On the path from xylem hydraulic failure to downstream cell death.

Xylem hydraulic failure (HF) has been identified as a ubiquitous factor in triggering drought-induced tree mortality through the damage induced by the progressive dehydration of plant living cells. However, fundamental evidence of the mechanistic link connecting xylem HF to cell death has not been identified yet. The main aim of this study was to evaluate, at the leaf level, the relationship between loss of hydraulic function due to cavitation and cell death under drought conditions and discern how this relationship varied across species with contrasting resistances to cavitation. Drought was induced by withholding water from potted seedlings, and their leaves were sampled to measure their relative water content (RWC) and cell mortality. Vulnerability curves to cavitation at the leaf level were constructed for each species. An increment in cavitation events occurrence precedes the onset of cell mortality. A variation in cells tolerance to dehydration was observed along with the resistance to cavitation. Overall, our results indicate that the onset of cellular mortality occurs at lower RWC than the one for cavitation indicating the role of cavitation in triggering cellular death. They also evidenced a critical RWC for cellular death varying across species with different cavitation resistance.

Role of hydraulic traits in stomatal regulation of transpiration under different vapour pressure deficits across five Mediterranean tree crops.

The differential stomatal regulation of transpiration among plant species in response to water deficit is not fully understood, although several hydraulic traits have been reported to influence it. This knowledge gap is partly due to a lack of direct and concomitant experimental data on transpiration, stomatal conductance, and hydraulic traits. We measured sap flux density (Js), stomatal conductance (gs), and different hydraulic traits in five crop species. Our aim was to contribute to establishing the causal relationship between water consumption and its regulation using a hydraulic trait-based approach. The results showed that the species-specific regulation of Js by gs was overall coordinated with the functional hydraulic traits analysed. Particularly relevant was the negative and significant relationship found between the Huber value (Hv) and its functional analogue ratio between maximum Js and gs (Jsmax/gsmax) which can be understood as a compensation to maintain the hydraulic supply to the leaves. The Hv was also significantly related to the slope of the relationship between gs and Js response to vapour pressure deficit and explained most of its variability, adding up to evidence recognizing Hv as a major trait in plant water relations. Thus, a hydraulic basis for regulation of tree water use should be considered.

Mechanisms of xylem hydraulic recovery after drought in Eucalyptus saligna.

The mechanisms by which woody plants recover xylem hydraulic capacity after drought stress are not well understood, particularly with regard to the role of embolism refilling. We evaluated the recovery of xylem hydraulic capacity in young Eucalyptus saligna plants exposed to cycles of drought stress and rewatering. Plants were exposed to moderate and severe drought stress treatments, with recovery monitored at time intervals from 24 h to 6 months after rewatering. The percentage loss of xylem vessels due to embolism (PLV) was quantified at each time point using microcomputed tomography with stem water potential (Ψx ) and canopy transpiration (Ec ) measured before scans. Plants exposed to severe drought stress suffered high levels of embolism (47.38% ± 10.97% PLV) and almost complete canopy loss. No evidence of embolism refilling was observed at 24 h, 1 week, or 3 weeks after rewatering despite rapid recovery in Ψx . Recovery of hydraulic capacity was achieved over a 6-month period by growth of new xylem tissue, with canopy leaf area and Ec recovering over the same period. These findings indicate that E. saligna recovers slowly from severe drought stress, with potential for embolism to persist in the xylem for many months after rainfall events.

Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.

Longitudinal preclinical evaluation of the novel radioligand [11C]CHDI-626 for PET imaging of mutant huntingtin aggregates in Huntington's disease.

PURPOSE: As several therapies aimed at lowering mutant huntingtin (mHTT) brain levels in Huntington's disease (HD) are currently being investigated, noninvasive positron emission tomography (PET) imaging of mHTT could be utilized to directly evaluate therapeutic efficacy and monitor disease progression. Here we characterized and longitudinally assessed the novel radioligand [11C]CHDI-626 for mHTT PET imaging in the zQ175DN mouse model of HD. METHODS: After evaluating radiometabolites and radioligand kinetics, we conducted longitudinal dynamic PET imaging at 3, 6, 9, and 13 months of age (M) in wild-type (WT, n = 17) and heterozygous (HET, n = 23) zQ175DN mice. Statistical analysis was performed to evaluate temporal and genotypic differences. Cross-sectional cohorts at each longitudinal time point were included for post-mortem [3H]CHDI-626 autoradiography. RESULTS: Despite fast metabolism and kinetics, the radioligand was suitable for PET imaging of mHTT. Longitudinal quantification could discriminate between genotypes already at premanifest stage (3 M), showing an age-associated increase in signal in HET mice in parallel with mHTT aggregate load progression, as supported by the post-mortem [3H]CHDI-626 autoradiography. CONCLUSION: With clinical evaluation underway, [11C]CHDI-626 PET imaging appears to be a suitable preclinical candidate marker to monitor natural HD progression and for the evaluation of mHTT-lowering therapies.

Biodistribution and dosimetry in human healthy volunteers of the PET radioligands [11C]CHDI-00485180-R and [11C]CHDI-00485626, designed for quantification of cerebral aggregated mutant huntingtin.

PURPOSE: Huntington's disease is caused by a trinucleotide expansion in the HTT gene, which leads to aggregation of mutant huntingtin (mHTT) protein in the brain and neurotoxicity. Direct in vivo measurement of mHTT aggregates in human brain parenchyma is not yet possible. In this first-in-human study, we investigated biodistribution and dosimetry in healthy volunteers of [11C]CHDI-00485180-R ([11C]CHDI-180R) and [11C]CHDI-00485626 ([11C]CHDI-626), two tracers designed for PET imaging of aggregated mHTT in the brain that have been validated in preclinical models. METHODS: Biodistribution and radiation dosimetry studies were performed in 3 healthy volunteers (age 25.7 ± 0.5 years; 2 F) for [11C]CHDI-180R and in 3 healthy volunteers (age 35.3 ± 6.8 years; 2 F) for [11C]CHDI-626 using sequential whole-body PET-CT. Source organs were delineated in 3D using combined PET and CT data. Individual organ doses and effective doses were determined using OLINDA 2.1. RESULTS: There were no clinically relevant adverse events. The mean effective dose (ED) for [11C]CHDI-180R was 4.58 ± 0.65 µSv/MBq, with highest absorbed doses for liver (16.9 µGy/MBq), heart wall (15.9 µGy/MBq) and small intestine (15.8 µGy/MBq). Mean ED for [11C]CHDI-626 was 5.09 ± 0.06 µSv/MBq with the highest absorbed doses for the gallbladder (26.5 µGy/MBq), small intestine (20.4 µGy/MBq) and liver (19.6 µGy/MBq). Decay-corrected brain uptake curves showed promising kinetics for [11C]CHDI-180R, but for [11C]CHDI-626 an increasing signal over time was found, probably due to accumulation of a brain-penetrant metabolite. CONCLUSION: [11C]CHDI-180R and [11C]CHDI-626 are safe for in vivo PET imaging in humans. The estimated radiation burden is in line with most 11C-ligands. While [11C]CHDI-180R has promising kinetic properties in the brain, [11C]CHDI-626 is not suitable for human in vivo mHTT PET due to the possibility of a radiometabolite accumulating in brain parenchyma. TRIAL REGISTRATION: EudraCT number 2020-002129-27. CLINICALTRIALS: gov NCT05224115 (retrospectively registered).

Modelling the Human Blood-Brain Barrier in Huntington Disease.

While blood-brain barrier (BBB) dysfunction has been described in neurological disorders, including Huntington's disease (HD), it is not known if endothelial cells themselves are functionally compromised when promoting BBB dysfunction. Furthermore, the underlying mechanisms of BBB dysfunction remain elusive given the limitations with mouse models and post mortem tissue to identify primary deficits. We established models of BBB and undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived brain-like microvascular endothelial cells (iBMEC) from HD patients or unaffected controls. We demonstrated that HD-iBMECs have abnormalities in barrier properties, as well as in specific BBB functions such as receptor-mediated transcytosis.

Linking xylem network failure with leaf tissue death.

Global warming is expected to dramatically accelerate forest mortality as temperature and drought intensity increase. Predicting the magnitude of this impact urgently requires an understanding of the process connecting atmospheric drying to plant tissue damage. Recent episodes of forest mortality worldwide have been widely attributed to dry conditions causing acute damage to plant vascular systems. Under this scenario vascular embolisms produced by water stress are thought to cause plant death, yet this hypothetical trajectory has never been empirically demonstrated. Here we provide foundational evidence connecting failure in the vascular network of leaves with tissue damage caused during water stress. We observe a catastrophic sequence initiated by water column breakage under tension in leaf veins which severs local leaf tissue water supply, immediately causing acute cellular dehydration and irreversible damage. By highlighting the primacy of vascular network failure in the death of leaves exposed to drought or evaporative stress our results provide a strong mechanistic foundation upon which models of plant damage in response to dehydration can be confidently structured.

Pharmacokinetics and metabolic disposition of a potent and selective kynurenine monooxygenase inhibitor, CHDI-340246, in laboratory animals.

The disposition of a novel kynurenine monooxygenase inhibitor, CHDI-340246, was investigated in vitro and in animals.In vitro, there was minimal metabolic turnover of CHDI-340246 in all species. The protein binding was higher in human plasma (99.7%) relative to other species.In all species, blood clearance was low (<20% of liver blood flow) and volume of distribution was small (<0.5 L/kg). The terminal half-life was longer in monkeys (9 hr) than in mice, rats, or dogs (1-2 hr). CHDI-340246 was orally bioavailable (>60%) in all species.In rats, [14C]CHDI-340246 showed wide distribution of radioactivity in all tissues except brain and testes. In rats, the parent drug was the major circulating moiety with minor amounts of a sulphate conjugate of an O-dealkylated metabolite. The elimination occurred via the urinary route and to a lesser extent by biliary route, but mostly as metabolites. In cynomolgus monkeys, the parent drug predominated in plasma with only trace amounts of metabolites detected.Acyl glucuronide conjugate of CHDI-340246 was not detected in plasma of rats or monkeys.Overall, the ADME profile of CHDI-340246 was favourable in rats and monkeys for potential evaluation of KMO inhibition in humans.

Analysis of HPV Integrations in Mexican Pre-Tumoral Cervical Lesions Reveal Centromere-Enriched Breakpoints and Abundant Unspecific HPV Regions.

Human papillomavirus (HPV) DNA integration is a crucial event in cervical carcinogenesis. However, scarce studies have focused on studying HPV integration (HPVint) in early-stage cervical lesions. Using HPV capture followed by sequencing, we investigated HPVint in pre-tumor cervical lesions. Employing a novel pipeline, we analyzed reads containing direct evidence of the integration breakpoint. We observed multiple HPV infections in most of the samples (92%) with a median integration rate of 0.06% relative to HPV mapped reads corresponding to two or more sequence breakages. Unlike cancer studies, most integrations events were unique (supported by one read), consistent with the lack of clonal selection. Congruent to other studies, we found that breakpoints could occur, practically, in any part of the viral genome. We noted that L1 had a higher frequency of rupture integration (25%). Based on host genome integration frequencies, we found previously reported integration sites in cancer for genes like FHIT, CSMD1, and LRP1B and putatively many new ones such as those exemplified in CSMD3, ROBO2, and SETD3. Similar host integrations regions and genes were observed in diverse HPV types within many genes and even equivalent integration positions in different samples and HPV types. Interestingly, we noted an enrichment of integrations in most centromeres, suggesting a possible mechanism where HPV exploits this structural machinery to facilitate integration. Supported by previous findings, overall, our analysis provides novel information and insights about HPVint.

Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.

Declining root water transport drives stomatal closure in olive under moderate water stress.

Efficient water transport from soil to leaves sustains stomatal opening and steady-state photosynthesis. The aboveground portion of this pathway is well-described, yet the roots and their connection with the soil are still poorly understood due to technical limitations. Here we used a novel rehydration technique to investigate changes in the hydraulic pathway between roots and soil and within the plant body as individual olive plants were subjected to a range of water stresses. Whole root hydraulic resistance (including the radial pathway from xylem to the soil-root interface) constituted 81% of the whole-plant resistance in unstressed plants, increasing to > 95% under a moderate level of water stress. The decline in this whole root hydraulic conductance occurred in parallel with stomatal closure and contributed significantly to the reduction in canopy conductance according to a hydraulic model. Our results demonstrate that losses in root hydraulic conductance, mainly due to a disconnection from the soil during moderate water stress in olive plants, are profound and sufficient to induce stomatal closure before cavitation occurs. Future studies will determine whether this core regulatory role of root hydraulics exists more generally among diverse plant species.

Visual and hydraulic techniques produce similar estimates of cavitation resistance in woody species.

Hydraulic failure of the plant vascular system is a principal cause of forest die-off under drought. Accurate quantification of this process is essential to our understanding of the physiological mechanisms underpinning plant mortality. Imaging techniques increasingly are applied to estimate xylem cavitation resistance. These techniques allow for in situ measurement of embolism formation in real time, although the benefits and trade-offs associated with different techniques have not been evaluated in detail. Here we compare two imaging methods, microcomputed tomography (microCT) and optical vulnerability (OV), to standard hydraulic methods for measurement of cavitation resistance in seven woody species representing a diversity of major phylogenetic and xylem anatomical groups. Across the seven species, there was strong agreement between cavitation resistance values (P50 ) estimated from visualization techniques (microCT and OV) and between visual techniques and hydraulic techniques. The results indicate that visual techniques provide accurate estimates of cavitation resistance and the degree to which xylem hydraulic function is impacted by embolism. Results are discussed in the context of trade-offs associated with each technique and possible causes of discrepancy between estimates of cavitation resistance provided by visual and hydraulic techniques.

Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction.

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.

Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor.

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ∼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for ∼8 h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.

Mapping xylem failure in disparate organs of whole plants reveals extreme resistance in olive roots.

The capacity of plant species to resist xylem cavitation is an important determinant of resistance to drought, mortality thresholds, geographic distribution and productivity. Unravelling the role of xylem cavitation vulnerability in plant evolution and adaptation requires a clear understanding of how this key trait varies between the tissues of individuals and between individuals of species. Here, we examine questions of variation within individuals by measuring how cavitation moves between organs of individual plants. Using multiple cameras placed simultaneously on roots, stems and leaves, we were able to record systemic xylem cavitation during drying of individual olive plants. Unlike previous studies, we found a consistent pattern of root > stem > leaf in terms of xylem resistance to cavitation. The substantial variation in vulnerability to cavitation, evident among individuals, within individuals and within tissues of olive seedlings, was coordinated such that plants with more resistant roots also had more resistant leaves. Preservation of root integrity means that roots can continue to supply water for the regeneration of drought-damaged aerial tissues after post-drought rain. Furthermore, coordinated variation in vulnerability between leaf, stem and root in olive plants suggests a strong selective pressure to maintain a fixed order of cavitation during drought.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).

Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, where the use of antioxidants as monotherapy has not yielded positive results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degradation. Recently, compounds that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochemical assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and analysis of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.

Most stomatal closure in woody species under moderate drought can be explained by stomatal responses to leaf turgor.

Reduced stomatal conductance (gs ) during soil drought in angiosperms may result from effects of leaf turgor on stomata and/or factors that do not directly depend on leaf turgor, including root-derived abscisic acid (ABA) signals. To quantify the roles of leaf turgor-mediated and leaf turgor-independent mechanisms in gs decline during drought, we measured drought responses of gs and water relations in three woody species (almond, grapevine and olive) under a range of conditions designed to generate independent variation in leaf and root turgor, including diurnal variation in evaporative demand and changes in plant hydraulic conductance and leaf osmotic pressure. We then applied these data to a process-based gs model and used a novel method to partition observed declines in gs during drought into contributions from each parameter in the model. Soil drought reduced gs by 63-84% across species, and the model reproduced these changes well (r(2) = 0.91, P < 0.0001, n = 44) despite having only a single fitted parameter. Our analysis concluded that responses mediated by leaf turgor could explain over 87% of the observed decline in gs across species, adding to a growing body of evidence that challenges the root ABA-centric model of stomatal responses to drought.