Deprivation of exogenous and endogenous glutamine synergistically promotes neuronal cell death.

<p>A–D) Primary neurons were grown in four different culture media. A) normal; B) normal glutamine in the presence of the glutamine synthetase (GS) inhibitor, methionine sulfoxide (MSO); C) glutamine-free; D) glutamine-free plus MSO. After 2–3 days cells were immunostained for Map2 (red) and cleaved caspase-3 (green), pictures were taken at a magnification of 200×. E) Cell counts reveal the ratio of apoptotic neurons (Caspase-3-stained) to total cell counts (DAPI staining). Error bars correspond to standard deviations; * denotes p<0.05, ** denotes p<0.01.</p

Glutamine supplementation protects neurons in AD mouse models <i>in vivo</i>.

<p>R1.40 and 8.9 mouse models of AD were treated with LPS as an immune challenge to enhance the degenerative phenotype. A) Microglia activation is dampened in mice supplemented with glutamine. Two different brain regions are shown as indicated at the top of each column. Microglia was stained with Iba-1 antibody. Activated microglia has thicker and shorter processes compared to resting microglia. B) Western blots show protective effect of glutamine supplementation (Gln+): lower tau phosphorylation (AT8), less cell cycle reentry (PCNA), less ATM activation (P-ATM) and more expression of synaptic maintenance proteins (VAMP2 and synaptophysin).C) Western blots shown in panel B were quantified and analyzed for statistical significance between control and glutamine supplementation, * denotes p<0.05. D) Immunofluorescent images show less cyclin A staining in cortical neurons of animals supplemented with glutamine.</p

Stress responses are compromised without glutamine.

<p>A) N2a cells were treated with different cellular stressors for 30 minutes, and then collected and analyzed on Western blots. The various stress response proteins are indicated at the right and are described in more detail in the text. P-ATM = ^PS1981-ATM. B) N2a cells were treated with 5 µM etoposide for 30 minutes to induce DNA damage. Etoposide was removed and fresh medium with or without glutamine was added. Cells were collected at indicated time points for Western blot analysis. Gln− = glutamine-free medium; Gln+ = 2 mM glutamine containing medium. C) Comet assays were performed on a portion of the samples collected for experiments shown in panel B. In each panel, the arrowhead points to a representative cell that is shown at higher magnification in the inset. There are few comet tails in control cultures. After 30 min in etoposide, however, the typical cell had well developed comet tails indicative of DNA damage. After 4 hours recovery, most comet tails disappeared in cultures with or without glutamine, but more pycnotic nuclei are found in glutamine deprived cultures. D) Counts of pycnotic nuclei as a measure of cell death. For each condition, 250 randomly selected cells were examined under a microscope; cells were separated into four categories: normal nucleus, blebbing nucleus, condensing nucleus, and disappearance of nuclear material. E) Cell death evaluated by LDH assay. N2a cells treated with 1 mM H₂O₂ for 30 minutes, and allowed to recover in fresh media for 6 hours. LDH activity was measured by the reactions absorbance at 490 nM, using absorbance at 650 nm as a reference. ** denotes p<0.01, error bars = standard deviation. F) Cell viability evaluated by MTT assay. Cells were treated as E, but allowed to recover for 24 hours before MTT assay (absorbance at 570 nm, reference at 650 nm). ** denotes p<0.01, error bars = standard deviation.</p

Glutamine deprivation up-regulates GS expression.

<p>A) GS immune-positive hippocampal neurons were detected in AD patients but not in control samples. Immunopositive astrocytes are found in both. B) GS levels in AD frontal cortex are significantly higher than in control samples. One representative blot at two different exposure times is shown. C) Quantification and statistics of 12 AD and 8 control cases. GS levels in controls were arbitrarily set as 100%. Error bars denote standard deviations. P values were calculated via Student's T-test. D) Removing glutamine from culture medium or inhibiting GS activity with MSO induces GS expression in primary neurons. E) Removing glutamine from the culture medium promotes GS expression in N2a cells.</p

Overexpression of GS enhances neurodegeneration in the absence of exogenous glutamine.

<p>A) GS overexpression causes the degeneration of processes in differentiated N2a cells, while the low activity mutant GS^R341C appears to have no same effect. B) Both wild type and mutant GS overexpression increases the levels of apoptotic markers (cleavage of caspase-3 and caspase-6) in basal conditions and under stress tested including 0.1 mM H₂O₂, 100 nM Aβ and 20 µM etoposide. Mutant GS^R341C had consistently less effect than GS^WT. C) Western blot shows the effects of GS overexpression in the presence or absence of free glutamine in the culture medium. The phospho-RB antibody provides evidence of both enhanced cell cycle (increased full length P-Rb) and cell death (caspase-3-cleaved Rb, P-Rb*) simultaneously. D) Quantification of 4 repetitions of the experiment illustrated in panel C. Error bars denotes standard deviations. * = p<0.05; ** = p<0.01 (by Student's T-test). E) Cell death evaluated by LDH assay. N2a cells overexpressing wild type GS and GS(R341C) were treated with 1 mM H₂O₂ for 30 minutes then allowed to recover in fresh media with or without glutamine for 6 hours before LDH assay. Error bars denote standard deviations. * = p<0.05.</p

Neurons in low glutamine are more sensitive to the neurotoxic effect of Aβ.

<p>A) ATR levels are significantly reduced in neurons cultured without glutamine. ATM and 53BP1 are reduced by a similar amount, but the variability in the assay kept these change at the level of a trend rather than a significant difference. Average of 3 repeat experiments; error bar denotes standard deviation, * = p<0.05. B) Neurons grown for 9 days (DIV9) in Neurobasal medium with or without glutamine were treated with 20 µM etoposide for 30 minutes, then immunostained for activated caspase 6 staining (green) and counterstained with DAPI (blue). C) Aβ-induced tau phosphorylation in primary neurons depends on glutamine status in the culture medium. D) Tau phosphorylation is suppressed by glutamine in a concentration-dependent manner, even though levels of total tau (3R) remain unchanged. E) Aged neurons treated with Aβ have more processes degeneration (beaded Map2 stained dendrites) in the absence of glutamine. F) Synaptophysin staining is increased in the presence of glutamine. The numbers in each panel refer to the concentration of glutamine (mM) in the culture medium.</p

Breaking the Linear Scaling Relationship of the Reverse Water–Gas–Shift Reaction via Construction of Dual-Atom Pt–Ni Pairs

Oxide-supported single-atom catalysts hold great potential for reverse water–gas–shift (RWGS) reactions. Nevertheless, it remains challenging to break the linear scaling relationships between the adsorption and desorption capability of catalysts. Herein, we report the design of ZrO2-anchored dual-atom Pt–Ni pairs for the RWGS reaction. The dual-atom material delivers a CO selectivity as high as 99.8% and a space-time yield of 157.2 μmolCO gcat–1 s–1 at atmospheric pressure. Theoretical calculations reveal that the dual-atom Pt–Ni pairs could direct the dual electronic transfer paths (dxz and dyz) to the 2π* orbitals of CO2 in the RWGS reaction, which achieve strong hybridization between them to enable efficient activation of CO2. Moreover, the delocalized charge in dual-atom Pt–Ni may lead to a facile desorption of the CO product

Tree-Guided Rare Feature Selection and Logic Aggregation with Electronic Health Records Data

Statistical learning with a large number of rare binary features is commonly encountered in analyzing electronic health records (EHR) data, especially in the modeling of disease onset with prior medical diagnoses and procedures. Dealing with the resulting highly sparse and large-scale binary feature matrix is notoriously challenging as conventional methods may suffer from a lack of power in testing and inconsistency in model fitting, while machine learning methods may suffer from the inability of producing interpretable results or clinically-meaningful risk factors. To improve EHR-based modeling and use the natural hierarchical structure of disease classification, we propose a tree-guided feature selection and logic aggregation approach for large-scale regression with rare binary features, in which dimension reduction is achieved through not only a sparsity pursuit but also an aggregation promoter with the logic operator of “or”. We convert the combinatorial problem into a convex linearly-constrained regularized estimation, which enables scalable computation with theoretical guarantees. In a suicide risk study with EHR data, our approach is able to select and aggregate prior mental health diagnoses as guided by the diagnosis hierarchy of the International Classification of Diseases. By balancing the rarity and specificity of the EHR diagnosis records, our strategy improves both prediction and interpretation. We identify important higher-level categories and subcategories of mental health conditions and simultaneously determine the level of specificity needed for each of them in associating with suicide risk. Supplementary materials for this article are available online, including a standardized description of the materials available for reproducing the work.</p

Breaking the Linear Scaling Relationship of the Reverse Water–Gas–Shift Reaction via Construction of Dual-Atom Pt–Ni Pairs

Oxide-supported single-atom catalysts hold great potential for reverse water–gas–shift (RWGS) reactions. Nevertheless, it remains challenging to break the linear scaling relationships between the adsorption and desorption capability of catalysts. Herein, we report the design of ZrO2-anchored dual-atom Pt–Ni pairs for the RWGS reaction. The dual-atom material delivers a CO selectivity as high as 99.8% and a space-time yield of 157.2 μmolCO gcat–1 s–1 at atmospheric pressure. Theoretical calculations reveal that the dual-atom Pt–Ni pairs could direct the dual electronic transfer paths (dxz and dyz) to the 2π* orbitals of CO2 in the RWGS reaction, which achieve strong hybridization between them to enable efficient activation of CO2. Moreover, the delocalized charge in dual-atom Pt–Ni may lead to a facile desorption of the CO product

Table_1_Genetic diversity and salt tolerance assessment of 51 alfalfa (Medicago sativa) varieties under saline soil conditions.XLSX

IntroductionSalt stress significantly impacts plant growth worldwide. Although alfalfa exhibits some tolerance to salt-alkali soils, the specific salt tolerance levels across different alfalfa varieties remain inadequately understood. For this purpose, we conducted a study to investigate the diversity of agronomic traits of alfalfa (Medicago sativa) under different soil environments in order to elucidate the salt tolerance of 51 alfalfa varieties.MethodsWe chose three different soil conditions for our study: normal conditions in Yantai as control, saline conditions in Yantai with soil salt concentrations ranging from 0.25 to 0.33%, and saline-alkali conditions in Dongying with soil salt concentrations ranging from 0.28 to 0.32%. We chose 51 alfalfa varieties from different sources as our experimental materials. Planting was conducted following a completely randomized block design with three replicates for each variety. Harvesting was performed when the plants reached 50% flowering, cutting them at a height of 5 cm above the ground. Comprehensive assessments encompassed 10 parameters, namely fresh weight, dry weight, stem-leaf ratio, branch count, plant height, crude protein content, crude fat content, acid detergent fiber, neutral detergent fiber, and ash content.ResultsOur findings revealed that the 51 studied alfalfa varieties displayed significant generalized heritability and coefficient of variation, indicating a high level of genetic diversity. Using principal component and cluster analyses, we categorized the varieties into three distinct clusters based on their agronomic traits. Grey correlation degree analysis revealed the commendable performance of Ying st, PI 672734, and Mei zuo across diverse environments. Genotype emerged as a predominant determinant of all parameters, except crude fat, acid detergent fiber, and neutral detergent fiber. Through the application of membership function analysis, PI 672768 emerged as a variety that demonstrated strong tolerance in both saline soil contexts.DiscussionDespite extensive previous studies indicating Zhong Mu No. 1 as a salt-tolerant alfalfa variety, its performance in this experiment did not distinguish itself. The findings of this study provide a fundamental basis for improving the management of saline-alkali lands and advancing alfalfa cultivation practices.</p