Synthesis of 4,5-Diazaspiro[2.3]hexanes and 1,2-Diazaspiro[3.3]heptanes as Hexahydropyridazine Analogues.

4,5-Diazaspiro[2.3]hexanes are made by dihalocarbene addition across the exocyclic double bond of readily accessible 3-alkylidene-1,2-diazetidines. Using difluorocarbene, generated from TMSCF3/NaI, these spirocycles were produced in yields up to 97% by stereospecific addition across the alkene. Lower yields (up to 64%) were observed using more reactive dichlorocarbene, due to competitive insertion of the carbene into the N-N bond. Larger 1,2-diazaspiro[3.3]heptanes are produced by [2 + 2] cycloaddition of 3-alkylidene-1,2-diazetidines with tetracyanoethylene (TCNE) in up to 99% yield.

Antibody and T-cell responses associated with experimental human malaria infection or vaccination show limited relationships.

This study examined specific antibody and T-cell responses associated with experimental malaria infection or malaria vaccination, in malaria-naive human volunteers within phase I/IIa vaccine trials, with a view to investigating inter-relationships between these types of response. Malaria infection was via five bites of Plasmodium falciparum-infected mosquitoes, with individuals reaching patent infection by 11-12 days, having harboured four or five blood-stage cycles before drug clearance. Infection elicited a robust antibody response against merozoite surface protein-119 , correlating with parasite load. Classical class switching was seen from an early IgM to an IgG1-dominant response of increasing affinity. Malaria-specific T-cell responses were detected in the form of interferon-γ and interleukin-4 (IL-4) ELIspot, but their magnitude did not correlate with the magnitude of antibody or its avidity, or with parasite load. Different individuals who were immunized with a virosome vaccine comprising influenza antigens combined with P. falciparum antigens, demonstrated pre-existing interferon-γ, IL-2 and IL-5 ELIspot responses against the influenza antigens, and showed boosting of anti-influenza T-cell responses only for IL-5. The large IgG1-dominated anti-parasite responses showed limited correlation with T-cell responses for magnitude or avidity, both parameters being only negatively correlated for IL-5 secretion versus anti-apical membrane antigen-1 antibody titres. Overall, these findings suggest that cognate T-cell responses across a range of magnitudes contribute towards driving potentially effective antibody responses in infection-induced and vaccine-induced immunity against malaria, and their existence during immunization is beneficial, but magnitudes are mostly not inter-related.

The discovery of potent, orally bioavailable pyrazolo and triazolopyrimidine CXCR2 receptor antagonists.

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrazolopyrimidine 2-methyl-5-((phenylthio)methyl)pyrazolo[1,5-a]pyrimidin-7-ol 1, resulting in the discovery of CXCR2 receptor antagonist 2-benzyl-5-(((2,3-difluorophenyl)thio)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 14. The SAR was investigated by systematic variation of the pendant thiol, alkyl and pyrimidinol groups. Replacement of the pyrazolopyrimidine core with a triazolo alternative led to a dual series of antagonists with favourable biological and pharmacokinetic properties.

The discovery of potent, orally bioavailable pyrimidine-5-carbonitrile-6-alkyl CXCR2 receptor antagonists.

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrimidine 2-((2,6-dichlorobenzyl)thio)-5-isocyano-6-phenylpyrimidin-4-ol 4, resulting in the discovery of CXCR2 receptor antagonist 2-((2,3-difluorobenzyl)thio)-6-(2-(hydroxymethyl)cyclopropyl)-5-isocyanopyrimidin-4-ol 24. The SAR was investigated by systematic variation of the aromatic group at c-6, the linker between c-2 and the halogenated ring, and the c-5 nitrile moiety.

Comparison of modeling methods to determine liver-to-blood inocula and parasite multiplication rates during controlled human malaria infection.

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.

Breast cancer detection with upstream data fusion, machine learning, and automated registration: initial results.

Purpose: To develop an artificial intelligence algorithm for the detection of breast cancer by combining upstream data fusion (UDF), machine learning (ML), and automated registration, using digital breast tomosynthesis (DBT) and breast ultrasound (US). Approach: Our retrospective study included examinations from 875 women obtained between April 2013 and January 2019. Included patients had a DBT mammogram, breast US, and biopsy proven breast lesion. Images were annotated by a breast imaging radiologist. An AI algorithm was developed based on ML for image candidate detections and UDF for fused detections. After exclusions, images from 150 patients were evaluated. Ninety-five cases were used for training and validation of ML. Fifty-five cases were included in the UDF test set. UDF performance was evaluated with a free-response receiver operating characteristic (FROC) curve. Results: Forty percent of cases evaluated with UDF (22/55) yielded true ML detections in all three images (craniocaudal DBT, mediolateral oblique DBT, and US). Of these, 20/22 (90.9%) produced a UDF fused detection that contained and classified the lesion correctly. FROC analysis for these cases showed 90% sensitivity at 0.3 false positives per case. In contrast, ML yielded an average of 8.0 false alarms per case. Conclusions: An AI algorithm combining UDF, ML, and automated registration was developed and applied to test cases, showing that UDF can yield fused detections and decrease false alarms when applied to breast cancer detection. Improvement of ML detection is needed to realize the full benefit of UDF.

Multiple functions of human T cells generated by experimental malaria challenge.

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming.

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

Towards CNN-Based Registration of Craniocaudal and Mediolateral Oblique 2-D X-ray Mammographic Images.

We investigate methodologies for the automated registration of pairs of 2-D X-ray mammographic images, taken from the two standard mammographic angles. We present two exploratory techniques, based on Convolutional Neural Networks, to examine their potential for co-registration of findings on the two standard mammographic views. To test algorithm performance, our analysis uses a synthetic, surrogate data set for performing controlled experiments, as well as real 2-D X-ray mammogram imagery. The preliminary results are promising, and provide insights into how the proposed techniques may support multi-view X-ray mammography image registration currently and as technology evolves in the future.

Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

Formation of surface-bound acyl groups by reaction of acyl halides on ge(100)-2x1.

We have investigated the reaction of a series of acyl halides, including acetyl chloride, acetyl bromide, acetyl-d3 chloride, benzoyl chloride, and pivaloyl chloride, on Ge(100)-2x1 with multiple internal reflection infrared (MIR-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT). Infrared spectra following saturation exposures of acetyl chloride and acetyl bromide to Ge(100)-2x1 at 310 K are nearly identical, both exhibiting strong nu(C=O) stretching peaks near 1685 cm-1 and no vibrational modes in the nu(Ge-H) region. These data provide strong evidence for the presence of a surface-bound acetyl group on Ge(100)-2x1, which results from a C-X dissociation reaction (where X=Cl, Br). For acetyl chloride, DFT calculations predict that the barrier to C-Cl dissociation is only 1 kcal/mol above a chlorine-bound precursor state and is considerably smaller than barriers leading to the [2+2] C=O cycloaddition and alpha-CH dissociation products. In addition to the C-X dissociation product, both infrared and photoelectron results point to the presence of a second structure for acetyl halides where the oxygen of the surface-bound acetyl group donates charge to a nearby surface atom. This interaction is not observed for benzoyl chloride and pivaloyl chloride.

[Erratum] Effects of glucose-dependent insulinotropic polypeptide receptor knockout and a high-fat diet on cognitive function and hippocampal gene expression in mice.

RACHAEL R. LENNOX, CHARLOTTE MOFFETT, DAVID W. PORTER, NIGEL IRWIN, VICTOR A. GAULT and PETER R. FLATT Mol Med Rep 12:[Related article:] 1544­1548, 2015; DOI: 10.3892/mmr.2015.3447 After the publication of the article, the authors noted that there was an error regarding the author list. RACHAEL R. LENNOX and CHARLOTTE MOFFETT should actually be listed as RACHAEL LENNOX and R. CHARLOTTE MOFFETT. We apologize for the mistake and possible misunderstanding. The correct author list is as follows: RACHAEL LENNOX, R. CHARLOTTE MOFFETT, DAVID W. PORTER, NIGEL IRWIN, VICTOR A. GAULT and PETER R. FLATT.

Effects of glucose-dependent insulinotropic polypeptide receptor knockout and a high-fat diet on cognitive function and hippocampal gene expression in mice.

It has been previously demonstrated that compromise of glucose-dependent insulinotropic polypeptide receptor (GIPR) action and chronic consumption of a high-fat diet can independently impair memory and learning ability, however, the underlying pathology remain to be elucidated. The present study investigated the effects of GIPR knockout (KO), alone and in combination with a high-fat diet, on aspects of cognitive function and hippocampal gene expression in mice. In object recognition tests, normal mice exhibited effective memory, preferring to investigate the novel over the familiar object. However, wild-type (WT) mice fed a high-fat diet and GIPR KO mice fed a standard or high-fat diet demonstrated no such discrimination, suggesting the impairment of memory function. This decline in cognitive function was associated with marked changes in the expression levels of hippocampal genes involved in memory and learning. The chronic consumption of a high-fat diet decreased the hippocampal gene expression levels of mammalian target of rapamycin (mTOR), neurotrophic tyrosine kinase receptor type 2 (NTRK2) and synaptophysin. Notably, the GIPR KO mice fed a high-fat diet exhibited no reduction in the hippocampal expression of synaptophysin expression, however, the GIPR KO mice fed a standard rodent maintenance diet exhibited reduced hippocampal expression of mTOR compared with the WT controls. These data highlighted the importance of intact GIPR signalling and dietary composition in modulating memory and learning, and hippocampal pathways involved in the maintenance of synaptic plasticity, including mTOR and NTRK2, appear to be key in this regard.

Concise asymmetric synthesis of (-)-sparteine.

A six-step asymmetric synthesis of natural (-)-sparteine from ethyl 7-iodohept-2-enoate is reported, involving a connective Michael addition of an amino ester-derived enolate to an alpha,beta-unsaturated amino ester.

Comparison of the independent and combined effects of sub-chronic therapy with metformin and a stable GLP-1 receptor agonist on cognitive function, hippocampal synaptic plasticity and metabolic control in high-fat fed mice.

Cognitive dysfunction is more common in individuals with type 2 diabetes (T2DM). Currently, glucagon-like peptide-1 (GLP-1) and metformin are important therapeutic options for patients with T2DM. However, their potential effects on cognitive function, including underlying mechanisms, are yet to be fully determined. We have compared the individual and combined effects of treatment for 20 days with (Val(8))GLP-1(GluPAL), an enzymatically stable GLP-1-receptor agonist, and metformin on metabolic control and aspects of learning and memory in high fat fed mice. (Val(8))GLP-1(GluPAL) treatment for 20 days alone, or in combination with metformin, improved (p < 0.05) the recognition index in high fat mice, indicating enhanced learning and memory. In addition, these mice exhibited a complete reversal of the deleterious effects of prolonged high-fat feeding on long-term potentiation in the hippocampal CA1 region. This was linked to reduced hippocampal levels of 8-oxoguanine (p < 0.01) and glial fibriallary acidic protein (p < 0.001), indicating decreased oxidative stress and inflammation; respectively. Expression of fundamental hippocampal genes including mTOR, VEGF, NTRK2 and SIRT1 was also increased significantly (p < 0.001) by all treatments. (Val(8))GLP-1(GluPAL) monotherapy, or in combination with metformin, reduced circulating glucose (p < 0.05) and increased insulin (p < 0.05 to p < 0.01) concentrations, as well as improving glucose tolerance (p < 0.001) and glucose-stimulated insulin secretion (p < 0.05 to p < 0.01). Insulin sensitivity and measurements of energy regulation and metabolic rate were not altered. These studies highlight the neuroprotective properties of (Val(8))GLP-1(GluPAL), alone and in combination with metformin, in T2DM.

(Val(8))GLP-1-Glu-PAL: a GLP-1 agonist that improves hippocampal neurogenesis, glucose homeostasis, and ß-cell function in high-fat-fed mice.

This study examined the biological properties of a novel GLP-1 peptide, (Val(8))GLP-1-Glu-PAL, engineered with an Ala(8)→Val(8) substitution and additional incorporation of a C(16) fatty acid moiety at Lys(26) via a glutamic acid linker. GLP-1 underwent 75 % degradation by DPP-IV over 8 h, whereas (Val(8))GLP-1 and (Val(8))GLP-1-Glu-PAL remained intact. All GLP-1 peptides significantly stimulated insulin secretion at 5.6 mM (1.3- to 4.9-fold, p<0.01 to p<0.001) and 16.7 mM glucose (1.5- to 2.3-fold, p<0.001). At higher concentrations (Val(8))GLP-1-Glu-PAL was significantly more potent at stimulating insulin secretion (1.2- to 1.3-fold, p<0.05). In high-fat-fed mice, all GLP-1 peptides significantly lowered plasma glucose concentrations (41-66 % decrease, p<0.05 to p<0.001), with (Val(8))GLP-1-Glu-PAL eliciting protracted glucose-lowering actions (32-59 % decrease, p<0.05 to p<0.01) when administered 8 h prior to a glucose load. Twice-daily administration of (Val(8))GLP-1-Glu-PAL in high-fat-fed mice for 21 days had no effect on bodyweight or food intake, but significantly lowered non-fasting plasma glucose (43-46 % decrease, p<0.05). (Val(8))GLP-1-Glu-PAL markedly decreased glycemic excursion following intraperitoneal glucose (32-48 % decrease, p<0.05), enhanced insulin response to glucose (2- to 2.3-fold, p<0.05 to p<0.01), and improved insulin sensitivity (25-38 % decrease in plasma glucose, p<0.05). O(2) consumption, CO(2) production, RER, and energy expenditure were not altered by (Val(8))GLP-1-Glu-PAL therapy. Treatment with (Val(8))GLP-1-Glu-PAL resulted in a significant increase in BrdU-positive cells (1.3-fold, p<0.05) in the granule cell layer of the dentate gyrus. These data demonstrate that (Val(8))GLP-1-Glu-PAL is a long-acting GLP-1 peptide that significantly improves hippocampal neurogenesis, glucose homeostasis, and insulin secretion in high-fat-fed mice.

Chemo- and enantioselective Rh-catalysed hydrogenation of 3-methylene-1,2-diazetidines: application to vicinal diamine synthesis.

Rhodium catalysed hydrogenation of 3-methylene-1,2-diazetidines with a range of chiral ligands is reported. Using Mandyphos, excellent levels of chemo- and enantioselectivity (up to 89% ee) can be achieved. Reductive cleavage of the derived 3-substituted 1,2-diazetidine with LiDBB provides the enantioenriched biscarbamate protected 1,2-diamine.

Effect of vaccine dose on the safety and immunogenicity of a candidate TB vaccine, MVA85A, in BCG vaccinated UK adults.

PURPOSE: A non-randomised, open-label, Phase I safety and immunogenicity dose-finding study to assess the safety and immunogenicity of the candidate TB vaccine Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A) from Mycobacterium tuberculosis (MTB) in healthy adult volunteers previously vaccinated with BCG. METHODS: Healthy BCG-vaccinated volunteers were vaccinated with either 1×10(7) or 1×10(8)PFU of MVA85A. All adverse events were documented and antigen specific T cell responses were measured using an ex vivo IFN-γ ELISPOT assay. Safety and immunogenicity were compared between the 2 dose groups and with a previous trial in which a dose of 5×10(7)PFU MVA85A had been administered. RESULTS: There were no serious adverse events recorded following administration of either 1×10(7) or 1×10(8)PFU of MVA85A. Systemic adverse events were more frequently reported following administration of 1×10(8)PFU of MVA85A when compared to either 5×10(7) or 1×10(7)PFU of MVA85A but were mild or moderate in severity and resolved completely within 7 days of immunisation. Antigen specific T cell responses as measured by the IFN-γ ELISPOT were significantly higher following immunisation in adults receiving 1×10(8)PFU compared to the 5×10(7) and 1×10(7) doses. Additionally, a broader range of Ag85A epitopes are detected following 1×10(8)PFU of MVA85A. CONCLUSION: A higher dose of 1×10(8)PFU of MVA85A is well-tolerated, increases the frequency of IFN-γ secreting T cells detected following immunisation and broadens the range of Ag85A epitopes detected.

Comparison of sub-chronic metabolic effects of stable forms of naturally occurring GIP(1-30) and GIP(1-42) in high-fat fed mice.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid hormone secreted from intestinal K-cells, which exhibits a number of actions including stimulation of insulin release. A truncated form, GIP(1-30), has recently been demonstrated in intestine and islet α-cells. To evaluate the potential physiological significance of this naturally occurring form of GIP, the present study has examined and compared the bioactivity of enzymatically stabilised forms, [D-Ala²]GIP(1-30) and [D-Ala²]GIP(1-42), in high-fat fed mice. Twice-daily injection of GIP peptides for 42 days had no significant effect on food intake or body weight. However, non-fasting glucose levels were significantly lowered, and insulin levels were elevated in both treatment groups compared to saline controls. The glycaemic response to i.p. glucose was correspondingly improved (P < 0.05) in [D-Ala²]GIP(1-30)- and [D-Ala²]GIP(1-42)-treated mice. Furthermore, glucose-stimulated plasma insulin levels were significantly elevated in both treatment groups compared to control mice. Insulin sensitivity was not significantly different between any of the groups. Similarly, plasma lipid profile, O2 consumption, CO2 production, respiratory exchange ratio, and energy expenditure were not altered by 42 days twice-daily treatment with [D-Ala²]GIP(1-30) or [D-Ala²]GIP(1-42). In contrast, ambulatory activity was significantly (P < 0.05) elevated during the light phase in both GIP treatment groups compared to saline controls. The results reveal that sustained GIP receptor activation exerts a spectrum of beneficial metabolic effects in high-fat fed mice. However, no differences were discernable between the biological actions of the enzyme-resistant analogues of the naturally occurring forms, GIP(1-30) and GIP(1-42).