltage pulse, and hence by the ability to feed high-frequency, broadband THz pulses into the junction. Here, we report on the coupling of ultrabroadband (1-30 THz) single-cycle THz pulses from a spintronic THz emitter (STE) into a metallic STM junction. We demonstrate broadband phase-resolved detection of the THz voltage transient directly in the STM junction via THz-field-induced modulation of ultrafast photocurrents. Comparison to the unperturbed far-field THz waveform reveals the antenna response of the STM tip. Despite tip-induced low-pass filtering, frequencies up to 15 THz can be detected in the tip-enhanced near-field, resulting in THz transients with a half-cycle period of 115 fs. We further demonstrate simple polarity control of the THz bias via the STE magnetization and show that up to 2 V THz bias at 1 MHz repetition rate can be achieved in the current setup. Finally, we find a nearly constant THz voltage and waveform over a wide range of tip-sample distances, which by comparison to numerical simulations confirms the quasi-static nature of the THz pulses. Our results demonstrate the suitability of spintronic THz emitters for ultrafast THz-STM with unprecedented bandwidth of the THz bias and provide insight into the femtosecond response of defined nanoscale junctions.

Dramatic Enhancement of Tip-Enhanced Raman Scattering Mediated by Atomic Point Contact Formation.

Tip-enhanced Raman scattering (TERS) in ångström-scale plasmonic cavities has drawn increasing attention. However, Raman scattering at vanishing cavity distances remains unexplored. Here, we show the evolution of TERS in transition from the tunneling regime to atomic point contact (APC). A stable APC is reversibly formed in the junction between an Ag tip and ultrathin ZnO or NaCl films on the Ag(111) surface at 10 K. An abrupt increase of the TERS intensity occurs upon APC formation for ZnO, but not for NaCl. This remarkable observation is rationalized by a difference in hybridization between the Ag tip and these films, which determines the contribution of charge transfer enhancement in the fused plasmonic junction. The strong hybridization between the Ag tip and ZnO is corroborated by the appearance of a new vibrational mode upon APC formation, whereas it is not observed for the chemically inert NaCl.

Orf Virus-Based Vaccine Vector D1701-V Induces Strong CD8+ T Cell Response against the Transgene but Not against ORFV-Derived Epitopes.

The potency of viral vector-based vaccines depends on their ability to induce strong transgene-specific immune response without triggering anti-vector immunity. Previously, Orf virus (ORFV, Parapoxvirus) strain D1701-V was reported as a novel vector mediating protection against viral infections. The short-lived ORFV-specific immune response and the absence of virus neutralizing antibodies enables repeated immunizations and enhancement of humoral immune responses against the inserted antigens. However, only limited information exists about the D1701-V induced cellular immunity. In this study we employed major histocompatibility complex (MHC) ligandomics and immunogenicity analysis to identify ORFV-specific epitopes. Using liquid chromatography-tandem mass spectrometry we detected 36 ORFV-derived MHC I peptides, originating from various proteins. Stimulated splenocytes from ORFV-immunized mice did not exhibit specific CD8+ T cell responses against the tested peptides. In contrast, immunization with ovalbumin-expressing ORFV recombinant elicited strong SIINFEKL-specific CD8+ T lymphocyte response. In conclusion, our data indicate that cellular immunity to the ORFV vector is negligible, while strong CD8+ T cell response is induced against the inserted transgene. These results further emphasize the ORFV strain D1701-V as an attractive vector for vaccine development. Moreover, the presented experiments describe prerequisites for the selection of T cell epitopes exploitable for generation of ORFV-based vaccines by reverse genetics.

12(S)-HETE mediates diabetes-induced endothelial dysfunction by activating intracellular endothelial cell TRPV1.

Patients with diabetes develop endothelial dysfunction shortly after diabetes onset that progresses to vascular disease underlying the majority of diabetes-associated comorbidities. Increased lipid peroxidation, mitochondrial calcium overload, and mitochondrial dysfunction are characteristics of dysfunctional endothelial cells in diabetic patients. We here identified that targeting the lipid peroxidation product 12(S)-hydroxyeicosatetraenoic acid-induced [12(S)-HETE-induced] activation of the intracellularly located cation channel transient receptor potential vanilloid 1 (TRPV1) in endothelial cells is a means to causally control early-stage vascular disease in type I diabetic mice. Mice with an inducible, endothelium-specific 12/15-lipoxygenase (12/15Lo) knockout were protected similarly to TRPV1-knockout mice from type 1 diabetes-induced endothelial dysfunction and impaired vascular regeneration following arterial injury. Both 12(S)-HETE in concentrations found in diabetic patients and TRPV1 agonists triggered mitochondrial calcium influx and mitochondrial dysfunction in endothelial cells, and 12(S)-HETE effects were absent in endothelial cells from TRPV1-knockout mice. As a therapeutic consequence, we found that a peptide targeting 12(S)-HETE-induced TRPV1 interaction at the TRPV1 TRP box ameliorated diabetes-induced endothelial dysfunction and augmented vascular regeneration in diabetic mice. Our findings suggest that pharmacological targeting of increased endothelial lipid peroxidation can attenuate diabetes-induced comorbidities related to vascular disease.