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Neuroinflammation within the superficial dorsal horn (SDH) of the spinal cord induces inflammatory pain with symptoms of hyperalgesia and allodynia. Glial activation and production of inflammatory mediators (e.g. cytokines) is associated with modulation of nociceptive signalling. In this context, medicinal signalling cells, e.g. obtained from adipose tissue (AdMSCs), gained attention due to their capacity to modulate the inflammatory response in several diseases, e.g. spinal cord injury. We applied the recently established mixed neuroglial primary cell culture of the rat SDH to investigate effects of AdMSCs on the inflammatory response of SDH cells. Following establishment of a co-cultivation system, we performed specific bioassays for tumour necrosis factor alpha (TNFα) and interleukin (IL)-6, RT-qPCR and immunocytochemistry to detect changes in cytokine production and glial activation upon inflammatory stimulation with lipopolysaccharide (LPS). LPS-induced expression and release of pro-inflammatory cytokines (TNFα, IL-6) by SDH cells was significantly attenuated in the presence of AdMSCs. Further evidence for anti-inflammatory capacities of AdMSCs derived from a blunted LPS-induced TNFα/IL-10 expression ratio and suppressed nuclear translocation of the inflammatory transcription factor nuclear factor kappa B (NFκB) in SDH microglial cells. Expression of IL-10, transforming growth factor beta (TGF-β) and TNFα-stimulated gene-6 (TSG-6) was detected in AdMSCs, which are putative candidates for anti-inflammatory capacities of these cells. We present a novel co-cultivation system of AdMSCs with neuroglial primary cultures of the SDH to investigate immunomodulatory effects of AdMSCs at a cellular level.

Chemotherapy-induced cognitive impairment (CICI) has been observed in a large fraction of cancer survivors. Although many of the chemotherapeutic drugs do not cross the blood-brain barrier, following treatment, the structure and function of the brain are altered and cognitive dysfunction occurs in a significant number of cancer survivors. The means by which CICI occurs is becoming better understood, but there still remain unsolved questions of the mechanisms involved. The hypotheses to explain CICI are numerous. More than 50% of FDA-approved cancer chemotherapy agents are associated with reactive oxygen species (ROS) that lead to oxidative stress and activate a myriad of pathways as well as inhibit pathways necessary for proper brain function. Oxidative stress triggers the activation of different proteins, one in particular is tumor necrosis factor alpha (TNFα). Following treatment with various chemotherapy agents, this pro-inflammatory cytokine binds to its receptors at the blood-brain barrier and translocates to the parenchyma via receptor-mediated endocytosis. Once in brain, TNFα initiates pathways that may eventually lead to neuronal death and ultimately cognitive impairment. TNFα activation of the c-jun N-terminal kinases (JNK) and Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathways may contribute to both memory decline and loss of higher executive functions reported in patients after chemotherapy treatment. Chemotherapy also affects the brain's antioxidant capacity, allowing for accumulation of ROS. This review expands on these topics to provide insights into the possible mechanisms by which the intersection of oxidative stress and TNFΑ are involved in chemotherapy-induced cognitive impairment.

We present a computational analysis of hexaphenylethane derivatives with heavier tetrels comprising the central bond. In stark contrast to parent hexaphenylethane, the heavier tetrel derivatives can readily be prepared. In order to determine the origin of their apparent thermodynamic stability against dissociation as compared to the carbon case, we employed local energy decomposition analysis (LED) and symmetry‐adapted perturbation theory (SAPT) at the DLPNO‐CCSD(T)/def2‐TZVP and sSAPT0/def2‐TZVP levels of theory. We identified London dispersion (LD) interactions as the decisive factor for the molecular stability of heavier tetrel derivatives. This stability is made possible owing to the longer (than C−C) central bonds that move the phenyl groups out of the heavily repulsive regime so they can optimally benefit from LD interactions., Carbon‐Carbon bonds are exceptional as demonstrated for the hexaphenylditetrels where attractive London dispersion interactions are the decisive factor for the thermodynamic stabilities of tetrels other than carbon. Structural and energetic comparisons show that even though hexaphenylethane displays the largest dispersion energy between the two molecular halves, it has remained elusive because of even larger Pauli repulsion of the phenyl moieties that are forced in close contact due to the C−C bond that is the shortest in this series.

Objective Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. Methods Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. Results At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. Conclusion A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

Pentelidene complexes of the type [Cp*E{W(CO)5}2] (Cp* = C5Me5; 1a: E = P, 1b: E = As) were reacted with the dichalcogenides R2Ch2 (R = Ph, Mes, Tipp; Ch = S, Se, Te; Mes = 2,4,6-trimethylphenyl; Tipp = 2,4,6-triisopropylphenyl) and the chalcogenols PhChH (Ch = S, Se). It has been shown that the formation of new E–Ch bonds proceeds under elimination of the Cp* substituent. The resulting chalcogenopentelidene complexes, which have been isolated and fully characterised, represent a novel class of phosphinidene complexes which can be synthesised through this general synthetic route.

The optimized superconducting stellarator device Wendelstein 7-X (with major radius , minor radius , and plasma volume) restarted operation after the assembly of a graphite heat shield and 10 inertially cooled island divertor modules. This paper reports on the results from the first high-performance plasma operation. Glow discharge conditioning and ECRH conditioning discharges in helium turned out to be important for density and edge radiation control. Plasma densities of with central electron temperatures were routinely achieved with hydrogen gas fueling, frequently terminated by a radiative collapse. In a first stage, plasma densities up to were reached with hydrogen pellet injection and helium gas fueling. Here, the ions are indirectly heated, and at a central density of a temperature of with was transiently accomplished, which corresponds to with a peak diamagnetic energy of and volume-averaged normalized plasma pressure . The routine access to high plasma densities was opened with boronization of the first wall. After boronization, the oxygen impurity content was reduced by a factor of 10, the carbon impurity content by a factor of 5. The reduced (edge) plasma radiation level gives routinely access to higher densities without radiation collapse, e.g. well above line integrated density and central temperatures at moderate ECRH power. Both X2 and O2 mode ECRH schemes were successfully applied. Core turbulence was measured with a phase contrast imaging diagnostic and suppression of turbulence during pellet injection was observed.

Background and aims Plant growth affects soil moisture, mineral N and organic C availability in soil, all of which influence denitrification. With increasing plant growth, root exudation may stimulate denitrification, while N uptake restricts nitrate availability. Methods We conducted a double labeling pot experiment with either maize (Zea mays L.) or cup plant (Silphium perfoliatum L.) of the same age but differing in size of their shoot and root systems. The 15N gas flux method was applied to directly quantify N2O and N2 fluxes in situ. To link denitrification with available C in the rhizosphere, 13CO2 pulse labeling was used to trace C translocation from shoots to roots and its release by roots into the soil. Results Plant water and N uptake were the main factors controlling daily N2O + N2 fluxes, cumulative N emissions, and N2O production pathways. Accordingly, pool-derived N2O + N2 emissions were 30–40 times higher in the treatment with highest soil NO3− content and highest soil moisture. CO2 efflux from soil was positively correlated with root dry matter, but we could not detect any relationship between root-derived C and N2O + N2 emissions. Conclusions Root-derived C may stimulate denitrification under small plants, while N and water uptake become the controlling factors with increasing plant and root growth.

Objectives The combination of lenalidomide + rituximab (R2) has shown complementary clinical activity and is a tolerable regimen in both untreated and relapsed or refractory (R/R) indolent non-Hodgkin lymphoma (NHL), as well as mantle cell lymphoma (MCL), an uncommon but aggressive form of NHL. The MAGNIFY phase 3 trial previously reported an ORR of 54% in patients with R/R MCL (Sharman J, et al. Hematol Oncol. 2019). Presented here are updated analyses from this trial. Materials and methods MAGNIFY is a multicenter, phase 3b trial (NCT01996865) in patients with R/R follicular lymphoma (FL) grades 1–3b, transformed FL, marginal zone lymphoma, and MCL. Lenalidomide 20 mg on d 1–21 of a 28-d cycle + rituximab 375 mg/m2/wk cycle 1 and then every 8 wk starting with cycle 3 (R2) is given for 12 cycles followed by 1:1 randomization in patients with stable disease, partial response, or complete response/complete response unconfirmed (CR/CRu) to R2 vs rituximab maintenance for 18 mo. The primary end point is progression-free survival (PFS) by 1999 International Working Group (IWG) criteria. Secondary end points include safety, CR rate, duration of response (DOR), duration of CR (DOCR), time-to-response (TTR), time-to-next antilymphoma therapy, and overall survival. This analysis evaluates the interim primary endpoint of overall response rate (ORR) by 1999 IWG criteria and safety of R2 induction in patients with MCL in the induction intention-to-treat population. Results As of August 28, 2020, 73 patients with MCL were enrolled (median age, 70.0 y [range, 51–88]); 89% had stage III/IV disease, and 41% had bulky disease (> 7 cm or > 3 cm ×3 lymph nodes). All patients had received prior rituximab-containing therapy, with 25 (34%) rituximab refractory (progression ≤ 6 mo after last rituximab dose). Seven patients (10%) had received prior ibrutinib. Median follow-up was 31.7 mo for patients still alive. ORR was 51%, with 34% CR rate (CR + CRu). Response rates were similar in patients refractory to rituximab (ORR = 48%, CR/CRu = 32%) and patients not refractory to rituximab (ORR = 52%, CR/CRu = 35%). Median DOR was 31.6 mo; median DOCR was not reached; median TTR was 2.8 mo, and median PFS was 28.0 mo, with 1-year PFS rate of 57%. The most common treatment emergent adverse events (TEAEs) of any grade were neutropenia (51%), fatigue (44%), diarrhea (32%), constipation (28%), cough (28%), dyspnea (26%), and nausea (26%). Grade 3/4 neutropenia was 46%; all other grade 3/4 TEAEs were ≤ 11%. Discussion R2 is an active and tolerated regimen with durable responses among patients with R/R MCL and mostly naive to Bruton tyrosine kinase inhibitor therapy. Conclusions These results suggest that R2 should be considered as a therapeutic option for patients with R/R MCL.

The nature of the bonding and magnetic exchange pathways of the water-oxidizing complex of photosystem 2 is explored using broken symmetry density functional theory. The electronic structure and superexchange pathways are illustrated and analyzed using corresponding orbitals and intrinsic bond orbitals. These demonstrate a dominating influence on the bonding and magnetic interactions by both the geometrical structure of the Mn4CaO5 core complex and the ionic interactions of the oxo bridges with the neighboring Ca2+ ion. The demonstrated ionic nature of the Ca2+ bonds is proposed to contribute to the stabilization of the oxygen atoms participating in O-O bond formation.

The standard of care for indolent non-Hodgkin lymphoma (iNHL) is rituximab, an anti-CD20 antibody, with/without chemotherapy. However, multiple relapses are common in these patients. This phase 3, randomized study compared outcomes of a combination of ofatumumab (a second-generation anti-CD20 antibody) and bendamustine, with bendamustine alone in patients unresponsive to prior rituximab-based treatment. Overall, 346 patients were randomized to receive either the combination or bendamustine alone. Bendamustine was given for ≤8 cycles and ofatumumab for ≤12 cycles. The primary end-point was progression-free survival (PFS) after 215 protocol-defined events assessed by independent review committee (IRC). Median IRC-assessed PFS was 16·7 and 13·8 months in the combination and monotherapy arms respectively [hazard ratio (HR) = 0·82; P = 0·1390]. Median overall survival (OS) was 58·2 and 51·8 months in the combination and monotherapy arms respectively (HR = 0·89, P = 0·4968). The safety profile was consistent with previous reports. Overall, 73% and 80% of patients in the combination and monotherapy arms, respectively, experienced a ≥grade 3 adverse event. The study did not meet its primary end-point. No significant improvement in PFS and OS was seen with the combination of ofatumumab and bendamustine as compared with bendamustine alone in rituximab-refractory iNHL (NCT01077518). ispartof: BRITISH JOURNAL OF HAEMATOLOGY vol:193 issue:6 pages:1123-1133 ispartof: location:England status: published

BACKGROUND: Eltrombopag is an oral, small-molecule, non-peptide thrombopoietin receptor agonist (TPO-RA) that increases hematopoiesis by inducing proliferation and differentiation of early bone marrow progenitor cells leading to increased platelet production (Erickson-Miller et al., 2010, Sun et al., 2012). Eltrombopag has demonstrated efficacy in adult and pediatric patients with immune thrombocytopenia (ITP) and has a well-established safety profile. It is approved in Europe for the treatment of thrombocytopenia, from 6 months following diagnosis, in patients with primary ITP who are refractory to other treatments (e.g. corticosteroids, immunoglobulins). The efficacy of eltrombopag for achieving hemostatic platelet counts (≥ 50 × 109/L) in previously treated adult ITP patients with more than 6 months' disease duration is around 80% (Wong et al., 2017). However, there is insufficient data on safety and efficacy of TPO-RAs in newly diagnosed ITP patients. AIM: The aim of this trial (NCT04346654; CETB115JDE01) is to compare the ability of eltrombopag in combination with a short course of high-dose dexamethasone to induce a sustained response off treatment in comparison to dexamethasone monotherapy in newly diagnosed primary ITP patients. METHODS: This is a Phase II, multicenter, randomized (1:1), open-label study (see Figure 1). Arm A: Eltrombopag + short course of high-dose dexamethasoneArm B: 1-3 cycles of high-dose dexamethasone Adult patients with newly diagnosed primary ITP who have platelet counts < 30 × 109/L and require treatment will be screened, and if eligible, will be randomized to either Arm A or Arm B. The study will be conducted in the following periods: Screening Period: Patients will be screened based on the inclusion and exclusion criteria specified below Treatment / Tapering Period: Arm A: Patients will receive eltrombopag in combination with a short course of high-dose dexamethasone beginning at day 1. The dose of dexamethasone will be 40 mg QD (daily; quaque die) for 4 consecutive days and limited to 1 cycle. The starting dose of eltrombopag will be 50 mg QD in order to achieve the target platelet count of ≥ 50 × 109/L. Patients who reach platelet counts ≥ 30 × 109/L and maintain counts ≥ 30 × 109/L during the tapering phase (week 20 - week 26) will be eligible for treatment discontinuation starting from week 26. During the tapering phase, eltrombopag will be decreased by 25 mg every 2 weeks to a minimum dose of 25 mg every other day for all patients. Arm B: Treatment in the control arm consists of 1-3 cycles of high-dose dexamethasone administered orally at a dose of 40 mg QD for 4 consecutive days at 4 weeks intervals. Patients will be treated up to 12 weeks during the treatment period with dexamethasone. If the platelet counts are > 150 × 109/L no further course of dexamethasone will be given. Patients who reach platelet counts ≥ 30 × 109/L and maintain counts ≥ 30 × 109/L after 1-3 cycles of dexamethasone treatment will be eligible for treatment discontinuation. Observation period: After completion of the treatment period, patients will be observed for sustained response off treatment defined as: maintain platelet counts ≥ 30 × 109/L after treatment discontinuation and no bleeding events ≥ Grade II and without the use of any rescue therapy until week 52 and week 78 respectively, after study start The study is designed to include 106 adult patients with newly diagnosed primary ITP at 25 sites in Germany. Patients meeting any of the following criteria are not eligible for inclusion in this study: Previous history of treatment for ITPPatients with diagnosis of secondary thrombocytopeniaPatients who have life threatening bleeding complications per physician´s discretionPatients with a history of thromboembolic events or known risk factors for thromboembolism The primary objective is the rate of sustained responses off treatment at 52 weeks. Key secondary objectives include the duration of sustained response off treatment, the rate of sustained response off treatment at 78 weeks as well as patient-oriented outcomes for health-related quality of life. Currently, the study is recruiting patients, expected to be completed by 2021. CONCLUSION: This trial will evaluate the potential of eltrombopag in combination with steroids to increase the rate of sustained response off treatment in comparison to steroids alone in patients with previously untreated primary ITP. Disclosures Matzdorff: Novartis Oncology:Consultancy, Other: Honoraria paid to institution;Amgen GmbH:Consultancy, Other: Honoraria paid to institution;Grifols Deutschland GmbH:Consultancy, Other: Honoraria paid to institution;Swedish Orphan Biovitrium GmbH:Consultancy, Other: Honoraria paid to institution;UCB Biopharma SRL:Consultancy, Other: Honoraria paid to institution;Roche Pharma AG:Other: Family stockownership.Binder:DGHO:Honoraria, Other: Speaker Activity;Amgen GmbH:Honoraria, Other: Speaker Activity;Deutsche Krebsgesellschaft:Honoraria, Other: Speaker Activity;Merck Serono GmbH:Honoraria, Other: Speaker Activity;OSHO:Membership on an entity's Board of Directors or advisory committees;Medconcept GmbH:Honoraria, Other: Speaker Activity;Tumorzentrum Anhalt:Honoraria, Other: Speaker Activity;Janssen-Cilag GmbH:Honoraria, Other: Speaker Activity;DFG:Research Funding;Art tempi:Honoraria, Other;Sanofi-Aventis Deutschland:Honoraria, Other;Uniklinikum Hamburg:Honoraria, Other;event lab GmbH:Honoraria, Other: Speaker Activity.Nimmerjahn:Novartis Pharma GmbH:Honoraria.Meyer:Amgen GmbH:Honoraria;Novartis Pharma GmbH:Honoraria;Grifols Germany:Consultancy, Honoraria.Rummel:Roche:Honoraria;Janssen:Honoraria;Novartis Pharma GmbH:Honoraria;Celgene:Consultancy, Honoraria;Amgen GmbH:Honoraria.Tesanovic:Novartis Pharma GmbH:Current Employment.Sauer:Novartis Pharma GmbH:Current Employment. OffLabel Disclosure: Eltrombopag is an oral, small-molecule, non-peptide thrombopoietin receptor agonist (TPO-RA) that increases hematopoiesis by inducing proliferation and differentiation of early bone marrow progenitor cells leading to increased platelet production (Erickson-Miller et al 2010, Sun et al 2012). Eltrombopag has demonstrated efficacy in adult and pediatric patients with ITP and has a well-established safety profile. It is approved in Europe for the treatment of thrombocytopenia, from 6 months following diagnosis, in patients with primary ITP who are refractory to other treatments (e.g. corticosteroids, immunoglobulins).

Zymosan, an immunogenic cell wall extract of Saccharomyces cerevisiae has potential for use as an experimental pyrogen. However, the short-lived sickness responses noted with intraperitoneal and intra-articular administration of zymosan limits investigations on the long-term effectiveness of antipyretic drugs. Thus, there remains a need to establish an alternative route of zymosan administration that could induce long-lived fevers and inflammation. We injected male Sprague Dawley rats (250–300 g) subcutaneously with zymosan (30 or 300 mg/kg) or saline; n = 7–8. We measured core body temperature, cage activity, food intake and body mass for 24 h after injection. Blood and brain samples were collected at 2, 8, and 18 h after injection. Zymosan (300 mg/kg) induced fever, lethargy, and anorexia, which lasted for 24 h. Zymosan-induced sickness responses were accompanied by increased blood plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α; activation of inflammatory transcription factors (nuclear factor (NF) for IL-6, signal transducer and activator of transcription (STAT)-3, and NF-κB) in the hypothalamus and circumventricular organs; and increased hypothalamic mRNA expression of TNF-α, IL-1β, and IL-6 and rate-limiting enzymes for prostaglandin synthesis. Our results confirm the suitability of subcutaneous administration of zymosan for screening antipyretic and anti-inflammatory drugs in rats.

Hydrogels constitute an important class of polymeric materials, mainly due to their promising applications in the biomedical field. Particularly, mechanical properties are a limiting factor for applications, and reinforcement and improved wear resistance are major directions in current research. In here, graphitic carbon nitride (g-CN)-based hydrogels are presented. The hydrogels are formed in a two-step procedure. In the first step, a prepolymer is synthesized that leads to a significantly improved g-CN dispersibility and stability of g-CN dispersions. Hence, the g-CN content in the final hydrogels can be increased to obtain tough hydrogels. More importantly, hydrogels from charged monomers can be formed as well, which leads to lubricant properties. As such, the investigated route opens up new opportunities to fabricate functional hydrogels with significant toughness, compressibility and lubricity.

Warming or cooling of the median preoptic nucleus (MnPO) in-vivo evokes appropriate thermoregulatory responses. In the present study, we aimed to investigate whether single neurons (and astrocytes) of primary rat MnPO cell cultures maintain properties, which are consistent with their putative role within the central thermoregulatory structures. Using the fura-2 ratio imaging technique, we therefore measured changes of intracellular Ca concentrations ([Ca]i) in neurons of rat MnPO primary cultures stimulated by rapid cooling from 37 to 25°C, or warming from 37 to 45°C, or glutamate, the transmitter which transfers thermal information to MnPO neurons. In the first experiment, we tested the responses to external cooling in a group of 212 neurons. Overall, 165 of these neurons were responsive to stimulation with glutamate; just four of them responded to the cold-stimulus with an increase of [Ca]i, and only one of these neurons was responsive to stimulation with menthol. In the second experiment, 24 of 327 neurons and 23 of 241 astrocytes responded to external warming with quick and pronounced Ca signals. Another 33 (10%) neurons showed a moderate and slowly developing increase of [Ca]i during the warming, which reflected the temperature changes in the chamber. These data correspond to properties of MnPO neurons upon thermal stimulation obtained by other experimental approaches. Primary cultures derived from the rat MnPO can thus be used to investigate neuronal thermosensitive properties and their possible modulation by other stimuli.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic β-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery., The translocation domain (HN) of Botulinum neurotoxins (BoNTs) mediates the delivery of the BoNT light chain (LC) into neuronal cytosol. Here the authors provide insights into HN membrane insertion by determining the crystal structure of BoNT/A1 HN at acidic pH, which reveals a molecular switch in HN, where buried α-helices are transformed into surface-exposed hydrophobic β-hairpins.

Phage display technology (PD) is a powerful technique for the generation of tumor‑targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell‑based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell‑based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast‑binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor‑targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML‑binding phages during panning. Furthermore, suggesting a common design‑related mechanism using a 'single‑pot' PD library, such as the well‑known Tomlinson single‑chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast‑binding phage particles were isolated and soluble scFv‑Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv‑Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea, therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

The main prerequisites for denitrification are availability of nitrate (NO3-) and easily decomposable organic substances, and oxygen deficiency. Growing plants modify all these parameters and may thus play an important role in regulating denitrification. Previous studies investigating plant root effects on denitrification have found contradictive results. Both increased and decreased denitrification in the presence of plants have been reported and were associated with higher Corg or lower NO3- availability, respectively. Accordingly, it is still unclear whether growing plants stimulate denitrification through root exudation or restrict it through NO3- uptake. Furthermore, reliable measurements of N2 fluxes and N2O/(N2O+N2) ratios in the presence of plants are scarce.Therefore, we conducted a double labeling pot experiment with either maize (Zea mays L.) or cup plant (Silphium perfoliatum L.) of the same age but differing in size of their shoot and root systems. The 15N gas flux method was applied to directly quantify N2O and N2 fluxes in situ. To link denitrification with available C in the rhizosphere, 13CO2 pulse labeling was used to trace C translocation from shoots to roots and its release by roots into the soil.Plant water uptake was a main factor controlling soil moisture and, thus, daily N2O+N2 fluxes, cumulative N emissions, and N2O production pathways. However, N fluxes remained on a low level when NO3- availability was low due to rapid plant N uptake. Only when both N and water uptake were low, high NO3- availability and high soil moisture led to strongly increased denitrification-derived N losses.Total CO2 efflux was positively correlated with root dry matter, but there was no indication of any relationship between recovered 13C from root exudation and cumulative N emissions. We anticipate that higher Corg availability in pots with large root systems did not lead to higher denitrification rates, as NO3- was limiting denitrification due to plant N uptake. Overall, we conclude that root-derived C stimulates denitrification only when soil NO3- is not limited and low O2 concentrations enable denitrification. Thus, root-derived C may stimulate denitrification under small plants, while N and water uptake become the controlling factors with increasing plant and root growth.

Environmental science & technology 55(16), 11006-11018 (2021). doi:10.1021/acs.est.0c07875, Published by American Chemical Society, Columbus, Ohio

Nuclear-structure effects often provide an irreducible theory error that prevents using precision atomic measurements to test fundamental theory. We apply newly developed effective field theory tools to Hydrogen atoms, and use them to show that (to the accuracy of present measurements) all nuclear finite-size effects (e.g. the charge radius, Friar moments, nuclear polarizabilities, recoil corrections, Zemach moments {\it etc.}) only enter into atomic energies through exactly two parameters, independent of any nuclear-modelling uncertainties. Since precise measurements are available for more than two atomic levels in Hydrogen, this observation allows the use of precision atomic measurements to eliminate the theory error associated with nuclear matrix elements. We apply this reasoning to the seven atomic measurements whose experimental accuracy is smaller than 10 kHz to provide predictions for nuclear-size effects whose theoretical accuracy is not subject to nuclear-modelling uncertainties and so are much smaller than 1 kHz. Furthermore, the accuracy of these predictions can improve as atomic measurements improve, allowing precision fundamental tests to become possible well below the 'irreducible' error floor of nuclear theory., 5 pages, 2 tables; This paper summarizes our results from arXiv:2008.09718

Wound healing is a complex process, and disturbance of even a single mechanism can result in chronic ulcers developing after exposure to the alkylating agent sulfur mustard (SM). A possible contributor may be SM-induced chronic senescent mesenchymal stem cells (MSCs), unable to fulfil their regenerative role, by persisting over long time periods and creating a proinflammatory microenvironment. Here we show that senescence induction in human bone marrow derived MSCs was time- and concentration-dependent, and chronic senescence could be verified 3 weeks after exposure to between 10 and 40 µM SM. Morphological changes, reduced clonogenic and migration potential, longer scratch closure times, differences in senescence, motility and DNA damage response associated genes as well as increased levels of proinflammatory cytokines were revealed. Selective removal of these cells by senolytic drugs, in which ABT-263 showed initial potential in vitro, opens the possibility for an innovative treatment strategy for chronic wounds, but also tumors and age-related diseases. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-020-02946-5.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 μg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 μg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

Growing plants affect soil moisture, mineral N and organic C (Corg) availability in soil and may thus play an important role in regulating denitrification. The availability of the main substrates for denitrification (Corg and NO3-) is controlled by root activity and higher denitrification activity in rhizosphere soils has been reported. We hypothesized that (I) plant N uptake governs NO3- availability for denitrification leading to increased N2O and N2 emissions, when plant N uptake is low due to smaller root system or root senescence. (II) Denitrification is stimulated by higher Corg availability from root exudation or decaying roots increasing total gaseous N emissions while decreasing their N2O/(N2O+N2) ratios.We tested these assumptions in a double labeling pot experiment with maize (Zea mays L.) grown under three N fertilization levels S / M / L (no / moderate / high N fertilization) and with cup plant (Silphium perfoliatum L., moderate N fertilization). After 6 weeks, all plants were labeled with 0.1 g N kg-1 (Ca(15NO3)2, 60 at%), and the 15N tracer method was applied to estimate plant N uptake, N2O and N2 emissions. To link denitrification with available C in the rhizosphere, 13CO2 pulse labeling (5 g Na213CO3, 99 at%) was used to trace C translocation from shoots to roots and its release by roots into the soil. CO2 evolving from soil was trapped in NaOH for δ13C analyses, and gas samples were taken for analysis of N2O and N2 from the headspace above the soil surface every 12 h.Although pots were irrigated, changing soil moisture through differences in plant water uptake was the main factor controlling daily N2O+N2 fluxes, cumulative N emissions, and N2O production pathways. In addition, total N2O+N2 emissions were negatively correlated with plant N uptake and positively with soil N concentrations. Recently assimilated C released by roots (13C) was positively correlated with root dry matter, but we could not detect any relationship with cumulative N emissions. We anticipate that higher Corg availability in pots with large root systems did not lead to higher denitrification rates as NO3- was limited due to plant uptake. In conclusion, plant growth controlled water and NO3- uptake and, subsequently, formation of anaerobic hotspots for denitrification.

Denitrification usually takes place under anoxic conditions and over short periods of time and depends on readily available nitrate and carbon sources. Variations in CO2 and N2O emissions from soils amended with plant residues have mainly been explained by differences in their decomposability. Another factor rarely considered so far is water-extractable organic matter (WEOM) released into soil during residue decomposition. Here, we examined the potential effect of plant residues on denitrification with special emphasis on WEOM. A range of fresh and leached plant residues was characterized by elemental analyses, 13C-NMR spectroscopy, and extraction with ultrapure water. The obtained solutions were analyzed for the concentration of organic carbon (OC), organic nitrogen (ON), and by UV-VIS spectroscopy. To test the potential denitrification induced by plant residues or three different OM solutions, these carbon sources were added to soil suspensions and incubated for 24 hours at 20 °C in the dark under anoxic conditions; KNO3 was added to ensure unlimited nitrate supply. Evolving N2O and CO2 were analyzed by gas chromatography and acetylene inhibition was used to determine denitrification and its product ratio. The production of all gases as well as the molar N2O+N2-N/CO2-C ratio was directly related to the water-extractable OC (WEOC) content of the plant residues and the WEOC increased with carboxylic/carbonyl C and decreasing OC/ON ratios of the plant residues. Incubation of OM solutions revealed that the molar N2O+N2-N/CO2-C ratio and share of N2O are influenced by the WEOM’s chemical composition. In conclusion, the effect of plant residues on potential denitrification is governed by their composition and the related production of WEOM.

Silicones have many applications and are produced in large quantities. Despite their potential toxicity, information on their environmental mineralisation is scarce. Therefore, we investigated a group of five organosilicon compounds (o-MeOC 6H 4SiMe 3 (1), p-MeOC 6H 4SiMe 3 (2), (p-MeOC 6H 4) 2SiMe 2 (3), o-Me 2NC 6H 4SiMe 3 (4) and p-Me 2NC 6H 4SiMe 3 (5)), recently developed to be ‘benign by design’ based on their readily degradable core structure. Five different degradability tests were performed, one assessing hydrolytic and two analysing biological and photolytic stability, respectively. All substances, except (p-MeOC 6H 4) 2SiMe 2 (3), hydrolysed within 24 h to 50% indicating that this is one of the major pathways of their primary elimination. In agreement with previous research, none of the substances was readily biodegradable. In contrast, 100% of p-Me 2NC 6H 4SiMe 3 (5) was primarily eliminated by photolytic and hydrolytic processes. The elimination rates of the other substances ranged from 7% to 64%. Irradiation at shorter wavelengths increased both the extent and speed of photodegradation. Eleven transformation products of p-Me 2NC 6H 4SiMe 3 (5) were detected, all of which were completely eliminated within 64 min of irradiation with a Hg lamp (200–400 nm). The insertion of an electron-donating group on the benzene ring like in p-Me 2NC 6H 4SiMe 3 (5) clearly enhanced photolytic degradability but further research is necessary to achieve truly biodegradable silicones.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

Existing root viability methods tend to be time consuming, finicky, and sometimes subjective. Plate reader assays are rapid but often limited to homogeneous liquid samples. The aim of the study was to develop a rapid, reliable, and quantitative method to determine the viability of plant root samples using an area-scan plate reader. We modified existing fluorescein diacetate (FDA) methods, and optimized incubation time and root volume using pea roots (Pisum sativum) of known viability. We then compared our method to FDA microscopy and triphenyl tetrazolium chloride (TTC) reduction using pea roots and field-collected grass, forb, and tree roots. Finally, we used fungal hyphae to assess the suitability of our method to broader applications. Our method was as accurate and precise as FDA microscopy, more precise than TTC, and twice as fast as the other methods yet similar in cost. Field-collected root viability was similar between our method and FDA microscopy, but not TTC. We were able to detect viability of fungal hyphae with our method, although further development is needed.

In the interaction of cyclohexa-1,4-diene (1,4-CHD) with a mixture of biphenyl and metallic lithium or sodium in THF at 20 °C, three processes occur, viz., disproportionation of 1,4-CHD to form benzene and cyclohexene, dehydrogenation of 1,4-CHD to form benzene and molecular hydrogen, and dehydrogenation of 1,4-CHD to form benzene and lithium or sodium hydride. In the case of lithium on the use of an equimolar amount of biphenyl, the isomerization of 1,4-CHD to cyclohexa-1,3-diene is also observed. When the molar ratio Li(Na): Ph2 increases from 1 : 1 to 2 : 1, i.e., when the reaction is carried out in the presence of an alkali metal solid phase, the overall conversion of 1,4-CHD into benzene and cyclohexene increases. The use of mixtures of lithium and sodium leads to acceleration of the processes of the formation of benzene and cyclohexene. The possible mechanism of the synergistic effect found is discussed.

Brewer’s yeast, derived from the yeast species Saccharomyces cerevisiae (S. cerevisiae), is commonly used for inducing pyrexia in pharmacological studies screening antipyretics in rats. Despite its widespread use, the peripheral and central inflammatory response associated with Brewer’s yeast-induced fever and sickness behavior in rats has not been investigated. Thus, we injected male Sprague-Dawley rats (150–200 g) subcutaneously with a high (4 g/kg, n = 9), medium (2 g/kg, n = 5) or low (0.4 g/kg, n = 6) dose of Brewer’s yeast solution or saline (0.9%, n = 6) and measured core body temperature, cage activity, food intake and body mass for six days after injection. Blood and brain samples were collected at 2, 8, 18 and 72 h after injection; n = 5–7 per time point. Brewer’s yeast administration dose-dependently induced fever, lethargy, anorexia and body mass stunting that was accompanied by increased blood plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and activation of inflammatory transcription factors (nuclear factor (NF) for interleukin-6, signal transducer and activator of transcription (STAT)-3, and NF-κB)) in the hypothalamus and circumventricular organs. The increased activation of transcription factors following Brewer’s yeast administration was accompanied by increased hypothalamic mRNA expression of TNF-α, IL-1β and IL-6 and rate-limiting enzymes for prostaglandin synthesis. Our results show that subcutaneous administration of S. cerevisae induces prolonged fever, anorexia and lethargy that is accompanied by a pronounced increase in the synthesis of pro-inflammatory cytokines, key prostaglandin synthesizing enzymes and transcription factors, in the periphery and brain.

A silicon heterostructure photovoltaic system fielded for 10 years has been investigated in detail. The system has shown degradation, but at a rate similar to an average Si system, and still within the module warranty level. The power decline is dominated by a nonlinear V oc loss rather than more typical changes in I sc or Fill Factor. Modules have been evaluated using multiple techniques including: dark and light I – V measurement, Suns- V oc, thermal imaging, and quantitative electroluminescence. All techniques indicate that recombination and series resistance in the cells have increased along with a decrease of factor 2 in minority carrier lifetime. Performance changes are fairly uniform across the module, indicating changes occur primarily within the cells.

Arsenic's high vapor pressure leads to thermal instability during high-temperature processing (>370 °C) of GaAs, contributing to the performance degradation of subsequently fabricated devices. The resulting surface damage also obfuscates the exact quantitative characterization of the diffusion process, a critical step in device manufacturing. In this experiment, an encapsulant-and-sacrificial-layer procedure is employed to reduce arsenic sublimation and preserve a smooth surface. A capped GaAs/InGaAs/GaAs quantum well structure is subjected to rapid thermal annealing, and AFM, SEM, and energy-dispersive x-ray spectroscopy are used to compare the surface qualities of the postannealed encapsulated GaAs against the reference GaAs. For the encapsulated substrate, a smooth surface with an average root-mean-squared value of 6.5 A is achieved after high-temperature processing. SIMS analysis is used to obtain the diffused indium atomic concentration profiles for a smooth and roughened GaAs surface and their corresponding diffusion parameters. The interdiffusion characterization process demonstrates in a GaAs/InGaAs system how precise diffusion parameter extraction requires preserving an atomically smooth surface when using surface-sensitive techniques.

The plasma fusion experiment Wendelstein 7-X (W7-X) uses a system of 50 nonplanar and 20 planar superconducting coils. These coils produce the magnetic field that is required to confine the plasma. Magnetic flux densities up to 3 tesla can be reached in the center of the plasma. Supercritical helium is used to cool down the coils to operating temperatures below 4 K. The coils are specified for currents up to 18 kA. A critical issue in all superconductors is the occurrence of quenches. These are unwanted local transitions from superconductivity to normal conductivity. If that happens, the coil current has to be discharged as fast as possible into a dump resistor. However, the strong current change will produce a self-induced high voltage (HV) with values up to several kilovolts. Therefore, the electrical coil insulation versus ground has to be HV proof to avoid a high-current arc in any circumstance. Worst case scenario is a quench that is induced by a loss of thermal insulation after a leak in the helium supply lines, because Paschen-minimum conditions could be given. This article describes some HV test procedures and techniques employed to test and qualify the coil system against that scenario. Some of these techniques are used today for the routine coil tests. Some other turned out as inefficient for daily use. Essential for a safe machine operation is the detection of insulation defects, but also on their localization along the superconductor, for the sake of a later repair.

Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memory formation; however, its contribution in the pathophysiology of depressive disorders is not fully understood. We investigated the involvement of MNK1/2 (MAPK-interacting serine/threonine-protein kinase 1 and 2) and their target, eIF4E (eukaryotic initiation factor 4E), in depression-like behavior in mice. Mice carrying a mutation in eIF4E for the MNK1/2 phosphorylation site (Ser209Ala, Eif4e ki/ki), the Mnk1/2 double knockout mice (Mnk1/2−/−), or mice treated with the MNK1/2 inhibitor, cercosporamide, displayed anxiety- and depression-like behaviors, impaired serotonin-induced excitatory synaptic activity in the prefrontal cortex, and diminished firing of the dorsal raphe neurons. In Eif4e ki/ki mice, brain IκBα, was decreased, while the NF-κB target, TNFα was elevated. TNFα inhibition in Eif4e ki/ki mice rescued, whereas TNFα administration to wild-type mice mimicked the depression-like behaviors and 5-HT synaptic deficits. We conclude that eIF4E phosphorylation modulates depression-like behavior through regulation of inflammatory responses., Translation of mRNA contributes to neuronal function and complex behaviours, and inflammation is thought to contribute to depression. Here the authors show that mice lacking phosphorylation sites in eIF4E (eukaryotic initiation factor 4E) display anxiety- and depression-like behaviour and decreased IkBα expression; furthermore TNFα delivery to the medial prefrontal cortex induces depression-like behaviour and deficits in serotonergic transmission.

Million of acres of U.S. wildlands are sprayed with herbicides to control invasive species, but relatively little is known about non-target effects of herbicide use. We combined greenhouse, field, and laboratory experiments involving the invasive forb spotted knapweed (Centaurea stoebe) and native bunchgrasses to assess direct and indirect effects of the forb-specific herbicide picloram on arbuscular mycorrhizal fungi (AMF), which are beneficial soil fungi that colonize most plants. Picloram had no effect on bunchgrass viability and their associated AMF in the greenhouse, but killed spotted knapweed and reduced AMF colonization of a subsequent host grown. Results were similar in the field where AMF abundance in bunchgrass-dominated plots was unaffected by herbicides one year after spraying based on 16:1ω5 phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) concentrations. In spotted-knapweed-dominated plots, however, picloram application shifted dominance from spotted knapweed, a good AMF host, to bulbous bluegrass (Poa bulbosa), a poor AMF host. This coincided with a 63% reduction in soil 16:1ω5 NLFA concentrations but no reduction of 16:1ω5 PLFA. Because 16:1ω5 NLFA quantifies AMF storage lipids and 16:1ω5 PLFA occurs in AMF membrane lipids, we speculate that the herbicide-mediated reduction in host quality reduced fungal carbon storage, but not necessarily fungal abundance after one year in the field. Overall, in greenhouse and field experiments, AMF were only affected when picloram altered host quantity and quality. This apparent lack of direct effect was supported by our in-vitro trial where picloram applied to AMF mycelia did not reduce fungal biomass and viability. We show that the herbicide picloram can have profound, indirect effects on AMF within one year. Depending on herbicide-mediated shifts in host quality, rapid interventions may be necessary post herbicide applications to prevent loss of AMF abundance. Future research should assess consequences of these potential shifts for the restoration of native plants that differ in mycorrhizal dependency.

Reactions between the 1,3-diphosphete complex [Cp'''Co(η4-P2C2tBu2)] (1) and Ag[Al{OC(CF3)3}4] (Ag[pftb]) were carried out. In CH2Cl2, the unprecedented 1,2-diphosphete isomerization product [Ag2{Cp'''Co(µ,η4:η1:η1-1,2-P2C2tBu2)}2{Cp'''Co(µ,η4:η1-1,2-P2C2tBu2)}2]·2[pftb] (2) could be isolated. In diffusion experiments of 1 in hexane with Ag[pftb] in CH2Cl2, the triphosphacobaltocenium complex [Cp'''Co(η5-P3C2tBu2)][pftb] (4) and the phosphirenylium complex [Cp'''Co(η3-PC2tBu2)][pftb] (5) were obtained, showing a ring expansion and a ring contraction, respectively, under mild conditions. Moreover, by addition of pyridine to the Ag complex 2, the novel 1,2-diphosphete complex [Cp'''Co(η4-1,2-P2C2tBu2)] (3) was released. The formation of 3 has also shown to be possible by thermolysis of 1. Compound 3 represents a rare example of the family of the 1,2-diphosphete complexes, which are thermodynamically more stable but also synthetically more elusive than their 1,3-isomer counterparts.

The presence of microplastic (MP) in the aquatic environment is recognized as a global-scale pollution issue. Secondary MP particles result from an ongoing fragmentation process governed by various biotic and abiotic factors. For a reliable risk assessment of these MP particles, knowledge about interactions with biota is needed. However, extensive testing with standard organisms under reproducible laboratory conditions with well-characterized MP suspensions is not available yet. As MP in the environment represents a mixture of particles differing in properties (e.g., size, color, polymer type, surface characteristics), it is likely that only specific particle fractions pose a threat towards organisms. In order to assign hazardous effects to specific particle properties, these characteristics need to be analyzed. As shown by the testing of particles (e.g. nanoparticles), characteristics other than chemical properties are important for the emergence of toxicity in organisms, and parameters such as surface area or size distribution need consideration. Therefore, the use of "well-defined" particles for ecotoxicological testing (i.e., standard particles) facilitates the establishment of causal links between physical-chemical properties of MP particles and toxic effects in organisms. However, the benefits of well-defined particles under laboratory conditions are offset by the disadvantage of the unknown comparability with MP in the environment. Therefore, weathering effects caused by biological, chemical, physical or mechanical processes have to be considered. To date, the characterization of the progression of MP weathering based on powder and suspension characterization methods is in its infancy. The aim of this commentary is to illustrate the prerequisites for testing MP in the laboratory from 3 perspectives: (i) knowledge of particle properties; (ii) behavior of MP in test setups involving ecotoxicological test organisms; and (iii) accordingly, test conditions that may need adjustment. Only under those prerequisites will reliable hazard assessment of MP be feasible. Integr Environ Assess Manag 2017;13:500-504. © 2017 SETAC.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins&rsquo, light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A&ndash, F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A&ndash, F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

BackgroundGabapentinoids are known to reduce neuropathic pain. The aim of this experimental study was to investigate whether gabapentinoids exert anti-inflammatory and/or anti-nociceptive effects at the cellular level using primary cultures of rat dorsal root ganglia (DRG).MethodsCells from rat DRG were cultured in the presence of gabapentin or pregabalin, and we tested the effects of subsequent stimulation with lipopolysaccharide (LPS) on the expression of genes (real-time polymerase chain reaction) and production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) by specific bioassays. Using Ca2+imaging, we further investigated in neurons the effects of gabapentinoids upon stimulation with the TRPV-1 agonist capsaicin.ResultsThere is a small influence of gabapentinoids on the inflammatory response to LPS stimulation, namely, a significantly reduced expression of IL-6. Pregabalin and gabapentin further seem to exert a moderate inhibitory influence on capsaicin-induced Ca2+signals in DRG neurons.ConclusionsAlthough the single inhibitory effects of gabapentinoids on inflammatory and nociceptive responses are moderate, a combination of both effects might provide an explanation for the proposed function of these substances as an adjuvant for the reduction of neuropathic pain.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins’ high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3–80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.