The impact of C-reactive protein levels on the effectiveness of upadacitinib in patients with rheumatoid arthritis: a 12-month prospective, non-interventional German study.

OBJECTIVES: We investigated whether the effectiveness of upadacitinib in rheumatoid arthritis (RA) treatment is affected by baseline CRP levels in a real-world setting. METHODS: UPwArds was a prospective, non-interventional study. Patients had moderate-to-severe RA and an inadequate response or intolerance to ≥1 disease-modifying anti-rheumatic drug (DMARD). The primary endpoint was clinical remission (Clinical Disease Activity Index [CDAI] ≤2.8) at 6 months. Secondary endpoints at 12 months included clinical remission and low disease activity assessed by CDAI and Simple Disease Activity Index criteria, DAS28-CRP <2.6/≤3.2, and patient-reported outcomes. The impact of baseline CRP levels (normal vs. above the upper limit of normal [ULN]) on primary and secondary endpoints was evaluated. The effect of concomitant MTX and prior inadequate response to biologic or targeted synthetic DMARDs (b/tsDMARD-IR) on the effectiveness of upadacitinib was also assessed. Safety was evaluated through 12 months. RESULTS: 518 patients were included in the effectiveness analyses. At 6 months, 24.4% of patients achieved the primary endpoint (CDAI ≤2.8). At 12 months, similar proportions of patients with normal CRP and CRP above the ULN at baseline achieved CDAI ≤2.8 (27.3% and 29.1%) and other key secondary endpoints. The effectiveness of upadacitinib was comparable with and without concomitant MTX and in b/tsDMARD-naive and b/tsDMARD-IR patients. The safety results were consistent with the known safety profile of upadacitinib; no new safety signals were identified. CONCLUSIONS: Upadacitinib therapy was effective for RA in a real-world setting. Baseline CRP levels had no significant impact on the effectiveness of upadacitinib.

Non-destructive fluorescence sensing for assessing microclimate, site and defoliation effects on flavonol dynamics and sugar prediction in Pinot blanc grapes.

In an era of growing international competition in modern viticulture, the study and implementation of innovative technologies to increase the production of high-quality grapes and wines are of critical importance. In this study, the non-destructive portable sensor Multiplex, based on fluorescence sensing technique, was applied to evaluate grape maturity parameters and flavonol content of the understudied Pinot blanc variety. The effects of environmental and agronomical factors on flavonol content of Pinot blanc grapes were investigated in eight vineyards characterised by different microclimatic and agronomic conditions. Furthermore, the direct impact of canopy management treatment on the flavonol dynamics of the grapes oriented in the four cardinal directions was assessed. Results highlight the positive role of moderate temperatures and direct sunlight exposure on Pinot blanc flavonol content; however, no direct vineyard-elevation effect was observed. The ability to modulate and evaluate the flavonol content in field represent crucial factors because of their potential effect on flavonoids-dependent wine characteristics, such as stability and ageing. In the present study, for the first time, two calibration curves were reported for pre- and post-veraison periods between flavonol indices and the berry skin flavonol content and a good correlation was observed between Multiplex measurement and the total polyphenolic content of grape juice. Moreover, the strong correlation between the chlorophyll index with grape juice sugar content and titratable acidity revealed the practical application of non-destructive sensors to predict the optimal harvest time for Pinot blanc grapes. In conclusion, the non-destructive fluorescence sensor Multiplex is a high-potential tool for innovative viticulture, for evaluating grape skin composition variables in white grape varieties.

Bioprocess development workflow: Transferable physiological knowledge instead of technological correlations.

Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters, the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology. Information processing condenses the data into physiological variables, while information mining condenses the variables further into physiological descriptors. This basis facilitates data analysis for a physiological explanation for observed phenomena in productivity. Targeting transferability, we demonstrate this workflow using an industrially relevant Escherichia coli process for recombinant protein production and substantiate the following three points: (1) The postinduction phase is independent in terms of productivity and physiology from the preinduction variables specific growth rate and biomass at induction. (2) The specific substrate uptake rate during induction phase was found to significantly impact the maximum specific product titer. (3) The time point of maximum specific titer can be predicted by an easy accessible physiological variable: while the maximum specific titers were reached at different time points (19.8 ± 7.6 h), those maxima were reached all within a very narrow window of cumulatively consumed substrate dSn (3.1 ± 0.3 g/g). Concluding, this contribution provides a workflow on how to gain a physiological view on the process and illustrates potential benefits. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:261-270, 2017.

Deterministic generation of multiparticle entanglement by quantum Zeno dynamics.

Multiparticle entangled quantum states, a key resource in quantum-enhanced metrology and computing, are usually generated by coherent operations exclusively. However, unusual forms of quantum dynamics can be obtained when environment coupling is used as part of the state generation. In this work, we used quantum Zeno dynamics (QZD), based on nondestructive measurement with an optical microcavity, to deterministically generate different multiparticle entangled states in an ensemble of 36 qubit atoms in less than 5 microseconds. We characterized the resulting states by performing quantum tomography, yielding a time-resolved account of the entanglement generation. In addition, we studied the dependence of quantum states on measurement strength and quantified the depth of entanglement. Our results show that QZD is a versatile tool for fast and deterministic entanglement generation in quantum engineering applications.

Telomerase activity impacts on Epstein-Barr virus infection of AGS cells.

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.

Activation of NF-κB via endosomal Toll-like receptor 7 (TLR7) or TLR9 suppresses murine herpesvirus 68 reactivation.

UNLABELLED: In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7-/- or tlr9-/- mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9-/- mice showed a higher frequency of reactivation than B cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE: A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.

Entangled states of more than 40 atoms in an optical fiber cavity.

Multiparticle entanglement enables quantum simulations, quantum computing, and quantum-enhanced metrology. Yet, there are few methods to produce and measure such entanglement while maintaining single-qubit resolution as the number of qubits is scaled up. Using atom chips and fiber-optical cavities, we have developed a method based on nondestructive collective measurement and conditional evolution to create symmetric entangled states and perform their tomography. We demonstrate creation and analysis of entangled states with mean atom numbers up to 41 and experimentally prove multiparticle entanglement. Our method is independent of atom number and should allow generalization to other entangled states and other physical implementations, including circuit quantum electrodynamics.

Surgery for metastasis to the pancreas: is it safe and effective?

BACKGROUND: Pancreatic metastases are rare and only sparse data exists on treatment options. After recent advances in pancreatic surgery, metastasectomies have become promising treatment alternatives. METHODS: Twenty-six patients underwent pancreatic metastasectomy between 1991 and 2010 at our institution. Data was evaluated retrospectively. RESULTS: Renal cell carcinoma was the most common origin of pancreatic metastases (n = 16; 62%). Other primaries include gall bladder carcinoma, leiomyosarcoma, colon cancer (all n = 2), and others. The median time interval between primary tumor and pancreatic resection was 5.3 years [0-24]. Eleven pancreatic head resections (42%), fourteen distal pancreatectomies (54%), and one total pancreatectomy were performed (4%). The estimated 3- and 5-year survival rates were 73.2% and 52.3%, respectively. The estimated median overall survival was 63 months (CI: 37.8-88.1 months). There' was no perioperative death. The complication rate and relaparotomy rate was 31% and 19%, respectively. Patients suffering from synchronous metastases at the time of pancreatic surgery had a statistically significant shorter median overall survival time (11 months vs. 64 months). CONCLUSIONS: Despite the operative risk involved, we believe that pancreatic resection should be considered in selected patients with good performance status, stable disease and isolated pancreatic metastases.

Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana.

In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by ß-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.

Improved sulfur nutrition provides the basis for enhanced production of sulfur-containing defense compounds in Arabidopsis thaliana upon inoculation with Alternaria brassicicola.

The antifungal activities of many sulfur-containing defense compounds suggest a connection between pathogen infection, primary sulfur metabolism and sulfate nutritional status of plants. This relationship was investigated using Arabidopsis thaliana plants that were cultivated under different sulfur regimes and challenged by Alternaria brassicicola. Plants grown with 500 µM sulfate were significantly less infected compared to plants grown on 50 µM sulfate. Upon infection, the formation of the sulfur-containing defense compound camalexin and the gene expression of the sulfur-rich defense peptide defensin were clearly enhanced in plants grown with an optimal compared to a sufficient sulfate supply in the growth medium. Elevated levels of sulfite and O-acetylserine and cysteine biosynthetic enzymes after infection indicated a stimulation of sulfur metabolism under the higher sulfate supply. The results suggest that, in addition to pathogen-triggered activation of sulfur metabolism and sulfur-containing defense compound formation, the sulfate nutritional status is sensed to contribute to plant defense.

Sulfite reductase defines a newly discovered bottleneck for assimilatory sulfate reduction and is essential for growth and development in Arabidopsis thaliana.

The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of (35)S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses.

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells.

Cysteine (Cys) synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase. Why these reactions occur in plastids, mitochondria, and cytosol of plants remained unclear. Expression of artificial microRNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis (Arabidopsis thaliana) plants demonstrates that mitochondria are the most important compartment for the synthesis of O-acetylserine (OAS), the precursor of Cys. Reduction of RNA levels, protein contents, SAT enzymatic activity, and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real-time polymerase chain reaction and microarray analyses. Application of radiolabeled serine to leaf pieces revealed severely reduced incorporation rates into Cys and even more so into glutathione. Accordingly, steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction-related genes is accompanied by an accumulation of sulfate in amiSAT3 lines. These results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data, the cytosol appears to be a major site of Cys synthesis, while plastids contribute reduced sulfur as sulfide. Thus, Cys synthesis in plants is significantly different from that in nonphotosynthetic eukaryotes at the cellular level.