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Background. Chronic hepatitis C virus (HCV) infection is characterized by activation of natural killer (NK) cells. Here, we asked whether HCV elimination by sofosbuvir-based direct-acting antivirals (DAAs) and the addition of ribavirin (RBV) improve NK cell function in liver transplant (LTx) recipients. Methods. We analyzed NK cell degranulation and interferon (IFN)γ-response along with STAT1 and STAT4 phosphorylation in 29 HCV-infected LTx recipients and 17 HCV-infected patients during DAA treatment. Results. Compared with uninfected LTx recipients, NK cells from HCV-infected LTx recipients were polarized toward cytotoxicity with increased CD107a-degranulation (10.1% versus 14.6%; P = 0.0263) and reduced capacity to produce IFNγ (43.0% versus 26.7%; P = 0.0002). The altered phenotype of NK cells in HCV-infected LTx recipients was accompanied by increased STAT1 (44.6% versus 87.4%; P < 0.0001) and STAT1 phosphorylation (0.7% versus 8.9%; P = 0.0005) compared with pSTAT4 IFNα-induction (29.9% versus 17.6%; P = 0.0014). Successful DAA therapy did not affect CD107a-degranulation but decreased STAT1. RBV cotreatment with DAA therapy for HCV increased CD56Bright NK cell IFNγ-responses in LTx recipients (70.9% versus 89.2%; P = 0.002), and this correlated to an increase in the inducibility of pSTAT4 (MFI 157 versus 173; P = 0.0002). Conclusions. RBV cotreatment of HCV infection improved pSTAT4-dependent IFNγ-production in NK cells. This is relevant especially for immunocompromised patients such as LTx recipients or patients with end-stage liver disease.

Most clonal lineages of Staphylococcus epidermidis are commensals present on human skin and in the nose. However, some globally spreading healthcare-associated and methicillin-resistant S. epidermidis (HA-MRSE) clones are major causes of difficult-to-treat implant or bloodstream infections. The molecular determinants that alter the lifestyle of S. epidermidis have remained elusive, and their identification might provide therapeutic targets. We reasoned that changes in surface-exposed wall teichoic acid (WTA) polymers of S. epidermidis, which potentially shape host interactions, may be linked to differences between colonization and infection abilities of different clones. We used a combined epidemiological and functional approach to show that while commensal clones express poly-glycerolphosphate WTA, S. epidermidis multilocus sequence type 23, which emerged in the past 15 years and is one of the main infection-causing HA-MRSE clones, contains an accessory genetic element, tarIJLM, that leads to the production of a second, Staphylococcus aureus-type WTA (poly-ribitolphosphate (RboP)). Production of RboP-WTA by S. epidermidis impaired in vivo colonization but augmented endothelial attachment and host mortality in a mouse sepsis model. tarIJLM was absent from commensal human sequence types but was found in several other HA-MRSE clones. Moreover, RboP-WTA enabled S. epidermidis to exchange DNA with S. aureus via siphovirus bacteriophages, thereby creating a possible route for the inter-species exchange of methicillin resistance, virulence and colonization factors. We conclude that tarIJLM alters the lifestyle of S. epidermidis from commensal to pathogenic and propose that RboP-WTA might be a robust target for preventive and therapeutic interventions against MRSE infections.

Reaction of [NiCl2(PnH)4] (1) (PnH=6‐tert‐butyl‐pyridazine‐3‐thione) with NiCl2 affords the binuclear paddlewheel (PW) complex [Ni2(Pn)4] (2). Diamagnetic complex 2 is the first example of a PW complex capable of reversibly binding and releasing NH3. The NH3 ligand in [Ni2(Pn)4(NH3)] (2⋅NH3) enforces major spectroscopic and magnetic susceptibility changes, thus displaying vapochromic properties (λ max(2)=532 nm, λ max(2⋅NH3)=518 nm) and magnetochemical switching (2: S=0; 2⋅NH3: S=1). Upon repeated adsorption/desorption cycles of NH3 the PW core remains intact. Compound 2 can be embedded into thin polyurethane films (2 P) under retention of its sensing abilities. Therefore, 2 qualifies as reversible optical probe for ammonia. The magnetochemical switching of 2 and 2⋅NH3 was studied in detail by SQUID measurements showing that in 2⋅NH3, solely the Ni atom coordinated the NH3 molecule is responsible for the paramagnetic behavior., The Ni2 paddlewheel complex can reversibly bind ammonia. This triggers spin‐state switching from dia‐ to paramagnetic and a change of color. Coordination of NH3 is fully reversible in solution, in solid state as well as encapsulated in a polyurethane foil.

The previously published oxidorhenium(V) complex [ReOCl(L1)2] (2), equipped with the bidentate phenol-dimethyloxazoline ligand HL1 (2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)-phenol), shows catalytic activity for the reduction of nitrate to nitrite under mild, ambient and aqueous conditions. The reaction operates under an oxygen atom transfer (OAT) mechanism, using dimethyl sulfide SMe2 (DMS) as oxygen acceptor. Experiments with catalytic amounts of 2 and labelled 15NO3– proved the full reduction of 15NO3– to 15NO2– by 15N NMR spectroscopy. For the second reduction step of nitrite NO2–, we have evidence for a single electron reduction to yield paramagnetic NO, as from one nitrate reduction experiment the paramagnetic cis-dioxidorhenium(VI) complex [Re(O)2(L1)2] (3) could be isolated and characterized by single-crystal X-ray diffraction analysis. Such a single electron reduction of nitrite NO2– would yield NO and complex 3 as the oxidation product. In a stoichiometric experiment of 2, 15NO3– and DMS, nitrous oxide 15N2O could be detected as the only 15N containing product by 15N NMR spectroscopy, proving that further reduction beyond NO is possible with pre-catalyst 2. The rhenium species responsible for the reduction to N2O is currently unknown. Most likely, N2O is formed via an intermediate rhenium nitrosyl complex. Experimental data was gathered by 1H and 15N NMR, IR- and UV–Vis spectroscopy, HR-ESI mass spectrometry, X-ray crystallography, and supported by theoretical computations (DFT).

Inspired by the proposed inner-sphere mechanism of the tungstoenzyme acetylene hydratase, we have designed tungsten acetylene complexes and investigated their reactivity. Here, we report the first intermolecular nucleophilic attack on a tungsten-bound acetylene (C2H2) in bioinspired complexes employing 6-methylpyridine-2-thiolate ligands. By using PMe3 as a nucleophile, we isolated cationic carbyne and alkenyl complexes., We report the first intermolecular nucleophilic attack on a tungsten-bound C2H2 in two bioinspired complexes differing only by the oxidation state of the metal center and one ligand. By using PMe3 as a nucleophile, we isolated cationic carbyne and alkenyl complexes.

Ethoxyquin (EQ) is commonly used as an antioxidant in animal feeds. Although EQ is not permitted for usage in food products for humans within the EU, residues of EQ and its transformation products could be determined in food of animal origin. Despite its widespread use and concerns on its toxicological profile, no information about the systemic exposure to EQ in the general population is available. Hence, we developed a human biomonitoring (HBM) method for EQ. Our approach included a metabolism study with five subjects, who were administered an oral dose of 0.005 mg EQ/kg body weight. Unchanged EQ and the major metabolite 2,2,4-trimethyl-6(2H)-quinolinone (EQI) were identified as urinary excretion products of EQ. While small amounts of EQ could be determined in high concentrated samples from the metabolism study only, 28.5% of the orally applied EQ dose could be recovered as EQI. Toxicokinetic parameters were determined for EQI, the potential biomarker of exposure. In addition, an analytical method for EQI (LOQ = 0.03 µg/L) in urine based on UHPLC-MS/MS comprising enzymatic glucuronide hydrolysis and salt-assisted liquid-liquid extraction was developed, validated and applied to 53 urine samples from the general population. EQI could be quantified in 11 (21%) of the samples in levels up to 1.7 µg/L urine, proving the suitability of the developed method for the intended purpose.

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment‐resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot‐scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost., The benefits of bulk: Future use of psilocybin as a medication will entail increased demand. We present a gram‐scale synthetic/biocatalytic approach in which the mushroom kinase PsiK replaces tedious chemical phosphorylation. A pilot‐scale protocol to make three‐digit milligram amounts of pure and active recombinant PsiK available is also described.

To assess plasma trace mineral (TM) concentrations, the acute phase protein response, and behavior in response to a lipopolysaccharide (LPS) challenge, 96 Angus cross steers (average initial body weight [BW]: 285 ± 14.4 kg) were sorted into two groups by BW (heavy and light; n = 48/group), fitted with an ear-tag–based accelerometer (CowManager SensOor; Agis, Harmelen, Netherlands), and stagger started 14 d apart. Consecutive day BW was recorded to start the 24-d trial (days −1 and 0). Dietary treatments began on day 0: common diet with either 30 (Zn30) or 100 (Zn100) mg supplemental Zn/kg DM (ZnSO4). On day 17, steers received one of the following injection treatments intravenously to complete the 2 × 3 factorial: 1) SALINE (~2–3 mL of physiological saline), 2) LOWLPS: 0.25 µg LPS/kg BW, or 3) HIGHLPS: 0.375 µg LPS/kg BW. Blood, rectal temperature (RT), and BW were recorded on day 16 (−24 h relative to injection), and BW was used to assign injection treatment. Approximately 6, 24 (day 18), and 48 (day 19) h after treatment, BW, RT, and blood were collected, and final BW recorded on day 24. Data were analyzed in Proc Mixed of SAS with fixed effects of diet, injection, diet × injection; for BW, RT, dry matter intake (DMI), plasma TM, and haptoglobin-repeated measures analysis were used to evaluate effects over time. Area under the curve analysis determined by GraphPad Prism was used for analysis of accelerometer data. Body weight was unaffected by diet or injection (P ≥ 0.16), but there was an injection × time effect for DMI and RT (P < 0.05), where DMI decreased in both LPS treatments on day 16, but recovered by day 17, and RT was increased in LPS treatments 6 h post-injection. Steers receiving LPS spent less time highly active and eating than SALINE (P < 0.01). Steers in HIGHLPS spent lesser time ruminating, followed by LOWLPS and then SALINE (P < 0.001). An injection × time effect (P < 0.001) for plasma Zn showed decreased concentrations within 6 h of injection and remained decreased through 24 h before recovering by 48 h. A tendency for a diet × time effect (P = 0.06) on plasma Zn suggests plasma Zn repletion occurred at a greater rate in Zn100 compared to Zn30. These results suggest that increased supplemental Zn may alter the rate of recovery of Zn status from an acute inflammatory event. Additionally, ear-tag–based accelerometers used in this study were effective at detecting sickness behavior in feedlot steers, and rumination may be more sensitive than other variables.

Electrolyte decomposition of lithium-ion battery as a consequence of thermal stress was investigated in Part 1 of this two-part study. The focus of Part 2 is on the influence of the battery cell op...

Bioinspired complexes employing the ligands 6‐tert‐butylpyridazine‐3‐thione (SPn) and pyridine‐2‐thione (SPy) were synthesized and fully characterized to mimic the tungstoenzyme acetylene hydratase (AH). The complexes [W(CO)(C2H2)(CHCH‐SPy)(SPy)] (4) and [W(CO)(C2H2)(CHCH‐SPn)(SPn)] (5) were formed by intramolecular nucleophilic attack of the nitrogen donors of the ligand on the coordinated C2H2 molecule. Labelling experiments using C2D2 with the SPy system revealed the insertion reaction proceeding via a bis‐acetylene intermediate. The starting complex [W(CO)(C2H2)(SPy)2] (6) for these studies was accessed by the new acetylene precursor mixture [W(CO)(C2H2)n(MeCN)3−nBr2] (n=1 and 2; 7). All complexes represent rare examples in the field of W−C2H2 chemistry with 4 and 5 being the first of their kind. In the ongoing debate on the enzymatic mechanism, the findings support activation of acetylene by the tungsten center., C2H2 under attack! Bioinspired model complexes for acetylene hydratase demonstrate that nucleophilic attack on a W−C2H2 adduct is feasible. With the insertion of the enzyme's substrate acetylene into the W−N bond of the biomimetic ligand, data, which support the still disputed mechanism involving a W‐η2‐C2H2 intermediate, is provided (see scheme).

Background During an infection with hepatitis B virus (HBV), infectious particles (Dane particles) can be detected in addition to aggregates of the subviral particles (SVP) which is considered an immune escaping mechanism for the virus. Dendritic cells (DCs) are a specialized type of antigen-presenting cell (APC) that can activate native T-cells to prime an immune response controlling HBV infection. The aim of this study was to characterize the interaction between HBVsvp and DCs in vitro. Methods HBVsvp that comprises surface and core proteins were produced in vitro by HepG2.2.15 as a culturing system; DCs derived from the bone marrow of mice were pulsed by HBVsvp. A different pattern of cytokines secreted by bone-marrow-derived dendritic cells from C56BL/6 mice pulsed with HBVsvp were analyzed. The interactions between HBVsvp and DCs were characterized using FACS analysis, protein assay, Western blot, and immunofluorescence staining. Results Pulsation of DCs with HBVsvp resulted in strong activation and higher secretion of DC cytokines including INF-α, INF-γ, TNF-α, IL-1α, IL-10, and IL-12; but not for IL-1β, IL-2, IL-6, and IL-15. The production of CXCL-10/IP-10 was increased during the observation period and reached the maximal secretion after 24 hrs (p < 0.001). In total protein assay, we found significantly higher protein concentration in HBVsvp stimulated DC groups compared to not activated DCs (p < 0.001). Both 24 kDa small surface antigen (HBVs) and the 21 kDa core protein (HBVc) were detected in activated DCs. For DCs immunofluorescence staining, our data showed clear differences in the morphology of DCs between negative control and those pulsed with HBVsvp. Conclusion Result demonstrates a significant complex interaction between HBVsvp and DCs, in vitro.

Introduction: Autologous serum eye drops (ASED) are used in the treatment of most severe stages of dry eye. Once introduced, it is currently considered impossible to return to other lubricating eye drops or other commercially available therapeutic regimen. Materials and Methods: In a randomized study, non-preserved high-molecular-weight hyaluronic acid eye drops were offered as an alternative to 11 patients using autologous serum treatment for at least 3 months. The control group (n = 5) continued their treatment with ASED. The verum group (n = 6) used very-high-molecular-weight hyaluronic eye drops (Comfort Shield®) instead of the ASED. Results: From four of initially six patients in the verum group that finished the study, 2 (50%) preferred to stay with the very-high-molecular-weight hyaluronic acid eye drops beyond the trial period, the other two returned to the earlier therapy with ASED. The control group continued their treatment as before and finished the study after 8 weeks. Conclusion: For the first time, artificial eye drops, i.e., high-molecular-weight hyaluronic acid eye drops, offered an acceptable alternative to ASED. Some patients perceived these drops as even better than the patient’s own serum. This is the first evidence that optimization of the molecular structure of hyaluronic acid can be used to create eye drops that are perceived to be better than other tested tear substitutes and even patients’ own serum. This offers a new treatment perspective for patients with very severe dry eye disease.

This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.

The synthesis and structural determination of four tungsten alkyne complexes coordinated by the bio-inspired S,N-donor ligand 2-(4',4'-dimethyloxazoline-2'-yl)thiophenolate (S-Phoz) is presented. A previously established protocol that involved the reaction of the respective alkyne with the bis-carbonyl precursor [W(CO)2 (S-Phoz)2 ] was used for the complexes [W(CO)(C2 R2 )(S-Phoz)2 ] (R=H, 1 a; Me, 1 b; Ph, 1 c). Oxidation with pyridine-N-oxide gave the corresponding W-oxo species [WO(C2 R2 )(S-Phoz)2 ] (R=H, 2 a; Me, 2 b; Ph, 2 c). All W-oxo-alkyne complexes (2 a, b, c) were found to be capable of alkyne release upon light irradiation to afford five-coordinate [WO(S-Phoz)2 ] (3). The photoinduced release of the alkyne ligand was studied in detail by in situ 1 H NMR measurements, which revealed correlation of the photodissociation rate constant (2 b>2 a>2 c) with the elongation of the alkyne C≡C bond in the molecular structures. Oxidation of [WO(S-Phoz)2 ] (3) with pyridine-N-oxide yielded [WO2 (S-Phoz)2 ] (4), which shows highly fluxional behavior in solution. Variable-temperature 1 H NMR spectroscopy revealed three isomeric forms with respect to the ligand arrangement versus each other. Furthermore, compound 4 rearranges to tetranuclear oxo compound [W4 O4 (μ-O)6 (S-Phoz)4 ] (5) and dinuclear [{WO(μ-O)(S-Phoz)}2 ] (6) over time. The latter two were identified by single-crystal X-ray diffraction analyses.

Context: Rhodiola rosea L. (Crassulaceae) is well-known to contain flavonoids such as the herbacetin derivative rhodiosin. However, flavonoids are not typically used in quality control. Objective: This study analyses two flavonoids of R. rosea rhizomes and roots for their potential as analytical markers. Materials and methods: Two constituents were isolated from ethanolic extracts via HPLC, identified via NMR and quantified via RP-HPLC. Presence and content variation was investigated according to extraction (solvent and repetitions), drying (temperature and duration) and sample origin (homogenously cultivated plants of different provenance, commercial samples). Results: Rhodiosin was identified as a main flavonoid, accompanied by 10-fold lower concentrated herbacetin. Both compounds were best extracted with 70–90% ethanol, but were also detectable in more aqueous extracts. Different drying conditions had no effect on the flavonoid content. These two flavonoids were consistently identified in rhizome and root extracts of over 100 R. rosea samples. Rhizomes tend to contain less flavonoids, with average ratios of rosavins to flavonoids of 1.4 (rhizomes) and 0.4 (roots). Provenance differences were detected in the range (rhodiosin plus herbacetin) of 760–6300 µg/mL extract corresponding to a maximum of approximately 0.5–4.2% (w/w) in the dry drug. Conclusions: For the first time, two main flavonoids present in R. rosea were quantified systematically. Rhodiosin and herbacetin can be detected simultaneously to phenylpropenoids or salidroside in authentic samples, influenced by the plant part examined and the plant origin. Rhodiosin and herbacetin may serve as additional marker to guarantee a consistent content of R. rosea products.

A new solid-phase peptide synthesis and bioprofiling of the antimicrobial activity of lugdunin, a fibupeptide, enable a comprehensive structure–activity relationship (SAR) study (MRSA Staphylococcu...

Staphylococcus aureus is the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond to invading S. aureus and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, sense S. aureus through their pattern-recognition receptor langerin, triggering a proinflammatory response. Langerin recognizes the β-1,4-linked N-acetylglucosamine (β1,4-GlcNAc) but not α-1,4-linked GlcNAc (α1,4-GlcNAc) modifications, which are added by dedicated glycosyltransferases TarS and TarM, respectively, on the cell wall glycopolymer wall teichoic acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified, which also modifies WTA with β-GlcNAc but at the C-3 position (β1,3-GlcNAc) of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of β-GlcNAc linkage position for langerin binding and LC activation. Using genetically modified S. aureus strains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA, yet tarP-expressing S. aureus induced increased cytokine production and maturation of in vitro-generated LCs compared to tarS-expressing S. aureus. Chemically synthesized WTA molecules, representative of the different S. aureus WTA glycosylation patterns, were used to identify langerin-WTA binding requirements. We established that β-GlcNAc is sufficient to confer langerin binding, thereby presenting synthetic WTA molecules as a novel glycobiology tool for structure-binding studies and for elucidating S. aureus molecular pathogenesis. Overall, our data suggest that LCs are able to sense all β-GlcNAc-WTA producing S. aureus strains, likely performing an important role as first responders upon S. aureus skin invasion.

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.

The research in storage and conversion of energy is an everlasting process. The use of fuel cells is very tempting but up to now there are still several conceptual challenges to overcome. Especially, the requirement of liquid water causes difficulties due to the temperature limit. Therefore, imidazoles and triazoles are increasingly investigated in a manifold of experimental and theoretical publications as they are both very promising in overcoming this problem. Recently, triazoles were found to be superior to imidazoles in proton conduction. An ab-initio molecular dynamics simulation of pure triazole phases for investigating the behavior of both tautomer species of the triazole molecule has never been done. In this work, we investigate the structural and dynamical properties of two different solid phases and the liquid phase at two different temperatures. We are able to show how the distinct tautomers contribute to the mechanism of proton conduction, to compute dynamical properties of the four systems and to suggest a mechanism of reorientation in solid phase.

Staphylococcus aureusis the leading cause of skin and soft tissue infections. It remains incompletely understood how skin-resident immune cells respond toS. aureusinvasion and contribute to an effective immune response. Langerhans cells (LCs), the only professional antigen-presenting cell type in the epidermis, senseS. aureusthrough their pattern-recognition receptor langerin, triggering a pro-inflammatory response. Langerin specifically recognizes the β-1,4-linkedN-acetylglucosamine (β-GlcNAc) modification, which requires the glycosyltransferase TarS, on the cell wall glycopolymer Wall Teichoic Acid (WTA). Recently, an alternative WTA glycosyltransferase, TarP, was identified in methicillin-resistantS. aureusstrains belonging to clonal complexes (CC) 5 and CC398. TarP also modifies WTA with β-GlcNAc but at the C-3 position of the WTA ribitol phosphate (RboP) subunit. Here, we aimed to unravel the impact of β-GlcNAc linkage position for langerin binding and LC activation. In addition, we performed structure-binding studies using a small panel of unique chemically-synthesized WTA molecules to assess langerin-WTA binding requirements. Using FITC-labeled recombinant human langerin and genetically-modifiedS. aureusstrains, we observed that langerin similarly recognized bacteria that produce either TarS- or TarP-modified WTA. Furthermore, using chemically-synthesized WTA, representative of the differentS. aureusWTA glycosylation patterns, established that β-GlcNAc is sufficient to confer langerin binding. Functionally,tarP-expressingS. aureusinduce increased cytokine production and maturation ofin vitro-generated LCs compared totarSexpressingS. aureus. Overall, our data suggest that LCs are able to sense all β-GlcNAc-WTA producingS. aureusstrains, likely performing an important role as first responders uponS. aureusskin invasion.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 A. The ligand with an 80 A linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.

This work presents a photodissociation study of the diamondoid adamantane using extreme ultraviolet femtosecond pulses. The fragmentation dynamics of the dication is unraveled by the use of advanced ion and electron spectroscopy giving access to the dissociation channels as well as their energetics. To get insight into the fragmentation dynamics, we use a theoretical approach combining potential energy surface determination, statistical fragmentation methods and molecular dynamics simulations. We demonstrate that the dissociation dynamics of adamantane dications takes place in a two-step process: barrierless cage opening followed by Coulomb repulsion-driven fragmentation, This research was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research and the Crafoord Foundation. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 641789 MEDEA and under grant agreement no 654148 Laserlab-Europe. The research was conducted in the framework of the International Associated Laboratory (LIA) Fragmentation DYNAmics of complex MOlecular systems - DYNAMO, funded by the Centre National de la Recherche Scientifique (CNRS). The authors acknowledge the generous allocation of computer time at the Centro de Computación Científica at the Universidad Autónoma de Madrid (CCC-UAM). This work was partially supported by the project CTQ2016-76061-P of the Spanish Ministerio de Economía y Competitividad (MINECO). Financial support from the MINECO through the "María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377) is also acknowledged

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers, either linked to peptidoglycan, wall teichoic acids (WTA), or to membrane glycolipids, lipoteichoic acids (LTA). In some bacteria, includingBacillus subtilisstrain 168, WTA and LTA both are glycerolphosphate polymers, yet are synthesized by different pathways and have distinct, although not entirely understood morphogenetic functions during cell elongation and division. We show here that the exo-lyticsn-glycerol-3-phosphodiesterase GlpQ can discriminate betweenB. subtilisWTA and LTA polymers. GlpQ completely degrades WTA, lacking modifications at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ is unable to cleave unmodified LTA. LTA can only be hydrolyzed by GlpQ when the polymer is partially pre-cleaved, thereby allowing GlpQ to get access to the end of the polymer that is usually protected by a connection to the lipid anchor. This indicates that WTA and LTA are enantiomeric polymers: WTA is made ofsn-glycerol-3-phosphate and LTA is made ofsn-glycerol-1-phosphate. Differences in stereochemistry between WTA and LTA were assumed based on differences in biosynthesis precursors and chemical degradation products, but so far had not been demonstrated directly by differential, enantioselective cleavage of isolated polymers. The discriminative stereochemistry impacts the dissimilar physiological and immunogenic properties of WTA and LTA and enables independent degradation of the polymers, while appearing in the same location; e.g. under phosphate limitation,B. subtilis168 specifically hydrolyzes WTA and synthesizes phosphate-free teichuronic acids in exchange.

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

Calcium bis[bis(trimethylsilyl)amide] (Ca(HMDS)2) is a widely used reagent in diverse stoichiometric and catalytic applications. These processes necessitate a straightforward and large-scale access of this complex. Calcium does not react with primary and secondary amines, but the addition of excess bromoethane to a mixture of calcium turnings and amines in THF at room temperature yields the corresponding calcium bis(amides), calcium bromide and ethane. This in situ Grignard metalation method (iGMM) allows the preparation of calcium bis(amides) from secondary and primary trialkylsilyl-substituted amines and anilines on a multigram scale.1 Background2 The In Situ Grignard Metalation Method (iGMM)3 Properties of [(thf)2M(HMDS)2]4 Applications and Perspective

Silica mono- and bilayer films on Ru(0001) can be physisorbed or chemisorbed, with ordered or vitreous structures, depending on the particular preparation procedures applied. Using the SMART spectro-microscope at BESSY-II with its capabilities for μ-spectroscopy, μ-diffraction, and LEEM imaging with lateral resolution below 5 nm, in situ and in real time and applied to identical areas, we have investigated the formation of these layers, defined and characterized their properties and their connected morphology, and followed their evolution. Two distinct chemisorbed monolayers and three bilayers (physisorbed crystalline and vitreous, and chemisorbed zigzag phases), and some transitions between them, have been studied. We found that, apart from the deposited silicon amount, the most important parameter for steering the evolution to a particular well-defined layer is the oxygen content at the Ru interface. Nucleation and growth of all layers are homogeneous on the scale of our resolution, leading to rather sm...

Doped barium hexaferrite films prepared on alumina substrates by means of the sol-gel method, intended for utilization in self-biased nonreciprocal components, are presented in this letter. To cover a broad range of potential applications, the ferromagnetic resonant frequency (FMRF) of the material is controlled by substituting iron for scandium and aluminum. The films are deposited on coplanar waveguides to permit broadband measurements of the FMRF. As shown from measurements, the resonance can be shifted from $\text {43.2}$ GHz for the undoped sample to $\text {27.1}$ GHz and $\text{65.1}$ GHz for the films with the highest tested scandium and aluminum doping, respectively. X-ray diffraction analysis is used to evaluate the formation of secondary phases in the film. Measurement results for the hysteresis curves are obtained by vibrating sample magnetometer measurements. The results for doped and undoped samples are discussed in this letter.

Bloodstains on textiles can provide useful information for the forensic reconstruction of a crime. Surprisingly, little is known about the applicability of bloodstain traces after a textile was machine washed. In this study, we investigated the effect of machine washing on bloodstains on both cotton and polyester cloths. The influence of the washing detergent, the type of washing machine, the washing temperature, and the duration of drying of the bloodstain prior to washing as well as the drying temperature was investigated. Additionally, the molecular analyses of a subsample of the experiments were conducted. We found that although the primary morphology of the traces is often blurred, the presence of blood on the textiles can still be detected in many cases. Blood can also be transmitted to previously blood-free textiles during the washing process, leading to a positive Luminol or Combur® reaction of these samples. When traces of blood can be detected via the Luminol reaction, a molecular identification of the blood donor was successful in 28% of the cases.

Series of nickel and palladium complexes bearing amine-imine ligands in various ortho-aryl and backbone positions were prepared and investigated in ethene polymerization. Ethene polymerization initiated by symmetrically ortho-substituted nickel and palladium amine-imine catalysts is controlled. Mono-substitution in the ortho-aryl positions of nickel complexes is not as efficient in protecting centers from chain transfer as di-substitution. Both the central metal and the size of the ortho-aryl substituent have a significant effect on the polyethylene (PE) topology. Based on detailed characterization by high temperature SEC-IR-, SEC with multi-angle laser light scattering and C-13 NMR data, PEs prepared by nickel amine-imine complexes have a linear rather than dendritic topology. In contrast, palladium amine-imine complexes with small ortho-aryl substituents at low ethene pressure were shown for the first time to form dendritic PEs with topology comparable to PEs formed by -diimine palladium catalyst.

7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance widely used in cosmetic and hygiene products. Due to its large scope, 7-HC was selected for the development of a biomonitoring method suitable for the general population within the frame of the cooperation project between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). In a human study with 5 healthy subjects who received single dermal and oral doses 7-HC, suitable metabolites and their urinary excretion kinetics was investigated. Two metabolites of 7-hydroxycitronellal were identified in urinary fractions after dermal and oral dosing: The alcohol 7-hydroxycitronellol (7-HCO) and the corresponding acid 7-hydroxycitronellylic acid (7-HCA). Only 7-HCA proved to be a suitable biomarker of exposure to 7-HC, since 7-HCO was quantifiable in only a minority of urine samples collected from the general population. Quantification of 7-HCA was conducted by means of a newly developed UPLC–MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion of 7-HCA occurred between 3 and 5 h after oral application and about 10 h after dermal administration. Due to the limited skin absorption of 7-HC, 7-HCA concentrations after dermal application were much lower than levels after oral application. After 24 h, about 9% and 50% of the dermally and orally applied dose, respectively, were excreted as 7- HCA. With the conversion factors derived from the controlled human study, we estimated median exposure doses in a group of 40 human volunteers from the general population of approximately 93 μg 7-HC per day. In conclusion, the 7-HC metabolite 7-HCA in urine is a suitable biomarker of exposure and can be applied for biomonitoring of the general population.

The phenylpropenoid glycosides rosavin, rosarin, rosin summarised as ‘total rosavins’ are quality indicators of Rhodiola rosea L. Here we report the systematic quantification of these glycosides and the aglycon cinnamyl alcohol in various provenances cultivated homogenously at 1580 m altitude. (I) We examined rhizomes versus roots in cultivation year 9 (20 provenances). Extracts (70% ethanol) contained 0.5–4.3 mg/mL (rhizomes) and 0.4–2.5 mg/mL (roots) total rosavins. The rosavin/cinnamyl alcohol ratio (average rhizomes 8.1:1, roots 6.2:1), was more influenced by plant origin than plant part. We additionally analysed salidroside including its aglycon tyrosol. The rosavins/salidroside ratios (including aglycons) of 6.4:1 (rhizome) and 5.5:1 (root) indicated authentic R. rosea independent of plant part and origin. (II) Rhizomes extracts (8 provenances) from various dates during years 6 and 7 contained 0.4–4.8 mg/mL total rosavins corresponding to 0.2–3.0% in the dry drug. Age (decreasing trend) and provenance impact were superposed by seasonal variation with a phenylpropenoid peak in spring. Rosavin/cinnamyl alcohol ratios in North European plants were 3–8:1, for Alpine/Pyrenean plants 9–14:1. (III) Compared to our previous dataset from cultivation in South England, on average, there was no major difference between phenylpropenoid values from cultivation at 65 m and 1580 m apart from moderately more cinnamyl alcohol at higher altitude. As per provenance, absolute values were site dependent but phenylpropenoid glycoside/aglycon ratios consistent. In conclusion, this is the first systematic investigation for a high altitude cultivation of R. rosea demonstrating the major extent plant part, season and age influence absolute phenylpropenoid values. In contrary, relative phenolic compound profiles are mostly origin-determined, independent of cultivation conditions and site, and could be used for authentication in quality control.

The synthesis, spectroscopic characterization, and catalytic activities of a series of PdII complexes bearing the monoanionic, bidentate ligand 2-(2-thiophenyl)-4,4-dimethyloxazoline (S-Phoz) are reported. These complexes were used as precatalysts for Suzuki–Miyaura coupling reactions under aqueous conditions. The dimers [{PdX(S-Phoz)}2] (X = Cl, Br, I; 1–3) were treated with the N-heterocyclic carbene (NHC) ligand 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (IMes) to afford the mononuclear complexes [PdX(S-Phoz)(IMes)] (X = Cl, Br, I; 4–6). The σ-donor/π-acceptor complexes [PdCl(S-Phoz)(EPh3)] (E = P, As, Sb; 7–9) were synthesized to evaluate the influence of a second donor ligand on the catalytic activity. Within the [PdCl(S-Phoz)L] series, the activity trend for L follows the trend PPh3 > IMes ≈ AsPh3 > SbPh3. The sulfur-bridged dinuclear complexes 1–3 are highly active for the benchmark coupling of p-bromoacetophenone with phenylboronic acid and exhibit turnover frequencies (TOFs) of up to 16000 h–1. DFT and G0W0 calculations were performed to rationalize the facile reduction and, hence, excellent activities of the dinuclear complexes.

The synthesis, characterization, and epoxidation catalysis of two neutral, dinuclear molybdenum(V) complexes [{MoO(Lx)(µ-O)}2] (x = 1: 1; x = 2: 2) bearing S,N-bidentate thiophenolate-oxazoline ligands (L1, L2) are described. Both complexes are formed under reduction of the metal with formation of disulfides (L1)2 and (L2)2. Each Mo center is coordinated by one bidentate thiophenolate-oxazoline ligand and one terminal and two bridging oxido ligands. The solid-state structures of NaL2 and complex 1 were confirmed by single-crystal X-ray diffraction. The five-coordinate nature of the molybdenum atoms in 1 and 2 leads to exceptionally high catalytic activities in the epoxidation of cyclooctene. The catalyst loading can be reduced to 0.001 mol-% (10 ppm), with a turnover number (TON) of 85000 and a maximum turnover frequency (TOF) of 62000 h–1 for 1, and a TON of 87400 and a TOF of 107400 h–1 for 2. For cyclooctene, the catalyst solutions can be recycled multiple times without significant loss of activity; however, other substrates (styrene, 1-octene, and limonene) showed conversions with lower selectivity. The fate of catalyst 1 was examined by stoichiometric addition experiments with tert-butyl hydroperoxide, which resulted in the formation of benzenesulfonate-oxazoline derivative HL3 and polyoxomolybdates.

Making use of a quartz crystal microbalance (QCM), concentrated solutions of therapeutic antibodies were studied with respect to their behavior under shear excitation with frequencies in the MHz range. At high protein concentration and neutral pH, viscoelastic behavior was found in the sense that the storage modulus, G', was nonzero. Fits of the frequency dependence of G'(ω) and G''(ω) (G'' being the loss modulus) using the Maxwell-model produced good agreement with the experimental data. The fit parameters were the relaxation time, τ, and the shear modulus at the inverse relaxation time, G* (at the "cross-over frequency" ωC = 1/τ). The influence of two different pharmaceutical excipients (histidine and citrate) was studied at variable concentrations of the antibody and variable pH. In cases, where viscoelasticity was observed, G* was in the range of a few kPa, consistent with entropy-driven interactions. τ was small at low pH, where the antibody carries a positive charge. τ increased with increasing pH. The relaxation time τ was found to be correlated with other parameters quantifying protein-protein interactions, namely the steady shear viscosity (η), the second osmotic virial coefficient as determined with both self-interaction chromatography (B22,SIC) and static light scattering (B22,SLS), and the diffusion interaction parameter as determined with dynamic light scattering (kD). While B22 and kD describe protein-protein interactions in diluted samples, the QCM can be applied to concentrated solutions, thereby being sensitive to higher-order protein-protein interactions.

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89Zr-Df-aTCRmu-F(ab')2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo. Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo. Local application of 89Zr-Df-aTCRmu-F(ab')2-labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x104 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89Zr-Df-aTCRmu-F(ab')2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x104 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

BackgroundFormyl-peptide receptors (FPRs) are important pattern recognition receptors that sense specific bacterial peptides. Formyl-peptide receptors are highly expressed on neutrophils and monocytes, and their activation promotes the migration of phagocytes to sites of infection. It is currently unknown whether FPRs may also influence subsequent processes such as bacterial phagocytosis and killing. Staphylococcus aureus, especially highly pathogenic community-acquired methicillin-resistant S aureus strains, release high amounts of FPR2 ligands, the phenol-soluble modulins.MethodsWe demonstrate that FPR activation leads to upregulation of complement receptors 1 and 3 as well as FCγ receptor I on neutrophils and, consequently, increased opsonic phagocytosis of S aureus and other pathogens.ResultsIncreased phagocytosis promotes killing of S aureus and interleukin-8 release by neutrophils.ConclusionsWe show here for the first time that FPRs govern opsonic phagocytosis. Manipulation of FPR2 activation could open new therapeutic opportunities against bacterial pathogens.

Introduction Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. Objectives The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. Methods Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 μM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. Results Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. Conclusions Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage Galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of S. lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.

The bacterium Staphylococcus aureus is an important cause of skin infections and is also associated with the occurrence and severity of eczema. Langerhans cells (LCs), a specific subset of skin immune cells, participate in the immune response to S. aureus, but it is yet unclear how LCs recognize S. aureus. Therefore, we investigated the molecular mechanism underlying the interaction between LCs and S. aureus. We identified that wall teichoic acid, an abundant polymer on the S. aureus surface, is recognized by langerin, a receptor unique to LCs. This interaction allows LCs to discriminate S. aureus from other related staphylococcal species and initiates a proinflammatory response similar to that observed in patients with eczema. Our data therefore provide important new insights into the relationship between S. aureus, LCs, and eczema., Staphylococcus aureus is a major cause of skin and soft tissue infections and aggravator of the inflammatory skin disease atopic dermatitis (AD [eczema]). Epicutaneous exposure to S. aureus induces Th17 responses through skin Langerhans cells (LCs), which paradoxically contribute to host defense but also to AD pathogenesis. The molecular mechanisms underlying the interaction between S. aureus and LCs are poorly understood. Here we demonstrate that human LCs directly interact with S. aureus through the pattern recognition receptor langerin (CD207). Human, but not mouse, langerin interacts with S. aureus through the conserved β-N-acetylglucosamine (GlcNAc) modifications on wall teichoic acid (WTA), thereby discriminating S. aureus from other staphylococcal species. Importantly, the specific S. aureus WTA glycoprofile strongly influences the level of proinflammatory cytokines that are produced by in vitro-generated LCs. Finally, in a murine epicutaneous infection model, S. aureus strongly upregulated transcripts of Cxcl1, Il6, and Il17, which required the presence of both human langerin and WTA β-GlcNAc. Our findings provide molecular insight into the unique proinflammatory capacities of S. aureus in relation to skin inflammation.

Multidrug-resistant bacterial pathogens are becoming increasingly prevalent, and novel strategies to treat bacterial infections caused by these organisms are desperately needed. Bacterial central metabolism is crucial for catabolic processes and provides precursors for anabolic pathways, such as the biosynthesis of essential biomolecules like amino acids or vitamins. However, most essential pathways are not regarded as good targets for antibiotic therapy since their products might be acquired from the environment. This issue raises doubts about the essentiality of such targets during infection. A putative target in bacterial anabolism is the methionine biosynthesis pathway. In contrast to humans, almost all bacteria carry methionine biosynthesis pathways which have often been suggested as putative targets for novel anti-infectives. While the growth of methionine auxotrophic strains can be stimulated by exogenous methionine, the extracellular concentrations required by most bacterial species are unknown. Furthermore, several phenotypic characteristics of methionine auxotrophs are only partly reversed by exogenous methionine. We investigated methionine auxotrophic mutants of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli (all differing in methionine biosynthesis enzymes) and found that each needed concentrations of exogenous methionine far exceeding that reported for human serum (∼30 µM). Accordingly, these methionine auxotrophs showed a reduced ability to proliferate in human serum. Additionally, S. aureus and P. aeruginosa methionine auxotrophs were significantly impaired in their ability to form and maintain biofilms. Altogether, our data show intrinsic defects of methionine auxotrophs. This result suggests that the pathway should be considered for further studies validating the therapeutic potential of inhibitors. IMPORTANCE New antibiotics that attack novel targets are needed to circumvent widespread resistance to conventional drugs. Bacterial anabolic pathways, such as the enzymes for biosynthesis of the essential amino acid methionine, have been proposed as potential targets. However, the eligibility of enzymes in these pathways as drug targets is unclear because metabolites might be acquired from the environment to overcome inhibition. We investigated the nutritional needs of methionine auxotrophs of the pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. We found that each auxotrophic strain retained a growth disadvantage at methionine concentrations mimicking those available in vivo and showed that biofilm biomass was strongly influenced by endogenous methionine biosynthesis. Our experiments suggest that inhibition of the methionine biosynthesis pathway has deleterious effects even in the presence of external methionine. Therefore, additional efforts to validate the effects of methionine biosynthesis inhibitors in vivo are warranted.