Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

Short-term Weight Trajectory of Severely Obese Individuals With and Without Pathogenic Satiety-Regulation Melanocortin 3/4 Receptor (MC3/4R) Mutations From a Multi-ethnic Asian Large Bariatric Surgery Program.

The melanocortin (3 or 4) receptor (MC3/4R) is involved in regulating satiety and body weight. Therefore, pathogenic mutation in MC3/4R is associated with severe obesity, for which bariatric surgery is one of the treatment options. However, there is limited data on whether individuals with MC3/4R mutation will have differential weight response to surgery, especially among the Asian populations-the epi-center of the evolving global obesity epidemic. From our large prospective Obesity-Metabolism & Intervention Cohort Study (OMICS; N = 654, recruited between 2007 and 2022), 5 individuals with pathogenic MC3/4R mutations ("case") were identified using candidate-genes panel next-generation sequencing (Illumina iSeq). These subjects were carefully propensity score-matched (baseline body mass index [BMI], age, sex, ethnicity, proportion with diabetes, type of bariatric surgery) in a 1:4 ratio to other controls. We performed linear mixed model analysis (for repeated measurements) to compare their longitudinal weight trajectories (percentage total weight loss, %TWL) over 12 months. The 5 cases with MC3/4R mutations were 48 ± 11 years, BMI 40.8 ± 11.2 kg/m2, 60% with diabetes, and all males. Their weight at baseline (pre-op), and 6 months and 12 months after surgery were 120 ± 38, 100 ± 31, and 101 ± 30 kg, respectively. Compared with propensity score-matched controls (N = 20), linear mixed model analysis suggested no difference in surgically induced %TWL (ß coefficient = -5.8 ± 3.7, P = .13) over 12 months between the groups. Therefore, we conclude that rare pathogenic MC3/4R mutations do not significantly modify weight change (%TWL) in response to bariatric surgery.

In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm.

Improving methods for human embryonic stem cell differentiation represents a challenge in modern regenerative medicine research. Using drug repurposing approaches, we discover small molecules that regulate the formation of definitive endoderm. Among them are inhibitors of known processes involved in endoderm differentiation (mTOR, PI3K, and JNK pathways) and a new compound, with an unknown mechanism of action, capable of inducing endoderm formation in the absence of growth factors in the media. Optimization of the classical protocol by inclusion of this compound achieves the same differentiation efficiency with a 90% cost reduction. The presented in silico procedure for candidate molecule selection has broad potential for improving stem cell differentiation protocols.

Evaluation of connectivity map shows limited reproducibility in drug repositioning.

The Connectivity Map (CMap) is a popular resource designed for data-driven drug repositioning using a large transcriptomic compendium. However, evaluations of its performance are limited. We used two iterations of CMap (CMap 1 and 2) to assess their comparability and reliability. We queried CMap 2 with CMap 1-derived signatures, expecting CMap 2 would highly prioritize the queried compounds; the success rate was 17%. Analysis of previously published prioritizations yielded similar results. Low recall is caused by low differential expression (DE) reproducibility both between CMaps and within each CMap. DE strength was predictive of reproducibility, and is influenced by compound concentration and cell-line responsiveness. Reproducibility of CMap 2 sample expression levels was also lower than expected. We attempted to identify the "better" CMap by comparison with a third dataset, but they were mutually discordant. Our findings have implications for CMap usage and we suggest steps for investigators to limit false positives.

Curation of over 10 000 transcriptomic studies to enable data reuse.

Vast amounts of transcriptomic data reside in public repositories, but effective reuse remains challenging. Issues include unstructured dataset metadata, inconsistent data processing and quality control, and inconsistent probe-gene mappings across microarray technologies. Thus, extensive curation and data reprocessing are necessary prior to any reuse. The Gemma bioinformatics system was created to help address these issues. Gemma consists of a database of curated transcriptomic datasets, analytical software, a web interface and web services. Here we present an update on Gemma's holdings, data processing and analysis pipelines, our curation guidelines, and software features. As of June 2020, Gemma contains 10 811 manually curated datasets (primarily human, mouse and rat), over 395 000 samples and hundreds of curated transcriptomic platforms (both microarray and RNA sequencing). Dataset topics were represented with 10 215 distinct terms from 12 ontologies, for a total of 54 316 topic annotations (mean topics/dataset = 5.2). While Gemma has broad coverage of conditions and tissues, it captures a large majority of available brain-related datasets, accounting for 34% of its holdings. Users can access the curated data and differential expression analyses through the Gemma website, RESTful service and an R package. Database URL: https://gemma.msl.ubc.ca/home.html.

A multicentre longitudinal study of flortaucipir (18F) in normal ageing, mild cognitive impairment and Alzheimer's disease dementia.

The advent of tau-targeted PET tracers such as flortaucipir (18F) (flortaucipir, also known as 18F-AV-1451 or 18F-T807) have made it possible to investigate the sequence of development of tau in relationship to age, amyloid-ß, and to the development of cognitive impairment due to Alzheimer's disease. Here we report a multicentre longitudinal evaluation of the relationships between baseline tau, tau change and cognitive change, using flortaucipir PET imaging. A total of 202 participants 50 years old or older, including 57 cognitively normal subjects, 97 clinically defined mild cognitive impairment and 48 possible or probable Alzheimer's disease dementia patients, received flortaucipir PET scans of 20 min in duration beginning 80 min after intravenous administration of 370 MBq flortaucipir (18F). On separate days, subjects also received florbetapir amyloid PET imaging, and underwent a neuropsychological test battery. Follow-up flortaucipir scans and neuropsychological battery assessments were also performed at 9 and 18 months. Fifty-five amyloid-ß+ and 90 amyloid-ß- subjects completed the baseline and 18-month study visits and had valid quantifiable flortaucipir scans at both time points. There was a statistically significant increase in the global estimate of cortical tau burden as measured by standardized uptake value ratio (SUVr) from baseline to 18 months in amyloid-ß+ but not amyloid-ß- subjects (least squared mean change in flortaucipir SUVr : 0.0524 ± 0.0085, P < 0.0001 and 0.0007 ± 0.0024 P = 0.7850, respectively), and a significant association between magnitude of SUVr increase and baseline tau burden. Voxel-wise evaluations further suggested that the regional pattern of change in flortaucipir PET SUVr over the 18-month study period (i.e. which regions exhibited the greatest change) also varied as a function of baseline global estimate of tau burden. In subjects with lower global SUVr, temporal lobe regions showed the greatest flortaucipir retention, whereas in subjects with higher baseline SUVr, parietal and frontal regions were increasingly affected. Finally, baseline flortaucipir and change in flortaucipir SUVr were both significantly (P < 0.0001) associated with changes in cognitive performance. Taken together, these results provide a preliminary characterization of the longitudinal spread of tau in Alzheimer's disease and suggest that the amount and location of tau may have implications both for the spread of tau and the cognitive deterioration that may occur over an 18-month period.

Predictability of human differential gene expression.

Differential expression (DE) is commonly used to explore molecular mechanisms of biological conditions. While many studies report significant results between their groups of interest, the degree to which results are specific to the question at hand is not generally assessed, potentially leading to inaccurate interpretation. This could be particularly problematic for metaanalysis where replicability across datasets is taken as strong evidence for the existence of a specific, biologically relevant signal, but which instead may arise from recurrence of generic processes. To address this, we developed an approach to predict DE based on an analysis of over 600 studies. A predictor based on empirical prior probability of DE performs very well at this task (mean area under the receiver operating characteristic curve, ∼0.8), indicating that a large fraction of DE hit lists are nonspecific. In contrast, predictors based on attributes such as gene function, mutation rates, or network features perform poorly. Genes associated with sex, the extracellular matrix, the immune system, and stress responses are prominent within the "DE prior." In a series of control studies, we show that these patterns reflect shared biology rather than technical artifacts or ascertainment biases. Finally, we demonstrate the application of the DE prior to data interpretation in three use cases: (i) breast cancer subtyping, (ii) single-cell genomics of pancreatic islet cells, and (iii) metaanalysis of lung adenocarcinoma and renal transplant rejection transcriptomics. In all cases, we find hallmarks of generic DE, highlighting the need for nuanced interpretation of gene phenotypic associations.

Relationships between flortaucipir PET tau binding and amyloid burden, clinical diagnosis, age and cognition.

The advent of tau-targeted positron emission tomography tracers such as flortaucipir (18F-AV-1451, also known as 18F-T807) have made it possible to investigate the sequence of development of tau and amyloid-ß in relationship to age, and to the development of cognitive impairment due to Alzheimer's disease. In this study, flortaucipir tau and florbetapir amyloid positron emission tomography were obtained for 217 subjects including 16 young and 58 older cognitively normal subjects, 95 subjects with mild cognitive impairment (Mini-Mental State Examination 24-30) and 48 subjects with clinically-defined possible or probable Alzheimer's disease (Mini-Mental State Examination >10). Images were evaluated visually and quantitatively by regional and voxel-based cortical to cerebellar standard uptake value ratios. For amyloid positron emission tomography positive (Aß+) subjects, flortaucipir neocortical standard uptake value ratio was significantly higher with more advanced clinical stage (Alzheimer's disease > mild cognitive impairment > older cognitively normal) and was significantly elevated for Aß+ mild cognitive impairment and Alzheimer's disease subjects relative to the respective Aß- subjects. In contrast, florbetapir Aß- older cognitively normal subjects showed an increase in flortaucipir standard uptake value ratios in mesial temporal lobe regions (amygdala, hippocampus/choroid plexus region of interest) compared to younger cognitively normal subjects, but no increased standard uptake value ratios in neocortical regions. Analysis of covariance with planned contrasts showed no differences in regional or composite posterior neocortical flortaucipir standard uptake value ratio as a function of diagnostic group among Aß- older cognitively normal or clinically diagnosed Alzheimer's disease or mild cognitive impairment subjects. The pattern of flortaucipir distribution among Aß+ subjects was reminiscent of the cross-sectional distribution of tau reported in post-mortem pathology studies, in that the most commonly affected regions were the inferior and lateral temporal lobes, the same regions where the first signs of increased retention appeared in Aß+ cognitively normal subjects. However, there was large variability in extent/density of flortaucipir tau binding among Aß+ subjects. Although high neocortical flortaucipir retention was consistently associated with an Aß+ florbetapir positron emission tomography scan, not all Aß+ subjects had elevated flortaucipir standard uptake value ratios. Finally, within the Aß+ group, increasing levels of flortaucipir tau binding were associated with increased cognitive impairment, as assessed by Mini-Mental State Examination and Alzheimer's Disease Assessment Scale. These results suggest development of tau beyond the mesial temporal lobe is associated with, and may be dependent on, amyloid accumulation. Further, the results are consistent with the hypothesis that cortical tau is associated with cognitive impairment.

Facile Route to 2-Fluoropyridines via 2-Pyridyltrialkylammonium Salts Prepared from Pyridine N-Oxides and Application to (18)F-Labeling.

Among known precursors for 2-[(18)F]fluoropyridines, pyridyltrialkylammonium salts have shown excellent reactivity; however, their broader utility has been limited because synthetic methods for their preparation suffer from poor functional group compatibility. In this paper, we demonstrate the regioselective conversion of readily available pyridine N-oxides into 2-pyridyltrialkylammonium salts under mild and metal-free conditions. These isolable intermediates serve as effective precursors to structurally diverse 2-fluoropyridines, including molecules relevant to PET imaging. In addition to providing access to nonradioactive analogues, this method has been successfully applied to (18)F-labeling in the radiosynthesis of [(18)F]AV-1451 ([(18)F]T807), a PET tracer currently under development for imaging tau.

Florbetapir f-18: a histopathologically validated Beta-amyloid positron emission tomography imaging agent.

Florbetapir F-18 is a molecular imaging agent combining high affinity for ß-amyloid, pharmacokinetic properties that allow positron emission tomography (PET) imaging within a convenient time after dose administration, and the wide availability of the radionuclide fluorine-18. Florbetapir F-18 is prepared by nucleophilic radiofluorination in approximately 60 minutes with a decay-corrected yield of 20%-40% and with a specific activity typically exceeding 100 Ci/mmol. The florbetapir F-18 dissociation constant (K(d)) for binding to ß-amyloid in brain tissue from Alzheimer's disease (AD) patients was 3.7 ± 0.3 nmol/L, and the maximum binding capacity (B(max)) was 8800 ± 1600 fmol/mg protein. Autoradiography studies have shown that florbetapir F-18 selectively binds to ß-amyloid aggregates in AD patient brain tissue, and the binding intensity is correlated with the density of ß-amyloid quantified by standard neuropathologic techniques. Studies in animals revealed no safety concerns and rapid and transient normal brain uptake (6.8% injected dose/g at 2 minutes and 1.9% injected dose/g at 60 minutes in the mouse). Florbetapir F-18 has been well-tolerated in studies of more than 2000 human subjects. Biodistribution studies in humans revealed predominantly hepatobiliary excretion. The whole body effective dose was 7 mSv from a dose of 370 MBq. The pharmacokinetic of florbetapir F-18 make it possible to obtain a PET image with a brief (10 minutes) acquisition time within a convenient time window of 30-90 minutes after dose administration. Clinical studies have demonstrated a clear correlation between in vivo PET imaging with florbetapir F-18 and postmortem histopathologic quantitation of ß-amyloid in the brain.

Preclinical properties of 18F-AV-45: a PET agent for Abeta plaques in the brain.

UNLABELLED: beta-amyloid plaques (Abeta plaques) in the brain, containing predominantly fibrillary Abeta peptide aggregates, represent a defining pathologic feature of Alzheimer disease (AD). Imaging agents targeting the Abeta plaques in the living human brain are potentially valuable as biomarkers of pathogenesis processes in AD. (E)-4-(2-(6-(2-(2-(2-(18)F-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine ((18)F-AV-45) is such as an agent currently in phase III clinical studies for PET of Abeta plaques in the brain. METHODS: In vitro binding of (18)F-AV-45 to Abeta plaques in the postmortem AD brain tissue was evaluated by in vitro binding assay and autoradiography. In vivo biodistribution of (18)F-AV-45 in mice and ex vivo autoradiography of AD transgenic mice (APPswe/PSEN1) with Abeta aggregates in the brain were performed. Small-animal PET of a monkey brain after an intravenous injection of (18)F-AV-45 was evaluated. RESULTS: (18)F-AV-45 displayed a high binding affinity and specificity to Abeta plaques (K(d), 3.72 +/- 0.30 nM). In vitro autoradiography of postmortem human brain sections showed substantial plaque labeling in AD brains and not in the control brains. Initial high brain uptake and rapid washout from the brain of healthy mice and monkey were observed. Metabolites produced in the blood of healthy mice after an intravenous injection were identified. (18)F-AV-45 displayed excellent binding affinity to Abeta plaques in the AD brain by ex vivo autoradiography in transgenic AD model mice. The results lend support that (18)F-AV-45 may be a useful PET agent for detecting Abeta plaques in the living human brain.

Squaramide hydroxamate-based chemidosimeter responding to iron(III) with a fluorescence intensity increase.

The synthesis and in vitro evaluation of a squarate hydroxamate-coumarin conjugate, 12, as a chemodosimeter for Fe(III) and other oxidants, such as Cr(VI) and Ce(IV), is described. As 12 was originally designed to become a chelation-enhanced fluorescence (CHEF)-type sensor for Fe(III), the competence of the squarate diamide platform to relay a CHEF response was demonstrated using a zinc-binding, cyclen-substituted squarate coumarin amide. Due to a photo electron transfer process, 12 possesses a low fluorescence yield. Upon exposure of 12 to Fe(III) (or other oxidants), an irreversible 9-fold fluorescence intensity increase is observed as the result of an oxidation/hydrolysis reaction. The (aminomethyl)coumarin portion of 12 is oxidized to an iminocoumarin that hydrolyzes to produce a highly fluorescent coumarinaldehyde. Fe(III) acts as a catalyst in this transformation, thereby enhancing the sensitivity of the system for the detection of Fe(III) down to 1 ppm in aqueous buffer solution. The identities of the major reaction products between 12 and Fe(III) were proven by independent synthesis.

Glucosamine conjugates bearing N,N,O-donors: potential imaging agents utilizing the [M(CO)3]+ core (M = Re, Tc).

The design rationale, synthesis and radiolabeling evaluation of four glucosamine conjugated ligands for the [(99m)Tc(CO)(3)](+) core is described. The capability to bind the tricarbonyl core is initially demonstrated using the cold surrogate [Re(CO)(3)](+). The four compounds are competent chelates in binding [(99m)Tc(CO)(3)](+) as labeling studies show, with yields ranging from 79 to 96% and the resulting complexes showing stability in the presence of competing chelates for 24 h at 37 degrees C. The rhenium complexes were tested for hexokinase-catalysed phosphorylation, and the technetium complexes were tested for GLUT-1 mediated cell uptake--they showed a small amount of uptake but it was not glucose dependent, suggesting that it was not via the GLUT-1 transporters.

Pyridine-tert-nitrogen-phenol ligands: N,N,O-Type tripodal chelates for the [M(CO)3]+ core (M = Re, Tc).

The design rationale, synthesis, and preliminary radiolabeling evaluation of new N,N,O-type pyridyl- tert-nitrogen-phenol ligands for the [M(CO) 3] (+) core, where M = (99m)Tc or Re, are described. The capability of the ligands to bind this technetium core is initially demonstrated by using the cold surrogate [Re(CO) 3] (+). NMR studies of the relevant rhenium tricarbonyl complexes indicate the formation of either a monomeric or a possible dimeric complex with each phenolic O atom bridging between two metal centers. Labeling with [ (99m)Tc(CO) 3] (+) provided further insight into the differences in complex formation on the dilute, no carrier added, level compared to the macroscopic scale at which the Re (I) counterparts were made. These new tridentate, monoanionic ligands are competent chelates in binding the [ (99m)Tc(CO) 3] (+) core because radiolabeling yields ranged from 85 to 99% and the resulting complexes were stable to cysteine and histidine challenges for as long as 24 h.

Coumarin-based chemosensors for zinc(II): toward the determination of the design algorithm for CHEF-type and ratiometric probes.

The synthesis of a series of coumarin-based chemosensor assemblies for zinc is detailed, using established and novel synthetic pathways. Variations of the nature of the chelating unit (DPA or cyclen), position of the attachment point of the chelating unit (3- or 4-position), and nature of the 7-substituent (-OH, -OAc, or -NR2) on the coumarin play a crucial role in whether, and to what extent, a CHEF-type or ratiometric response of the chemosensor is observed. Solvent effects are also discussed. The chemosensors were shown to be competent for detecting zinc pools in cultured rat pituitary (GH3) and hepatoma (H4IIE) cell lines. The work further defines the design algorithms for zinc-selective CHEF-type and ratiometric chemosensors.

Illuminating zinc in biological systems.

Zinc is the second most abundant transition metal in the human body, fulfilling a multitude of biological roles, but the mechanisms underlying its physiology are poorly understood. The lack of knowledge is, in part, due to the hitherto limited techniques available to track zinc in biological systems. The recent emergence of a number of zinc-specific molecular sensors has provided a new tool to image zinc in live cells and tissue samples. This contribution highlights the concepts behind using zinc-specific fluorescent molecular sensors to gain information about zinc action in biological samples, and provides representative examples of images recorded.

DPA-substituted coumarins as chemosensors for zinc(II): modulation of the chemosensory characteristics by variation of the position of the chelate on the coumarin.

The sensory capabilities of two novel di(2-picolyl)amine (DPA)-substituted coumarins are described and it is shown that the variation of the point of attachment of the DPA group to the coumarin framework controls their sensing behavior: the 4-substituted system is a CHEF-type sensor that shows a significant increase in fluorescence intensity upon Zn(2+) binding, whereas the 3-substituted system is a ratiometric sensor.

Squaric acid N-hydroxylamides: synthesis, structure, and properties of vinylogous hydroxamic acid analogues.

The synthesis of squaric acid N-hydroxylamide esters 5 and amides 6 from dimethyl squarate 2a is described. These derivatives are analogues of the naturally occurring iron(III) chelator hydroxamic acid. On the basis of a comparative reactivity study, a concerted retro-Cope mechanism for the formation of the N-hydroxylamide esters 5 by reaction of dimethyl squarate with hydroxylamines is proposed. A preliminary iron(III) binding study of these hydroxamic acid analogues is presented, demonstrating binding of iron(III) to amides 6 in aqueous solutions, while the esters 5 did not show any sign of metal ion binding. 13C NMR spectroscopic data (chemical shift and spin-lattice relaxation time determination) of these and related derivatives delineate the resonance structures predominant in these molecules. The resonance structures of the derivatives rationalize their spectroscopic data, chemical reactivity, and iron(III) binding properties. Single-crystal X-ray structure analyses of squaric acid N-hydroxylamide ester 5b and squaric acid N-hydroxylamide amide 6c confirm their connectivity and provide structural evidence supporting the spectroscopically derived conclusions. The squaric acid N-hydroxylamides are potentially useful in the construction of chemosensors for iron(III).

Synthesis of a fluorescent chemosensor suitable for the imaging of zinc(II) in live cells.

The synthesis of a coumarin-cyclen conjugate-based zinc-specific chemosensor and its ability to sense Zn(2+) in vitro is described. Using fluorescence microscopy, the chemosensor was shown to be capable of imaging Zn(2+) in live rat pituitary tumour cells.