Pulse Labeling Research Articles

Hybrid adiabatic pulse with asymmetry (HAPY): An asymmetric adiabatic pulse with an application in pulsed arterial spin labeling at 7T.

A new class of asymmetric adiabatic radiofrequency (RF) pulses, Hybrid Adiabatic Pulse with asYmmetry (HAPY), is designed to be used as the labeling pulse for Pulsed Arterial Spin labeling (PASL) at 7T to reduce overall specific absorption rate (SAR) while maintaining high labeling efficiency with and inhomogeneities. Realistic and distributions were extracted from multiple in vivo scans. The proposed class of asymmetric pulses was parameterized and optimized considering these conditions. Simulation and phantoms experiments were performed to compare the optimized pulses with HS-3, GOIA, and trFOCI pulses. In vivo experiments were conducted to demonstrate the application of HAPY in PICORE PASL at 7T, compared with the GOIA and trFOCI pulses. HAPYs with different amounts of pulse energy reduction are obtained by the proposed optimization framework. Both simulation and phantom experiments demonstrate that HAPY achieves high labeling efficiency and high selectivity along the critical side despite off-resonance and low amplitude. In vivo experiments reveal that HAPY is able to generate robust perfusion signal with less overall SAR or shorter pulse repetition time, compared to the GOIA and trFOCI pulses. The HAPY class of pulses, obtained via systematic optimization tailored to the application of PASL at 7T, reduces power deposition without affecting labeling efficiency, which provides a prospect of further exploiting the benefits of ultra-high field in ASL.

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Are There Wheat Cultivars Allowing Enhanced Carbon Allocation to Soils?

The transfer of atmospheric carbon (C) in soils is a possible strategy for climate change mitigation and for restoring land productivity. While some studies have compared the ability of existing crops to allocate C into the soil, the genetic variations between crop genotypes have received less attention. The objective of this study was to compare the allocation to the soil of atmospheric C by genetically diverse wheat genotypes under different scenarios of soil water availability. The experiments were set up under open-field and greenhouse conditions with 100 wheat genotypes sourced from the International Maize and Wheat Improvement Centre and grown at 25% (drought stressed) and 75% (non-stressed) field capacity, using an alpha lattice design with 10 incomplete blocks and 10 genotypes per block. The genotypes were analyzed for grain yield (GY), plant shoot and root biomass (SB and RB, respectively) and C content, and stocks in plant parts. Additionally, 13C pulse labeling was performed during the crop growth period of 10 selected genotypes for assessing soil C inputs. The average GY varied from 75 to 4696 g m−2 and total plant biomass (PB) from 1967 to 13,528 g m−2. The plant C stocks ranged from 592 to 1109 g C m−2 (i.e., an 87% difference) under drought condition and between 1324 and 2881 g C m−2 (i.e., 117%) under well-watered conditions. Atmospheric C transfer to the soil only occurred under well-drained conditions and increased with the increase in the root to shoot ratio for C stocks (r = 0.71). Interestingly, the highest transfer to the soil was found for LM-26 and LM-47 (13C/12C of 7.6 and 6.5 per mille, respectively) as compared to LM-70 and BW-162 (0.75; 0.85). More is to be done to estimate the differences in C fluxes to the soil over entire growing seasons and to assess the long-term stabilization of the newly allocated C. Future research studies also need to identify genomic regions associated with GY and soil C transfer to enable the breeding of “carbon-superior” cultivars.

Detection of ictal and periictal hyperperfusion with subtraction of ictal-interictal 1.5-Tesla pulsed arterial spin labeling images co-registered to conventional magnetic resonance images (SIACOM).

Our recent report showed that 1.5-T pulsed arterial spin labeling (ASL) magnetic resonance (MR) perfusion imaging (1.5-T Pulsed ASL [PASL]), which is widely available in the field of neuroemergency, is useful for detecting ictal hyperperfusion. However, the visualization of intravascular ASL signals, namely, arterial transit artifact (ATA), is more remarkable than that of 3-T pseudocontinuous ASL and is easily confused with focal hyperperfusion. To eliminate ATA and enhance the detectability of (peri) ictal hyperperfusion, we developed the subtraction of ictal-interictal 1.5-T PASL images co-registered to conventional MR images (SIACOM). We retrospectively analyzed the SIACOM findings in four patients who underwent ASL during both (peri) ictal and interictal states and examined the detectability for (peri) ictal hyperperfusion. In all patients, the ATA of the major arteries was almost eliminated from the subtraction image of the ictal-interictal ASL. In patients 1 and 2 with focal epilepsy, SIACOM revealed a tight anatomical relationship between the epileptogenic lesion and the hyperperfusion area compared with the original ASL image. In patient 3 with situation-related seizures, SIACOM detected minute hyperperfusion at the site coinciding with the abnormal electroencephalogram area. SIACOM of patient 4 with generalized epilepsy diagnosed ATA of the right middle cerebral artery, which was initially thought to be focal hyperperfusion on the original ASL image. Although it is necessary to examine several patients, SIACOM can eliminate most of the depiction of ATA and clearly demonstrate the pathophysiology of each epileptic seizure.

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

Role of root hair elongation in rhizosheath aggregation and in the carbon flow into the soil

One of the most prominent changes in the rhizospheric soil structure is associated with the formation of a strongly bound soil layer in the surroundings of the root, which is named rhizosheath. In this study, we investigated how root hair elongation, a ubiquitous root morphological trait, affect the stability of rhizosheath aggregates. Using 13CO2 pulse labeling, we tracked the fate of root-derived 13C inputted into the rhizosheath of two Zea mays L. genotypes with contrasting root hair elongation: a mutant with root hair defective elongation (rth3) and a corresponding wild type (WT). In addition, we also investigated the differences between two 13CO2 labeling approaches (single vs. multiple pulse labeling) in the distribution of 13C in the rhizosheath aggregates. We were able to demonstrate that the rhizosheath aggregate stability and the resulting aggregate size distribution follows the same mechanisms irrespective of the root hair elongation. This result reinforces the assumption that other soil properties are more decisive for the soil structure formation in the rhizosheath in comparison to root hair elongation. The majority of recently deposited root-derived C (57%) was found in the macroaggregates. Increasing the number of pulses (multiple pulse labeling approach) resulted in a higher 13C enrichment of the rhizosheath aggregates fractions in comparison to the application of a single pulse. While both labeling approaches resulted in a similar distribution of 13C in the rhizosheath aggregates, the higher enrichment given by multiple pulse labeling allowed the separation of significant differences between the genotypes in plant C allocation in the rhizosheath.

Modulation Effects of Repeated Transcranial Direct Current Stimulation at the Dorsolateral Prefrontal Cortex: A Pulsed Continuous Arterial Spin Labeling Study.

Transcranial direct current stimulation (tDCS) is a promising non-invasive method to modulate brain excitability. The aim of this study was to better understand the cerebral blood flow (CBF) changes during and after repeated tDCS at the right dorsolateral prefrontal cortex (DLPFC) in healthy participants using pulsed continuous arterial spin labeling (pCASL). Elucidating CBF changes associated with repeated tDCS may shed light on the understanding of the mechanisms underlying the therapeutic effects of tDCS. tDCS was applied for three consecutive days for 20 min at 2 mA, and MRI scans were performed on day 1 and 3. During anodal tDCS, increased CBF was detected in the bilateral thalamus on day 1 and 3 (12% on day 1 and of 14% on day 3) and in the insula on day 1 (12%). After anodal tDCS on day 1, increased CBF was detected in the cerebellum and occipital lobe (11.8%), while both cathodal and sham tDCS were associated with increased CBF in the insula (11% and 10%, respectively). Moreover, anodal tDCS led to increased CBF in the lateral prefrontal cortex and midcingulate cortex in comparison to the sham. These findings suggest that tDCS can modulate the CBF and different tDCS modes may lead to different effects.

The impact of hemodiafiltration on cognitive function in patients with end-stage renal disease.

Patients with end-stage renal disease are more likely to suffer cognitive impairment. Cognitive impairment may lead to long-term severe adverse consequences. To explore the impact of different blood purification therapy on cerebral blood flow and cognitive functions in end-stage renal disease. This prospective study evaluated patients with end-stage renal disease undergoing blood purification from January to March 2021. Matched healthy controls were also included. Participants performed neurocognitive measurements, including a mini-mental state examination, logical memory test-20-minutes delayed, verbal fluency test, digit span test, clock drawing test, and stroop color and word test C. In addition, we tested plasma amyloid-β protein levels, serum Fe and hemoglobin levels in blood samples. Cerebral blood flow was measured using pulsed pseudocontinuous arterial spin labeling. We analyzed and compared the correlation between cognitive function, biomarkers, and cerebral blood flow between patients and healthy subjects, as well as between patients with different treatments. A total of 44 patients with end-stage renal disease (mean age, 57.39 years ± 8.63) and 46 healthy controls (mean age, 56.15 years ± 6.40) were recruited. Patients receive hemodialysis three times a week, and 27 of them have been replaced hemodialysis for hemodiafiltration twice a month. The cognitive function of patients was worse than healthy controls (P < 0.05). The patients showed higher plasma concentrations of amyloid-β40, amyloid-β42, Tau, and pTau181 than healthy controls (P < 0.05). The group receiving both hemodialysis and hemodiafiltration had higher cerebral blood flow signal values in the left caudate nucleus (chuster-level P < 0.05, voxel-level P < 0.001). They also exhibited better verbal fluency function than the hemodialysis-only group (P < 0.05). Patients with the end-stage renal disease showed widespread cognitive declines. Cerebral blood flow generally decreased in the cerebral cortex and increased in subcortical regions. The hemodiafiltration may protect verbal function by increasing cerebral blood flow in the left caudate.

One-helix protein 2 is not required for the synthesis of photosystem II subunit D1 in Chlamydomonas.

In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization.

Comparison between 1.5-Tesla pseudocontinuous and pulsed arterial spin labeling in visualizing peri-ictal hyperperfusion: A case report

Arterial spin labeling (ASL) imaging with multiple postlabeling delays (PLD) can demonstrate the hemodynamics of peri-ictal hyperperfusion. Labeling methods include pulsed ASL (PASL) and pseudocontinuous ASL (pCASL). Although PASL is more widely used in daily clinical practice, it has several disadvantages because of lower delivery of labeled magnetization. We compared pCASL and PASL images acquired after a 24-h interval, using a 1.5-Tesla machine, in a 62-year-old man with non-convulsive status epilepticus. The pCASL during electrographic status epilepticus clearly demonstrated that ASL signals in the grey matter of the right hemisphere, particularly in the posteromedial part of the right temporal lobe, increased at PLDs of 1.5 s and 1.75 s and were almost washed out at 2.0 s. Although PASL, which was obtained during the ictal-interictal continuum when there were no changes in ictal findings on diffusion-weighted images, showed a similar trend, the increased signals were less pronounced than those of pCASL, with more contamination of arte-rial transit artifact in the subarachnoid space. Although the electroencephalographic findings were not the same when both ASLs were performed, the present study confirms that PASL is somewhat inferior to pCASL in detecting peri-ictal hyperperfusion because of the greater effect of arterial transient artifacts.

13CO2-labelling and Sampling in Algae for Flux Analysis of Photosynthetic and Central Carbon Metabolism.

The flux in photosynthesis can be studied by performing 13CO2 pulse labelling and analysing the temporal labelling kinetics of metabolic intermediates using gas or liquid chromatography linked to mass spectrometry. Metabolic flux analysis (MFA) is the primary approach for analysing metabolic network function and quantifying intracellular metabolic fluxes. Different MFA approaches differ based on the metabolic state (steady vs. non-steady state) and the use of stable isotope tracers. The main methodology used to investigate metabolic systems is metabolite steady state associated with stable isotope labelling experiments. Specifically, in biological systems like photoautotrophic organisms, isotopic non-stationary 113C metabolic flux analysis at metabolic steady state with transient isotopic labelling (13C-INST-MFA) is required. The common requirement for metabolic steady state, alongside its very short half-timed reactions, complicates robust MFA of photosynthetic metabolism. While custom gas chambers design has addressed these challenges in various model plants, no similar tools were developed for liquid photosynthetic cultures (e.g., algae, cyanobacteria), where diffusion and equilibration of inorganic carbon species in the medium entails a new dimension of complexity. Recently, a novel tailor-made microfluidics labelling system has been introduced, supplying short 13CO2 pulses at steady state, and resolving fluxes across most photosynthetic metabolic pathways in algae. The system involves injecting algal cultures and medium containing pre-equilibrated inorganic 13C into a microfluidic mixer, followed by rapid metabolic quenching, enabling precise seconds-level label pulses. This was complemented by a 13CO2-bubbling-based open labelling system (photobioreactor), allowing long pulses (minutes-hours) required for investigating fluxes into central C metabolism and major products. This combined labelling procedure provides a comprehensive fluxome cover for most algal photosynthetic and central C metabolism pathways, thus allowing comparative flux analyses across algae and plants.

Combining Metabolic Pulse Labeling and Quantitative Proteomics to Monitor Protein Synthesis Upon Viral Infection.

Viruses like influenza A virus (IAV) hijack host cells in order to replicate. To actively and abundantly synthesize viral proteins, they reprogram the cellular transcriptional and translational landscape. Here, we present a proteomic approach that allows us to quantify the differences in host and viral protein synthesis comparatively for different strains of IAV. The method is based on combining quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC) and bioorthogonal labeling with methionine analogs. This methodology accurately quantifies synthesis of host and viral proteins with high temporal resolution and faithfully detects global changes in cellular translation capacity. It thus provides unique insights into the dynamics of protein synthesis as the infection progresses.

Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance

The accumulation of α-synuclein (α-syn) in intracellular formations known as Lewy bodies (LBs) is associated with several neurodegenerative diseases including Parkinson’s disease and Lewy Body Dementia. There is still limited understanding of how α-syn and LB formation is associated with cellular dysfunction and degeneration in these diseases. To examine the clearance and production dynamics of α-syn we transduced organotypic murine brain slice cultures (BSCs) with recombinant adeno-associated viruses (rAAVs) to express Dendra2-tagged human wild-type (WT) and mutant A53T α-syn, with and without the addition of exogenous α-syn fibrillar seeds and tracked them over several weeks in culture using optical pulse labeling. We found that neurons expressing WT or mutant A53T human α-syn show similar rates of α-syn turnover even when insoluble, phosphorylated Ser129 α-syn has accumulated. Taken together, this data reveals α-syn aggregation and overexpression, pSer129 α-syn, nor the A53T mutation affect α-syn dynamics in this system. Prion-type seeding with exogenous α-syn fibrils significantly slows α-syn turnover, in the absence of toxicity but is associated with the accumulation of anti-p62 immunoreactivity and Thiazin Red positivity. Prion-type induction of α-syn aggregation points towards a potential protein clearance deficit in the presence of fibrillar seeds and the ease of this system to explore precise mechanisms underlying these processes. This system facilitates the exploration of α-syn protein dynamics over long-term culture periods. This platform can further be exploited to provide mechanistic insight on what drives this slowing of α-syn turnover and how therapeutics, other genes or different α-syn mutations may affect α-syn protein dynamics.

Long-Term Grazing Intensity Impacts Belowground Carbon Allocation and Mycorrhizas Revealed by 13CO2 Pulse Labeling

Despite the importance of grasslands for carbon storage and climate regulation, there is uncertainty about the effect of livestock grazing intensity on aboveground carbon assimilation and belowground carbon partitioning. Furthermore, the relationship between belowground carbon allocation and arbuscular mycorrhizal fungi, which serve as a conduit for carbon movement through the plant and soil, is unclear. To investigate this, we used an in situ 13C stable isotope pulse-chase labeling approach in plots under seven rates of sheep grazing intensity in a steppe grassland in northern China. We quantified the allocation of carbon to plants, soil, and soil-respired CO2 along with measurements of mycorrhizal hyphal density in the soil. With increasing grazing intensity, carbon assimilation per unit shoot biomass increased significantly, whereas carbon allocation to roots marginally decreased. Soil-respired CO2 appeared to be independent of grazing intensity. Mycorrhizal hyphal density decreased with increasing grazing intensity and was correlated significantly with new carbon input to roots 2 d after labeling and marginally related to that of soil 1 d after the 13C-CO2 pulse. Our study suggests that grazing intensity alters the distribution of carbon among different carbon pools within the plant-soil system. The results also underscored the key role of mycorrhizas as a fast route for carbon transfer from plant to soil.

Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation

Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods relying on pulsed metabolic labeling are popular to quantify turnover rates on a proteome-wide scale. Such data have traditionally been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a protein’s lifetime—a fundamentally different metric. This is due to interconversion of the measured proteoform species. Using this framework, we identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results thus allow tying PTMs to the age of the modified proteins.

Hippocampal inflammation is associated with vascular dysfunction in middle aged Non‐Latinx Black APOE‐ε4 carriers

AbstractBackgroundNeuroinflammation may be associated with global vascular dysfunction and damage. APOE‐ε4, a major genetic risk factor for late onset AD, is expressed in neuroinflammatory cells and may lead to increased pro‐inflammatory cytokine and nitric oxide production in response to injury. We examined the relationship of vascular dysfunction and damage with a magnetic resonance spectroscopy (MRS) marker of neuroinflammation in APOE‐ε4 carriers and non‐carriers.MethodSingle proton MRS, pulsed arterial spin labeling (PASL), and T2 fluid attenuated inversion recovery (FLAIR) were collected in a sample of racially/ethnically diverse, middle‐aged adults (n=313, women=67%, age=55±11, years of education=13±3, 72% Latinx/22% Non‐Latinx Black/6% Non‐Latinx White) and used to derive hippocampal Inositol/creatine ratios (Ins/Cr) as a measure of neuroinflammation. Global cerebral blood flow (CBF) was used as a measure of vascular function, and white matter hyperintensity (WMH) volume as a measure of small vessel cerebrovascular damage. APOE genotype was categorized as the presence or absence of at least one ε4 alleles. We estimated general linear models to test main effects and the interaction of APOE‐ε4 and Ins/Cr on WMH or CBF, adjusting for age and stratified by race/ethnicity due to differential prevalence and effects of APOE ε4.ResultsIn the whole sample, being an ε4 non‐carrier (β=‐12.0 95%CI: [‐24.8, 0.83], p=0.067) and having greater Ins/Cr (β=‐1.1[‐2.2, 0.1], p=0.062) were associated with lower CBF. Within Non‐Latinx Black participants, being an APOE‐ε4 non‐carrier (β=‐40.2[‐73.3, ‐7.1], p=0.02) and having greater Ins/Cr (β=‐3.7[‐73.3, ‐7.1], p=0.007) were associated with lower CBF. The negative association with Ins/Cr was stronger in APOE‐ε4 carriers compared to non‐carriers (β=3.9[0.65, 7.1], p=0.02). No associations of Ins/Cr were observed with WMH.ConclusionGlobal vascular dysfunction, but not vascular damage, was related to hippocampal inflammation in APOE‐ε4 carriers among Non‐Latinx Black middle‐aged adults. Neuroinflammation may be linked to vascular brain health which is particularly important for groups with higher APOE‐ε4 prevalence or with greater exposure to social determinants of health that can lead to greater inflammation.

Longitudinal spatial mapping of lipid metabolites reveals pre-symptomatic changes in the hippocampi of Huntington's disease transgenic mice

In Huntington's disease (HD), a key pathological feature includes the development of inclusion-bodies of fragments of the mutant huntingtin protein in the neurons of the striatum and hippocampus. To examine the molecular changes associated with inclusion-body formation, we applied MALDI-mass spectrometry imaging and deuterium pulse labelling to determine lipid levels and synthesis rates in the hippocampus of a transgenic mouse model of HD (R6/1 line). The R6/1 HD mice lacked inclusions in the hippocampus at 6 weeks of age (pre-symptomatic), whereas inclusions were pervasive by 16 weeks of age (symptomatic). Hippocampal subfields (CA1, CA3 and DG), which formed the highest density of inclusion formation in the mouse brain showed a reduction in the relative abundance of neuron-enriched lipids that have roles in neurotransmission, synaptic plasticity, neurogenesis, and ER-stress protection. Lipids involved in the adaptive response to ER stress (phosphatidylinositol, phosphatidic acid, and ganglioside classes) displayed increased rates of synthesis in HD mice relative to WT mice at all the ages examined, including prior to the formation of the inclusion bodies. Our findings, therefore, support a role for ER stress occurring pre-symptomatically and potentially contributing to pathological mechanisms underlying HD.

Carbon allocation in cassava is affected by water deficit and potassium application - A 13 C-CO2 pulse labelling assessment.

Cassava production faces challenges in a changing climate. Pulse labelling cassava with 13 C-CO2 has the potential to elucidate carbon allocation mechanisms of cassava under drought stress and with potassium application. Understanding these mechanisms could guide efforts to mitigate effects of drought in cassava cropping systems. Forty-eight cassava plants received a nutrient solution high or low in potassium. Water deficit was imposed on half of the plants at bulk root initiation stage, after which they were labelled for 8 h with 13 C-CO2 in a 15 m3 growth chamber. Plants were harvested 8 h, 9 days and 24 days after labelling, and separated into leaves, stems and roots. δ13 C values of the different parts were measured using an isotope ratio mass spectrometer, from which 13 C excess was calculated. Water deficit decreased transpiration (P< 0.001) and increased carbon respiration (P< 0.05). Potassium application increased assimilate distribution to the roots (P< 0.05) at 9 days after labelling, more strongly for plants under water deficit. The opposite was found at 24 days (P< 0.05) with the legacy of water deficit additionally increasing assimilate distribution to roots (P< 0.05). Youngest, fully expanded leaves contained up to 47% of initial 13 C excess at 24 days after labelling. Pulse labelling proved to be successful in shedding light on carbon allocation in relation to water and potassium availability. This technique, once adapted to field conditions, could further be used to improve fertilizer recommendations or change agronomic practices to cope with plant stress.

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Efficiency of Protein Renewal Is Limited by Feed Intake and Not by Protein Lifetime in Aging Caenorhabditis elegans.

Protein turnover maintains the proteome's functional integrity. Here, protein turnover efficiency over time in wild-type Caenorhabditis elegans was assessed using inverse [15N]-pulse labeling up to 7 days after the egg-laying phase at 20 °C. Isotopic analysis of some abundant proteins was executed favoring data quality over quantity for mathematical modeling. Surprisingly, isotopic enrichment over time reached an upper limit showing an apparent cessation of protein renewal well before death, with protein fractions inaccessible to turnover ranging from 14 to 83%. For life span modulation, worms were raised at different temperatures after egg laying. Mathematical modeling of isotopic enrichment points either to a slowdown of protein turnover or to an increasing protein fraction resistant to turnover with time. Most notably, the estimated time points of protein turnover cessation from our mathematical model were highly correlated with the observed median life span. Thrashing and pumping rates over time were linearly correlated with isotopic enrichment, therefore linking protein/tracer intake to protein turnover rate and protein life span. If confirmed, life span extension is possible by optimizing protein turnover rate through modulating protein intake in C. elegans and possibly other organisms. While proteome maintenance benefits from a high protein turnover rate, protein turnover is fundamentally energy-intensive, where oxidative stress contributes to damage that it is supposed to repair.