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Radiotherapy (RT) along with surgery is the mainstay of treatment in head and neck squamous cell carcinoma (HNSCC). Radioresistance represents a major source of treatment failure, underlining the urgent necessity to explore and implement effective radiosensitization strategies. The MET receptor widely participates in the acquisition and maintenance of an aggressive phenotype in HNSCC and modulates the DNA damage response following ionizing radiation (IR). Here, we assessed MET expression and mutation status in primary and metastatic lesions within a cohort of patients with advanced HNSCC. Moreover, we investigated the radiosensitization potential of the MET inhibitor tepotinib in a panel of cell lines, in vitro and in vivo, as well as in ex vivo patient-derived organotypic tissue cultures (OTC). MET was highly expressed in 62.4% of primary tumors and in 53.6% of lymph node metastases (LNM), and in 6 of 9 evaluated cell lines. MET expression in primaries and LNMs was significantly associated with decreased disease control in univariate survival analyses. Tepotinib abrogated MET phosphorylation and to distinct extent MET downstream signaling. Pretreatment with tepotinib resulted in variable radiosensitization, enhanced DNA damage, cell death, and G2–M-phase arrest. Combination of tepotinib with IR led to significant radiosensitization in one of two tested in vivo models. OTCs revealed differential patterns of response toward tepotinib, irradiation, and combination of both modalities. The molecular basis of tepotinib-mediated radiosensitization was studied by a CyTOF-based single-cell mass cytometry approach, which uncovered that MET inhibition modulated PI3K activity in cells radiosensitized by tepotinib but not in the resistant ones.

Supplementary Table 1 shows HNSCC patients' characteristics

Supplementary figure 1 provides data concerning establishment of organotypic tissue cultures from freshly-resected HNSCC specimen Supplementary figure 2 shows analyses of MET expression and mutational status in a HNSCC cohort Supplementary figure 3 shows impact of MET inhibition by tepotinib in HNSCC cell lines in vitro Supplementary figure 4 shows effects of combined treatment by tepotinib and ionizing radiation on HNSCC cell lines Supplementary figure 5 shows analyses of OTCs responses to treatment by tepotinib combined with ionizing radiation Supplementary figure 6 shows results of assays studying mechanisms underlying tepotinib-mediated radiosensitization of HNSCC cell lines

Supplementary Methods

Supplementary Table 2 provides odds ratio per protein (CyTOF measurements)

Dose-dependent effects of ketamine on processed electroencephalographic depth-of-hypnosis indices have been reported. Limited data are available for the NeuroSENSE WAVCNS index. Our aim was to establish the feasibility of closed-loop propofol-remifentanil anesthesia guided by the WAVCNS index in the presence of an analgesic dose of ketamine. Thirty ASA I-II adults, 18–54 years, requiring general anesthesia for anterior cruciate ligament surgery were randomized to receive: full-dose [ketamine, 0.5 mg kg−1 initial bolus, 10 mcg kg−1 min−1 infusion] (recommended dose for postoperative pain management); half-dose [ketamine, 0.25 mg kg−1 bolus, 5 mcg kg−1 min−1 infusion]; or control [no ketamine]. After the ketamine bolus, patients received 1.0 mcg kg−1 remifentanil over 30 s, then 1.5 mg kg−1 propofol over 30 s, followed by manually-adjusted propofol-remifentanil anesthesia. The WAVCNS was > 60 for 7/9 patients in the full-dose group at 7 min after starting the propofol infusion. This was inconsistent with clinical observations of depth-of-hypnosis and significantly higher than control (median difference [MD] 17.0, 95% confidence interval [CI] 11.4–26.8). WAVCNS was median [interquartile range] 49.3 [42.2–62.6] in the half-dose group, and not different to control (MD 5.1, 95% CI − 4.9 to 17.9). During maintenance of anesthesia, the WAVCNS was higher in the full-dose group compared to control (MD 14.7, 95% CI 10.2–19.2) and in the half-dose group compared to control (MD 11.4, 95% CI 4.7–20.4). The full-dose of ketamine recommended for postoperative pain management had a significant effect on the WAVCNS. This effect should be considered when using the WAVCNS to guide propofol-remifentanil dosing. Trial Registration ClinicalTrails.gov No. NCT02908945.

Background: Ondansetron is a highly effective antiemetic for the treatment of nausea and vomiting. However, this medication has also been associated with QT prolongation. Pharmacogenomic information on therapeutic response to ondansetron exists, but no investigation has been performed on genetic factors that influence the cardiac safety of this medication. Methods: Three patient groups receiving ondansetron were recruited and followed prospectively (pediatric post-surgical patients n = 101; pediatric oncology patients n = 98; pregnant women n = 62). Electrocardiograms were conducted at baseline, and 5- and 30-min post-ondansetron administration, to determine the effect of ondansetron treatment on QT interval. Pharmacogenomic associations were assessed via analyses of comprehensive CYP2D6 genotyping and genome-wide association study data. Results: In the entire cohort, 62 patients (24.1%) met the criteria for prolonged QT, with 1.2% of the cohort exhibiting unsafe QT prolongation. The most significant shift from baseline occurred at five minutes post-ondansetron administration (P = 9.8 × 10−4). CYP2D6 activity score was not associated with prolonged QT. Genome-wide analyses identified novel associations with a missense variant in TLR3 (rs3775291; P = 2.00 × 10−7) and a variant linked to the expression of SLC36A1 (rs34124313; P = 1.97 × 10−7). Conclusions: This study has provided insight into the genomic basis of ondansetron-induced cardiac changes and has emphasized the importance of genes that have been implicated in serotonin-related traits. These biologically-relevant findings represent the first step towards understanding this adverse event with the overall goal to improve the safety of this commonly used antiemetic medication.

The paper accompanying the OptiFit algorithm is now out in mSphere! https://journals.asm.org/doi/10.1128/msphere.00916-21 Assigning amplicon sequences to operational taxonomic units (OTUs) is an important step in characterizing microbial communities across large data sets. A notable difference betweende novoclustering and database-dependent reference clustering methods is that OTU assignments fromde novomethods may change when new sequences are added. However, one may wish to incorporate new samples to previously clustered data sets without clustering all sequences again, such as when comparing across data sets or deploying machine learning models. Existing reference-based methods produce consistent OTUs but only consider the similarity of each query sequence to a single reference sequence in an OTU, resulting in assignments that are worse than those generated byde novomethods. To provide an efficient method to fit sequences to existing OTUs, we developed the OptiFit algorithm. Inspired by thede novoOptiClust algorithm, OptiFit considers the similarity of all pairs of reference and query sequences to produce OTUs of the best possible quality. We tested OptiFit using four data sets with two strategies: (i) clustering to a reference database and (ii) splitting the data set into a reference and query set, clustering the references using OptiClust, and then clustering the queries to the references. The result is an improved implementation of reference-based clustering. OptiFit produces OTUs of a quality similar to that of OptiClust at faster speeds when using the split data set strategy. OptiFit provides a suitable option for users requiring consistent OTU assignments at the same quality as afforded byde novoclustering methods.

Dexmedetomidine is a highly selective α2-adrenergic agonist, which is increasingly used in pediatric anesthesia and intensive care. Potential adverse effects that have not been rigorously evaluated in children include its effects on myocardial repolarization, which is important given that the drug is listed as a possible risk factor for torsades de pointes. We investigated the effect of 3 different doses of dexmedetomidine on myocardial repolarization and transmural dispersion in children undergoing elective surgery with total IV anesthesia.Sixty-four American Society of Anesthesiologists I-II children 3-10 years of age were randomized to receive dexmedetomidine 0.25 µg/kg, 0.5 µg/kg, 0.75 µg/kg, or 0 µg/kg (control), as a bolus administered over 60 seconds, after induction of anesthesia. Pre- and postintervention 12-lead electrocardiograms were recorded. The interval between the peak and the end of the electrocardiogram T wave (Tp-e; transmural dispersion) and heart rate-corrected QT intervals (myocardial repolarization) were measured by a pediatric electrophysiologist blinded to group allocation. Data were analyzed using an analysis of covariance regression model. The study was powered to detect a 25-millisecond difference in Tp-e.Forty-eight children completed the study, with data analyzed from 12 participants per group. There were no instances of dysrhythmias. Tp-e values were unaffected by dexmedetomidine administration at any of the studied doses (F = 0.09; P = .96). Mean (99% CI) within-group differences were all2 milliseconds (-5 to 8). Postintervention, corrected QT interval increased in the control group, but decreased in some dexmedetomidine groups (F = 7.23; P.001), specifically the dexmedetomidine 0.5 and 0.75 µg/kg doses. Within groups, the mean (99% CI) differences between pre- and postintervention corrected QT interval were 12.4 milliseconds (-5.8 to 30.6) in the control group, -9.0 milliseconds (-24.9 to 6.9) for dexmedetomidine 0.25 µg/kg, -18.6 milliseconds (-33.7 to -3.5) for dexmedetomidine 0.5 µg/kg, and -14.1 milliseconds (-27.4 to -0.8) for dexmedetomidine 0.75 µg/kg.Of the bolus doses of dexmedetomidine studied, none had an effect on Tp-e and the dexmedetomidine 0.5 and 0.75 µg/kg doses shortened corrected QT intervals when measured at 1 minute after dexmedetomidine bolus injection during total IV anesthesia. There is no evidence for an increased risk of torsades de pointes in this context.

Dexmedetomidine is a highly selective α2-adrenergic agonist, which is increasingly used in pediatric anesthesia and intensive care. Potential adverse effects that have not been rigorously evaluated in children include its effects on blood glucose and serum potassium concentrations, which are relevant due to the associations of derangements of both parameters with undesired outcomes. We investigated the effects of 3 different doses of dexmedetomidine on these outcomes in a randomized controlled trial in children undergoing elective surgery.Sixty-four American Society of Anesthesiologists I-II children were randomized to receive either dexmedetomidine 0.25 µg/kg, dexmedetomidine 0.5 µg/kg, dexmedetomidine 0.75 µg/kg, or 0 µg/kg (control), as a bolus administered over 60 seconds after induction of anesthesia. Changes in plasma glucose and serum potassium concentrations were measured in venous blood sampled before and at 15 and 30 minutes after study drug administration. Data were plotted within and between groups and analyzed using a constrained longitudinal data approach.Forty-nine children completed the study. Mean glucose levels at 15 and 30 minutes were elevated with estimated changes from baseline of 0.37 mmol/L (95% CI, 0.29-0.45 mmol/L) and 0.05 mmol/L (95% CI, 0.00-0.10 mmol/L), respectively. At 15 minutes, there was a linear dose-response relationship (1.07 mmol/L/μg/kg [95% CI, 0.57-1.58 mmol/L/μg/kg]), but there was no appreciable effect of dexmedetomidine at 30 minutes (0.15 mmol/L/μg/kg [95% CI, -0.40 to 0.70 mmol/L/μg/kg]). Potassium levels were depressed relative to baseline, with a mean difference at 15 minutes of -0.20 mEq/L (95% CI, -0.28 to -0.12 mEq/L) and at 30 minutes of -0.12 mEq/L (95% CI, -0.15 to -0.08 mEq/L), but there was no appreciable effect of dexmedetomidine at either time.Small elevations in glucose and decreases in potassium were observed after induction of anesthesia in children. The elevation in glucose at 15 minutes depended on the dose of dexmedetomidine administered. These preliminary data warrant further investigation.

CuI complexes containing the bulky dialkylbiarylphosphane 2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl ( t BuXPhos, L) and an ancillary ligand (Cl−, Br−, I−, MeCN, ClO4 − or SCN−) have been structurally characterized, namely, chlorido[2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP]copper(I), [CuCl(C29H45P)], 1, bromido[2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP]copper(I), [CuBr(C29H45P)], 2, [2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP]iodidocopper(I), [CuI(C29H45P)], 3, (acetonitrile-κN)[2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP]copper(I) hexafluoridophosphate, [Cu(CH3CN)(C29H45P)]PF6, 4, [2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP](perchlorato-κO)copper(I), [Cu(ClO4)(C29H45P)], 5, and di-μ-thiocyanato-κ2 S:N;κ2 N:S-bis{[2-(di-tert-butylphosphanyl)-2′,4′,6′-triisopropylbiphenyl-κP]copper(I)}, [Cu2(NCS)2(C29H45P)2], 6. Iodide complex 3 shows significant CuI–arene interactions, in contrast to its chloride 1 and bromide 2 counterparts, which is attributed to the weaker interaction between the iodide ion and the CuI centre. When replacing iodide with an acetonitrile (in 4) or perchlorate (in 5) ligand, the reduced interaction between the CuI atom and the ancillary ligand results in stronger CuI–arene interactions. No CuI–arene interactions are observed in dimer 6, due to the tricoordinated CuI centre having sufficient electron density from the coordinated ligands.

Background Closed-loop control of anesthesia involves continual adjustment of drug infusion rates according to measured clinical effect. The NeuroSENSE monitor provides an electroencephalographic measure of depth of hypnosis (wavelet-based anesthetic value for central nervous system monitoring [WAVCNS]). It has previously been used as feedback for closed-loop control of propofol, in a system designed using robust control engineering principles, which implements features specifically designed to ensure patient safety. Closed-loop control of a second drug, remifentanil, may be added to improve WAVCNS stability in the presence of variable surgical stimulation. The objective of this study was to design and evaluate the feasibility of a closed-loop system for robust control of propofol and remifentanil infusions using WAVCNS feedback, with an infusion safety system based on the known pharmacological characteristics of these 2 drugs. Methods With Health Canada authorization, research ethics board approval, and informed consent, American Society of Anesthesiologists I-III adults, requiring general anesthesia for elective surgery, were enrolled in a 2-phase study. In both phases, infusion of propofol was controlled in closed loop during induction and maintenance of anesthesia, using WAVCNS feedback, but bounded by upper- and lower-estimated effect-site concentration limits. In phase I, remifentanil was administered using an adjustable target-controlled infusion and a controller was designed based on the collected data. In phase II, remifentanil was automatically titrated to counteract rapid increases in WAVCNS. Results Data were analyzed for 127 patients, of median (range) age 64 (22-86) years, undergoing surgical procedures lasting 105 (9-348) minutes, with 52 participating in phase I and 75 in phase II. The overall control performance indicator, global score, was a median (interquartile range) 18.3 (14.2-27.7) in phase I and 14.6 (11.6-20.7) in phase II (median difference, -3.25; 95% confidence interval, -6.35 to -0.52). The WAVCNS was within ±10 of the setpoint for 84.3% (76.6-90.6) of the maintenance of anesthesia in phase I and 88.2% (83.1-93.4) in phase II (median difference, 3.7; 95% confidence interval, 0.1-6.9). The lower propofol safety bound was activated during 30 of 52 (58%) cases in phase I and 51 of 75 (68%) cases in phase II. Conclusions Adding closed-loop control of remifentanil improved overall controller performance. This controller design offers a robust method to optimize the control of 2 drugs using a single sensor. The infusion safety system is an important component of a robust automated anesthesia system, but further research is required to determine the optimal constraints for these safe conditions.

Closed-loop control of anesthesia based on continuous feedback from processed electroencephalography adjusts drug dosing to target a desired depth of hypnosis during dynamic clinical circumstances, freeing the anesthesiologist to focus on more complex tasks. We describe a case of closed-loop control of total intravenous anesthesia in which a sudden loss of blood required immediate intervention. This case illustrates that closed-loop control of drug delivery maintained an appropriate depth of hypnosis during a rapidly changing surgical situation, and that processed electroencephalography may be a useful adjunct indicator for cerebral hypoperfusion.

We report the characteristics of the strained In

Tribenzylphosphane sulfide (SPBn3) reacts with [Cu(CH3CN)4]PF6 in a 2:1 ratio to give [Cu(CH3CN)(SPBn3)2]PF6 whereas with [Ag(CH3CN)4]PF6 in the same ratio it forms [Ag2(SPBn3)3](PF6)2. The crystal structure of the Cu(I) complex shows it to contain discrete 3-coordinate [Cu(CH3CN)(SPBn3)2]+ cations and [PF6]− anions with the Cu atom lying in the plane formed by the two coordinated sulfur atoms and the nitrogen from the coordinated acetonitrile molecule. The Ag complex is a dinuclear species with the one silver atom being 3-coordinate and the other 4-coordinate. Each of the SPBn3 molecules forms a Ag–C η1-bond with one of the aromatic rings thus blocking the coordination of further SPBn3 ligands. The two Ag centers are linked by a bridging sulfur and a weaker interaction with carbon from the aromatic ring of the same tribenzylphosphane sulfide.

Six complexes consisting of either 2,6-bis(1H-benzimidazol-2-yl)pyridine (bbp) or 2,2′:6′,2″-terpyridine (tpy) moieties coordinated to ruthenium(II) and attached to (pentaphenoxy)cyclotriphosphazene were synthesised and structurally characterised by single-crystal X-ray diffraction techniques. Two of the complexes are the first examples of structurally characterised mono-protonated Ru(bbp)(tpy) complexes. The new complexes were studied by NMR, electronic absorption and vibration spectroscopy to gain an understanding of their physical characteristics. Remarkably the mono-deprotonated form of the bbp ligand, but only when attached to (pentaphenoxy)cyclotriphosphazene by a pyridyl phosphoester linker, shows an equivalence of the benzimidazole/benzimidazolate moieties on the NMR time scale, but not on the electronic time scale, as evidenced by UV–Vis and resonance Raman spectroscopy.

To examine the relationship of transcallosal pathway microstructure and transcallosal inhibition (TCI) with motor function and impairment in chronic stroke.Diffusion-weighted magnetic resonance imaging and transcranial magnetic stimulation (TMS) data were collected from 24 participants with chronic stroke and 11 healthy older individuals. Post-stroke motor function (Wolf Motor Function Test) and level of motor impairment (Fugl-Meyer score) were evaluated.Fractional anisotropy (FA) of transcallosal tracts between prefrontal cortices and the mean amplitude decrease in muscle activity during the ipsilateral silent period evoked by TMS over the non-lesioned hemisphere (termed NL-iSPmean) were significantly associated with level of motor impairment and motor function after stroke (p0.05). A regression model including age, post-stroke duration, lesion volume, lesioned corticospinal tract FA, transcallosal prefrontal tract FA and NL-iSPmean accounted for 84% of variance in motor impairment (p0.01). Both transcallosal prefrontal tract FA (ΔR(2)=0.12, p=0.04) and NL-iSPmean (ΔR(2)=0.09, p=0.04) accounted for unique variance in motor impairment level.Prefrontal transcallosal tract microstructure and TCI are each uniquely associated with motor impairment in chronic stroke.Utilizing a multi-modal approach to assess transcallosal pathways may improve our capacity to identify important neural substrates of motor impairment in the chronic phase of stroke.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

The synthesis of a series of cyclotriphosphazene and polyphosphazene ruthenium(II) compounds is reported using 2,2′:6′,2″-terpyridine (terpy) and 2,6-di(1H-pyrazol-1-yl)pyridine (bpp) pendant ligands. X-ray crystallography, UV–Vis and resonance Raman spectroscopy have been employed to gain an insight into the physical and coordination behaviour of these complexes and indicate that both the small molecule and their polymeric analogues contain coordinated Ru in an octahedral ‘N6’ environment. The results reveal a difference between the chemistry of the ruthenium(II)-bpp-terpy and ruthenium(II)-bis-terpy complexes and demonstrate a means of grafting functional groups to a polyphosphazene backbone under mild conditions.

Background Direct-acting oral anticoagulants (DOACs), which have gained approval for stroke prevention in nonvalvular atrial fibrillation and treatment of venous thromboembolism, have become increasingly preferred over warfarin given their predictable pharmacodynamics, lack of required monitoring, and superior outcomes. Direct-acting oral anticoagulants have been shown to be associated with an increased frequency of gastrointestinal bleeding compared with warfarin, but the severity and characteristics of gastrointestinal bleeding in these patients is poorly understood. Methods We retrospectively evaluated electronic medical records of patients with gastrointestinal bleeding (n = 8496) from 2010-2016. We identified 61 patients with gastrointestinal bleeding episodes while treated with DOACs (rivaroxaban, dabigatran, or apixaban) and 123 patients with gastrointestinal bleeding while taking warfarin. We randomly selected a control group of 296 patients with gastrointestinal bleeding who were not receiving anticoagulation treatment from the same sample. Outcomes included the need for hospitalization, blood transfusion, endoscopic or surgical intervention, and 30-day mortality. Results The DOAC and warfarin groups were similar in terms of age and underlying comorbidity (assessed using the Charlson Comorbidity Index), but the DOAC group had greater concomitant aspirin use. Gastrointestinal bleeding was classified as upper (n = 186), lower (n = 88), anorectal (n = 183), small bowel (n = 9), and indeterminate (n = 14). After adjusting for differences in baseline variables, the DOAC group had fewer hospitalizations and required fewer transfusions than the warfarin group. The DOAC and control groups were not statistically different for all outcomes. There were no significant mortality differences among groups. Conclusion Although prior studies have shown a higher frequency of gastrointestinal bleeding in patients treated with DOACs compared with warfarin, our data suggest that gastrointestinal bleeding in patients taking DOACs may be less severe. These differences occurred despite significantly greater concomitant aspirin use in the DOAC group compared with warfarin users.

Sedation in the intensive care unit (ICU) is challenging, as both over- and under-sedation are detrimental. Current methods of assessment, such as the Richmond Agitation Sedation Scale (RASS), are measured intermittently and rely on patients’ behavioral response to stimulation, which may interrupt sleep/rest. A non-stimulating method for continuous sedation monitoring may be beneficial and allow more frequent assessment. Processed electroencephalography (EEG) monitors have not been routinely adopted in the ICU. The aim of this observational study was to assess the feasibility of using the NeuroSENSE™ monitor for EEG-based continuous sedation assessment. With ethical approval, ICU patients on continuous propofol sedation were recruited. Depth-of-hypnosis index (WAVCNS) values were obtained from the NeuroSENSE. Bedside nurses, blinded to the NeuroSENSE, performed regular RASS assessments and maintained the sedation regimen as per standard of care. Participants were monitored throughout the duration of their propofol infusion, up to 24 h. Fifteen patients, with median [interquartile range] age of 57 [52–62.5] years were each monitored for a duration of 9.0 [5.7–20.1] h. Valid WAVCNS values were obtained for 89% [66–99] of monitoring time and were widely distributed within and between individuals, with 6% [1–31] spent 90 (awake). Significant EEG suppression was detected in 3/15 (20%) participants. Observed RASS matched RASS goals in 36/89 (40%) assessments. The WAVCNS variability, and incidence of EEG suppression, highlight the limitations of using RASS as a standalone sedation measure, and suggests potential benefit of adjunct continuous brain monitoring.

Type 1 regulatory T (Tr1) cells play a pivotal role in restraining human T-cell responses toward environmental allergens and protecting against allergic diseases. Still, the precise molecular cues that underlie their transcriptional and functional specification remain elusive. Here, we show that the cytokine activin-A instructs the generation of CD4 + T cells that express the Tr1-cell–associated molecules IL-10, inducible T-Cell costimulator (ICOS), lymphocyte activation gene 3 protein (LAG-3), and CD49b, and exert strongly suppressive functions toward allergic responses induced by naive and in vivo-primed human T helper 2 cells. Moreover, mechanistic studies reveal that activin-A signaling induces the activation of the transcription factor interferon regulatory factor (IRF4), which, along with the environmental sensor aryl hydrocarbon receptor, forms a multipartite transcriptional complex that binds in IL-10 and ICOS promoter elements and controls gene expression in human CD4 + T cells. In fact, IRF4 silencing abrogates activin-A–driven IL10 and ICOS up-regulation and impairs the suppressive functions of human activin-A–induced Tr1-like (act-A–iTr1) cells. Importantly, using a humanized mouse model of allergic asthma, we demonstrate that adoptive transfer of human act-A–iTr1 cells, both in preventive and therapeutic protocols, confers significant protection against cardinal asthma manifestations, including pulmonary inflammation. Overall, our findings uncover an activin-A–induced IRF4-aryl hydrocarbon receptor (AhR)–dependent transcriptional network, which generates suppressive human Tr1 cells that may be harnessed for the control of allergic diseases.

In this paper, we present our findings on a series of copper(ii) 2,2′-bipyridyl (bipy) complexes that inhibit the oxidation of thiosulfate, a current problem in the gold leaching process. The formation of six complexes, five of which have been structurally characterized by X-ray crystallography, illustrate a thermally induced, controllable switching between oxidation states, which in turn inhibits the oxidation of thiosulfate. These findings give further insight and understanding into the rich chemistry of the coinage metals and the hydrolytic processes involved with gold leaching.

The neurophysiological mechanism of interhemispheric inhibition (IHI) between the human primary sensory cortices (S1s) is poorly understood. Here we used a paired median nerve somatosensory evoked potential protocol to observe S1-S1 IHI from the dominant to the nondominant hemisphere with electroencephalography. In 10 healthy, right-handed individuals, we compared mean peak-to-peak amplitudes of five somatosensory evoked potential components (P14/N20, N20/P25, P25/N30, N30/P40, and P40/N60) recorded over the right S1 after synchronous versus asynchronous stimulation of the right and left median nerves. Asynchronous conditioning + test stimuli (CS+TS) were delivered at interstimulus intervals of 15, 20, 25, 30, and 35 ms. We found that, in relation to synchronous stimulation, when a CS to the left S1 preceded a TS to the right S1 at the short intervals (15 and 20 ms) the amplitude of the cortical N20/P25 complex was significantly depressed, whereas at the longer intervals (25, 30, and 35 ms) significant inhibition was observed for the thalamocortical P14/N20 as well as the cortical N20/P25 components. We conclude that the magnitude of S1 IHI appears to depend on the temporal asynchrony of bilateral inputs and the specific timing is likely reflective of a direct transcallosal mechanism. Employing a method that enables direct S1 IHI to be reliably quantified may provide a novel tool to assess potential IHI imbalances in individuals with neurological damage, such as stroke.

Tris(2-cyanoethyl)phosphine (tcep) reacts with the copper(I) compounds, CuX (X = Cl, Br, I and SCN), in a 1:1 ratio to give 1:1 complexes, CuX(tcep), whereas it reacts with CuY (Y = PF 6 , ClO 4 , NO 3 , BH 4 , CN and CF 3 COO) in a 2:1 ratio to give the 2:1 complexes, CuY(tcep) 2 . Single crystal X-ray structures show that for the anions X = Br and SCN, the complexes are coordination polymers, [CuX(tcep)] n , with the Cu centres being bridged by the anion, and as well, one nitrile arm per tcep ligand coordinates intermolecularly to the Cu to give tetrahedral ‘PBr 2 N’ and ‘PSN 2 ’ coordination spheres respectively. The 2:1 compounds exhibit a variety of structures. For Y = ClO 4 , CN and CF 3 COO polymeric structures are formed except for Y = BH 4 where the compound is a discrete monomer, [Cu(BH 4 )(tcep) 2 ], with a chelating anion and two monodentate P-bound tcep ligands. Both the compounds obtained with Y = CN and CF 3 COO also contain coordinated anions and are formulated as [Cu(CN)(tcep) 2 ] n and [Cu(CF 3 COO)(tcep) 2 ] n respectively. In the case of Y = CN the anion is bridging and the tcep ligands are only P-bound giving a ‘P 2 NC’ coordination sphere. In contrast, for Y = CF 3 COO, the anion is an O-bound monodentate and the tcep ligands bridge to give a ‘P 2 NO’ environment for the copper. In the case of Y = ClO 4 , the anion is not coordinated but a polymeric structure, [Cu(tcep) 2 ] n (ClO 4 ) n , is formed via bridging tcep ligands linking Cu centres intermolecularly resulting in a ‘P 2 N 2 ’ coordination sphere.

Healthy, uninfected individuals harbor HIV-specific naive and memory CD4+ T cells, and many memory T cell epitopes are similar in sequence to peptides expressed by natural commensal bacteria, suggesting potential cross-reactivity., The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4+ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4+ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4+ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.

Mossbauer and electronic absorbance spectroscopy along with variable temperature magnetic moment measurements demonstrate that a cyclotriphosphazene substituted with an iron(II)-bis-2,6-di(1H-pyrazoly-yl)pyridine moiety ( 1 ) and its polyphosphazene analogue ( 2 ) differ significantly in magnetic behaviour.

Following antigen encounter and subsequent resolution of the immune response, a single naive T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single-cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T-cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T-cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.

The synthesis of the new cyclotriphosphazene (CTP) ligand substituted with a pendant 2,6-bis(benzimidazole-2-yl)pyridine (bbp), namely (pentaphenoxy)[4-{2,6-bis(benzimidazole-2-yl)pyridine-4-yl}phenoxy]cyclotriphosphazene L is reported. The single crystal structure of L shows that the bbp group is attached to the CTP via the oxygen. L reacts with FeX2 (X = ClO4− or BF4-) salts forming the [FeL2]X2 complexes 1 and 2 respectively. For [FeL2](BF4)2 (2), the single crystal structure shows an ‘N6’ coordination sphere around the iron atom. UV–Vis, resonance Raman and Mossbauer spectroscopies and magnetic susceptibility measurements, aided by density functional theory (DFT) calculations, determine the complexes are low spin below 300 K but display spin crossover (SCO) behavior above this temperature, hence showing that the addition of a phosphazene to a SCO moiety does not prevent SCO.

Alginates have been shown not only to be beneficial in their relief of symptomatic post-prandial gastroesophageal reflux, but there is also evidence that they may inhibit reflux, including bile acids and pepsin, and in turn attenuate and even prevent the activation and up-regulation of molecules associated with the development of metaplasia and cancer of the esophagus. Here we present the case of a patient with Barrett's esophagus with high-grade dysplasia (HGD) which was initially unresponsive to endoscopic radiofrequency ablation therapy and cryotherapy for highgrade dysplasia. Following initiation of therapy with an alginate solution in addition to twice daily proton pump inhibitor (PPI) therapy, the patient showed a favorable response and eventual complete eradication of his dysplastic Barrett's. The patient's course is suggestive that the alginate solution, through reflux inhibition, prevented persistent esophageal cell changes which occur secondary to gastric and bile acid exposure, thus allowing development of neosquamous epithelium. This case is intriguing with regard to the role of alginates in creating a favorable environment for esophageal healing when treating Barrett's esophagus with high-grade dysplasia.

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8 + T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8 + T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8 + T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8 + T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8 + T cells or the global CD8 + memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8 + T-cell compartment that illuminates the etiology of eBL.

The synthesis and characterization of the fluxional, d(10) cyclotriphosphazene complexes, [MLCl(2)] (M = Zn, Cd, and Hg; L = spiro-[(1,1'-biphenyl)-2,2'-dioxy]tetrakis(4-methyl-2-pyridyloxy)cyclotriphosphazene), are described. Single-crystal X-ray structures show that the zinc complex has crystallized into two crystal forms: one as a tetrahedral species, with a N(2)Cl(2) donor set in which a geminal pair of the pendant pyridyloxy nitrogen atoms binds to the zinc, and the other as a trigonal-bipyramidal (tbp) one, with an N(3)Cl(2) donor set. The third nitrogen atom comes from the phosphazene ring and the two pyridyl ligands are non-geminal. The asymmetric unit of the cadmium complex contains three structurally distinct molecules. One molecule has a tbp structure similar to that of the zinc complex. The second molecule has a six-coordinate, distorted octahedral geometry around the cadmium center with a N(4)Cl(2) donor set, with three of the nitrogen donor atoms coming from the pendant pyridyloxy arms. The third site contains a tbp complex and a distorted octahedral species with a relative occupancy of 3:1. The identification of these three different forms in the one crystal suggests that the energy difference between the tbp and distorted octahedral isomers is not large. Quantitative analysis of the (1)H NMR and variable-temperature (31)P NMR spectra of the zinc, cadmium, and mercury complexes in a CD(2)Cl(2) solution, coupled with the X-ray structural results, shows that an associative fluxional mechanism (ΔS(++) < -65 J mol(-1) K(-1)) is operating.

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).

Two new cyclotriphosphazene ligands with pendant 2,2':6',2″-terpyridine (Terpy) moieties, namely, (pentaphenoxy){4-[2,6-bis(2-pyridyl)]pyridoxy}cyclotriphosphazene (L(1)), (pentaphenoxy){4-[2,6-terpyridin-4-yl]phenoxy}cyclotriphosphazene (L(2)), and their respective polymeric analogues, L(1P) and L(2P), were synthesized. These ligands were used to form iron(II) complexes with an Fe(II)Terpy(2) core. Variable-temperature resonance Raman, UV-visible, and Mössbauer spectroscopies with magnetic measurements aided by density functional theory calculations were used to understand the physical characteristics of the complexes. By a comparison of measurements, the polymers were shown to behave in the same way as the cyclotriphosphazene analogues. The results showed that spin crossover (SCO) can be induced to start at high temperatures by extending the spacer length of the ligand to that in L(2) and L(2P); this combination provides a route to forming a malleable SCO material.

BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1-BARD1 heterodimer binds and ubiquitinates γ-tubulin to inhibit centrosome amplification and promote microtubule nucleation; however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N and C termini of BRCA1 are required for its centrosomal localization and that BRCA1 moves to the centrosome independently of BARD1 and γ-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1, it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes.

Lepidocrocite and ferrihydrite-seeded Al(OH) 3 crystallisation from supersaturated sodium aluminate liquor at 70 °C was investigated using in situ synchrotron X-ray diffraction. The presence of iron oxides and oxyhydroxides in the Bayer process has implications for the nucleation and growth of scale on process equipment, and a greater understanding of the effect they have on Al(OH) 3 crystallisation may allow for development of methods for Al(OH) 3 scale prevention. The early stages of both crystallisation reactions were characterised by nucleation of gibbsite on the seed material. This was followed by a rapid increase in gibbsite concentration, which coincided with the appearance of the bayerite and nordstrandite polymorphs of Al(OH) 3 . The lepidocrocite-seeded reaction then proceeded via a mechanism similar to that which has been observed previously for goethite, hematite and magnetite-seeded Al(OH) 3 crystallisation. Different behaviour was observed in the ferrihydrite-seeded experiment, with nucleation as well as growth occurring during the period of rapid increase in gibbsite concentration, followed by a period of diffusion controlled growth.

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time >500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~20s). The ~25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.