Transcriptome analysis of lipid biosynthesis during kernel development in two walnut (Juglans regia L.) varieties of 'Xilin 3' and 'Xiangling'.

BACKGROUND: Walnut is an oilseed tree species and an ecologically important woody tree species that is rich in oil and nutrients. In light of differences in the lipid content, fatty acid composition and key genes expression patterns in different walnut varieties, the key gene regulatory networks for lipid biosynthesis in different varieties of walnuts were intensively investigated. RESULTS: The kernels of two walnut varieties, 'Xilin 3' (X3) and 'Xiangling' (XL) were sampled at 60, 90, and 120 days post-anthesis (DPA) to construct 18 cDNA libraries, and the candidate genes related to oil synthesis were identified via sequencing and expression analysis. A total of 106 differentially expressed genes associated with fatty acid biosynthesis, fatty acid elongation, unsaturated fatty acid biosynthesis, triglyceride assembly, and oil body storage were selected from the transcriptomes. Weighted gene co-expression network analysis (WGCNA), correlation analysis and quantitative validation confirmed the key role of the FAD3 (109002248) gene in lipid synthesis in different varieties. CONCLUSIONS: These results provide valuable resources for future investigations and new insights into genes related to oil accumulation and lipid metabolism in walnut seed kernels. The findings will also aid future molecular studies and ongoing efforts to genetically improve walnut.

Caveolin Regulates the Transport Mechanism of the Walnut-Derived Peptide EVSGPGYSPN to Penetrate the Blood-Brain Barrier.

Bioactive peptides, derived from short protein fragments, are recognized for their neuroprotective properties and potential therapeutic applications in treating central nervous system (CNS) diseases. However, a significant challenge for these peptides is their ability to penetrate the blood-brain barrier (BBB). EVSGPGYSPN (EV-10) peptide, a walnut-derived peptide, has demonstrated promising neuroprotective effects in vivo. This study aimed to investigate the transportability of EV-10 across the BBB, explore its capacity to penetrate this barrier, and elucidate the regulatory mechanisms underlying peptide-induced cellular internalization and transport pathways within the BBB. The results indicated that at a concentration of 100 µM and osmotic time of 4 h, the apparent permeability coefficient of EV-10 was Papp = 8.52166 ± 0.58 × 10-6 cm/s. The penetration efficiency of EV-10 was influenced by time, concentration, and temperature. Utilizing Western blot analysis, immunofluorescence, and flow cytometry, in conjunction with the caveolin (Cav)-specific inhibitor M-ß-CD, we confirmed that EV-10 undergoes transcellular transport through a Cav-dependent endocytosis pathway. Notably, the tight junction proteins ZO-1, occludin, and claudin-5 were not disrupted by EV-10. Throughout its transport, EV-10 was localized within the mitochondria, Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, and cell membranes. Moreover, Cav-1 overexpression facilitated the release of EV-10 from lysosomes. Evidence of EV-10 accumulation was observed in mouse brains using brain slice scans. This study is the first to demonstrate that Cav-1 can facilitate the targeted delivery of walnut-derived peptide to the brain, laying a foundation for the development of functional foods aimed at CNS disease intervention.

JrPPO1/2 play distinct roles in regulating walnut fruit browning by different spatiotemporal expression and enzymatic characteristics.

Polyphenol oxidase (PPO) activity drives walnut fruit browning, but the roles of its only two-family genes, JrPPO1 and JrPPO2, remain unclear. This study explores the spatiotemporal expression and enzymatic characteristics of JrPPO1 and JrPPO2 in walnut. Treatment with the PPO activator CuSO4 and H2O2 accelerated fruit browning and up-regulated JrPPO1/2 expression, whereas treatment with the PPO inhibitor ascorbic acid delayed browning, down-regulating JrPPO1 and up-regulating JrPPO2 expression. Compared to mJrPPO1, mJrPPO2 can exhibited better enzyme activity at higher temperatures (47 °C) and in more acidic environments (pH 4.25). mJrPPO2 exhibited a higher substrate specificity over mJrPPO1, and the preferred substrates are catechol, chlorogenic acid, and epicatechin. Additionally, mJrPPO2 adapted better to low concentration of oxygen (as low as 1.0% O2) and slightly elevated CO2 levels compared to mJrPPO1. Subcellular localization and spatiotemporal expression patterns showed that JrPPO1 is only expressed in green tissues and located in chloroplasts, while JrPPO2 is also located in chloroplasts, partly associated with membranes, and is expressed in both green and non-green tissues. Silencing JrPPO1/2 with virus-induced gene silencing (VIGS) reduced fruit browning, maintained higher total phenols, and decreased MDA production. Notably, silencing JrPPO1 had a greater impact on browning than JrPPO2, indicating JrPPO1's greater contribution to PPO activity and fruit browning in walnut fruits. Consequently, JrPPO1 can be effectively regulated both at the molecular level and by manipulating environmental conditions, to achieve the objective of controlling fruit browning.

External browning mechanism in walnut kernel pellicles under different drying conditions based on the combination of widely-targeted and anthocyanin-targeted metabolomics.

There has been limited research on external browning (EB) of walnut. This work discovered 1888 metabolites and 34 anthocyanins in walnut pellicles (WPs) after three drying methods using widely-targeted and anthocyanin-targeted metabolomics. Based on OPLS-DA and correlation analysis, 64 temperature-responsive metabolites (TRMs; 13 anthocyanins and 51 flavonoids) were identified as critical components in relation to EB. Notably, 14 flavonoids exhibited a strong positive correlation (r > 0.9) with the browning index (BI), with upregulation of >60% after browning. Most of the identified anthocyanins were negatively linked with BI because of degradation (>45%), with correlation coefficients ranging from 0.75 to 0.97. Furthermore, anthocyanidin reductase and laccase were the two key enzymes involved in the EB of WPs, with their activities increasing by 10.57-fold and 1.32-fold, respectively, with increasing drying temperature. A metabolic pathway network of the TRM was built to provide insights into the potential mechanisms underlying EB in WPs.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Transcriptomic Analyses Reveal the Effects of Walnut Kernel Cake on Adipose Deposition in Pigs.

With the rising cost of animal feed protein, finding affordable and effective substitutes is crucial. Walnut kernel cake, a polyphenol-, fiber-, protein- and fat-rich byproduct of walnut oil extraction, has been underexplored as a potential protein replacement in pig feed. In this study, we found that feeding large Diqing Tibetan pigs walnut kernel cake promoted adipose deposition and improved pork quality during pig growth. Transcriptome analysis revealed the upregulation of genes ANGPTL8, CCNP, ETV4, and TRIB3, associated with adipose deposition. Pathway analysis highlighted enrichment in adipose deposition-related pathways, including PPAR, insulin, PI3K-Akt, Wnt, and MAPK signaling. Further analysis identified DEGs (differentially expressed genes) positively correlated with adipose-related traits, such as PER2 and PTGES. Single-cell transcriptome data pointed to the specific expression of CD248 and PTGES in adipocyte progenitor/stem cells (APSCs), pivotal for adipocyte differentiation and adipose deposition regulation. This study demonstrates walnut kernel cake's potential to substitute soybean cake in pig feed, providing high-quality protein and promoting adipose deposition. It offers insights into feed protein replacement, human functional food, fat metabolism, and related diseases, with marker genes and pathways supporting pig breeding and pork quality improvement.

Nutritional upgrade of olive mill stone waste, walnut shell and their mixtures by applying solid state fermentation initiated by Pleurotus ostreatus.

Present study concerns the transformation of the agro-industrial by-products olive mill stone waste (OMSW) and walnut shell (WS) to a protein-enriched animal feedstuff utilizing the solid state fermentation (SSF) technique. For this purpose, various mixtures of these by-products were exploited as substrates of the SSF process which was initiated by the P. ostreatus fungus. The respective results indicated that the substrate consisted of 80% WS and 20% OMSW afforded the product with the highest increase in protein content, which accounted the 7.57% of its mass (69.35% increase). In addition, a 26.13% reduction of lignin content was observed, while the most profound effect was observed for their 1,3-1,6 ß-glucans profile, which was increased by 3-folds reaching the 6.94% of substrate's mass. These results are indicative of the OMSW and WS mixtures potential to act as efficient substrate for the development of novel proteinaceous animal feed supplements using the SSF procedure. Study herein contributes to the reintegration of the agro-industrial by-products aiming to confront the problem of proteinaceous animal feed scarcity and reduce in parallel the environmental footprint of the agro-industrial processes within the context of circular economy.

Influence of Cold Stress on Physiological and Phytochemical Characteristics and Secondary Metabolite Accumulation in Microclones of Juglans regia L.

The current study investigated the impact of cold stress on the morphological, physiological, and phytochemical properties of Juglans regia L. (J. regia) using in vitro microclone cultures. The study revealed significant stress-induced changes in the production of secondary antioxidant metabolites. According to gas chromatography-mass spectrometry (GC-MS) analyses, the stress conditions profoundly altered the metabolism of J. regia microclones. Although the overall spectrum of metabolites was reduced, the production of key secondary antioxidant metabolites significantly increased. Notably, there was a sevenfold (7×) increase in juglone concentration. These findings are crucial for advancing walnut metabolomics and enhancing our understanding of plant responses to abiotic stress factors. Additionally, study results aid in identifying the role of individual metabolites in these processes, which is essential for developing strategies to improve plant resilience and tolerance to adverse conditions.

Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

Effect of bi-enzyme hydrolysis on the properties and composition of hydrolysates of Manchurian walnut dreg protein.

Walnut dreg (WD) active peptides are an important source of dietary antioxidants; however, the products of conventional hydrolysis have limited industrial output owing to poor flavour and low bioactivity. To this end, in this study, we aimed to employ bvLAP, an aminopeptidase previously identified in our research, as well as commercially available Alcalase for bi-enzyme digestion. The flavour, antioxidant activity, and structures of products resulting from various digestion methods were compared. The results showed that the bi-enzyme digestion products had enhanced antioxidant activity, increased ß-sheet content, and reduced bitterness intensity from 9.65 to 6.93. Moreover, bi-enzyme hydrolysates showed a more diverse amino acid composition containing 1640 peptides with distinct sequences. These results demonstrate that bi-enzyme hydrolysis could be a potential process for converting WD into functional food ingredients. Additionally, our results provide new concepts that can be applied in waste processing and high-value utilisation of WD.

The regulating mechanism of salt tolerance of black walnut seedlings was revealed by the physiological and biochemical integration analysis.

Salt stress is an important abiotic stress that seriously affects plant growth. In order to research the salt tolerance of walnut rootstocks so as to provide scientific basis for screening salt-tolerant walnut rootstocks, two kinds of black walnut seedlings, Juglans microcarpa L. (JM) and Juglans nigra L. (JN), were treated under salt stress with different concentrations of NaCl (0, 50, 100, and 200 mM) and the growth situation of seedlings were observed. The physiological indexes of JM and JN seedlings were also measured in different days after treatment. Our study showed salt stress inhibited seedlings growth and limited biomass accumulation. Walnut mainly increased osmotic adjustment ability by accumulation Pro and SS. Furthermore, with the duration of treatment time increased, SOD and APX activities decreased, TPC and TFC contents increased. Walnut accumulated Na mostly in roots and transported more K and Ca to aboveground parts. The growth and physiological response performance differed between JM and JN, specifically, the differences occurred in the ability to absorb minerals, regulate osmotic stress, and scavenge ROS. Salt tolerance of JM and JN was assessed by principal component analysis (PCA) and resulted in JN > JM. In conclusion, our results indicated that JN has higher salt tolerance than JM, and JN might be used as a potential germplasm resource for the genetic breeding of walnuts.

Therapeutic effects of a walnut-derived peptide on NLRP3 inflammasome activation, synaptic plasticity, and cognitive dysfunction in T2DM mice.

NLRP3 inflammasome activation plays a key role in the development of diabetes-induced cognitive impairment. However, strategies to inhibit NLRP3 inflammasome activation remain elusive. Herein, we evaluated the impact of a walnut-derived peptide, TWLPLPR (TW-7), on cognitive impairment in high-fat diet/streptozotocin-induced type 2 diabetes mellitus (T2DM) mice and explored its underlying mechanisms in high glucose-induced HT-22 cells. In the Morris water maze test, TW-7 alleviated cognitive deficits in mice; this was confirmed at the level of synaptic structure and dendritic spine density in the mouse hippocampus using transmission electron microscopy and Golgi staining. TW-7 increased the expression of synaptic plasticity-related proteins and suppressed the NEK7/NLRP3 inflammatory pathway, as determined by western blotting and immunofluorescence analysis. The mechanism of action of TW-7 was verified in an HT-22 cell model of high glucose-induced insulin resistance. Collectively, TW-7 could regulate T2DM neuroinflammation and synaptic function-induced cognitive impairment by inhibiting NLRP3 inflammasome activation and improving synaptic plasticity.

Screening and Constructing of Novel Angiotensin I-Converting Enzyme Inhibiting Peptides from Walnut Protein Isolate and Their Mechanisms of Action: A Merged In Silico and In Vitro Study.

Angiotensin I-converting enzyme (ACE)-inhibiting peptides were isolated from walnut protein isolate (WPI) using ultrasound-assisted extraction. This study aimed to assess the impact of ultrasonic pretreatment on the physicochemical properties of WPI. The optimal extraction conditions for WPI were determined as a 15-min ultrasonic treatment at 400 W. Subsequently, the hydrolysate exhibiting the highest in vitro ACE-inhibiting activity underwent further processing and separation steps, including ultrafiltration, ion exchange chromatography, liquid chromatography-tandem mass spectrometry, ADMET screening, and molecular docking. As a result of this comprehensive process, two previously unidentified ACE-inhibiting peptides, namely Tyr-Ile-Gln (YIQ) and Ile-Tyr-Gln (IYQ), were identified. In addition, a novel peptide, Ile-Lys-Gln (IKQ), was synthesized, demonstrating superior ACE-inhibiting activity and temperature stability. In silico analysis estimated an in vivo utilization rate of 21.7% for IKQ. These peptides were observed to inhibit ACE through an anti-competitive mechanism, with molecular docking simulations suggesting an interaction mechanism involving hydrogen bonding. Notably, both IYQ and IKQ peptides exhibited no discernible toxicity to HUVECs cells and promoted nitric oxide (NO) generation. These findings underscore the potential of ultrasonicated WPI in the separation of ACE-inhibiting peptides and their utility in the development of novel ACE inhibitors for functional food applications.

Antioxidant and Anti-Obesity Effects of Juglans mandshurica in 3T3-L1 Cells and High-Fat Diet Obese Rats.

Juglans mandshurica Maxim. walnut (JMW) is well-known for the treatment of dermatosis, cancer, gastritis, diarrhea, and leukorrhea in Korea. However, the molecular mechanism underlying its anti-obesity activity remains unknown. In the current study, we aimed to determine whether JMW can influence adipogenesis in 3T3-L1 preadipocytes and high-fat diet rats and determine the antioxidant activity. The 20% ethanol extract of JMW (JMWE) had a total polyphenol content of 133.33 ± 2.60 mg GAE/g. Considering the antioxidant capacity, the ABTS and DPPH values of 200 µg/ml of JMWE were 95.69 ± 0.94 and 79.38 ± 1.55%, respectively. To assess the anti-obesity activity of JMWE, we analyzed the cell viability, fat accumulation, and adipogenesis-related factors, including CCAAT-enhancer-binding protein alpha (C/EBPα), sterol regulatory element-binding protein-1c (SREBP1c), peroxisome proliferator-activated receptor-gamma (PPARγ), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). We found that total lipid accumulation and triglyceride levels were reduced, and the fat accumulation rate decreased in a dose-dependent manner. Furthermore, JMWE suppressed adipogenesis-related factors C/EBPα, PPARγ, and SREBP1c, as well as FAS and ACC, both related to lipogenesis. Moreover, animal experiments revealed that JMWE could be employed to prevent and treat obesity-related diseases. Hence, JMWE could be developed as a healthy functional food and further explored as an anti-obesity drug.

The Gastroprotective Effect of Walnut Peptides: Mechanisms and Impact on Ethanol-Induced Acute Gastric Mucosal Injury in Mice.

The objective of this research was to explore the protective impact of walnut peptides (WP) against ethanol-induced acute gastric mucosal injury in mice and to investigate the underlying defense mechanisms. Sixty male BALB-c mice were divided into five groups, and they were orally administered distilled water, walnut peptides (200 and 400 mg/kg bw), and omeprazole (20 mg/kg bw) for 24 days. Acute gastric mucosal injury was then induced with 75% ethanol in all groups of mice except the blank control group. Walnut peptides had significant protective and restorative effects on tissue indices of ethanol-induced gastric mucosal damage, with potential gastric anti-ulcer effects. Walnut peptides significantly inhibited the excessive accumulation of alanine aminotransferase (ALT), aspartate transferase (AST), and malondialdehyde (MDA), while promoting the expression of reduced glutathione (GSH), total antioxidant capacity (T-AOC), glutathione disulfide (GSSG), and mouse epidermal growth factor (EGF). Furthermore, the Western blot analysis results revealed that walnut peptides significantly upregulated the expression of HO-1 and NQO1 proteins in the Nrf2 signaling pathway. The defensive impact of walnut peptides on the gastric mucosa may be achieved by mitigating the excessive generation of lipid peroxides and by boosting cellular antioxidant activity.

Overexpression of JsFLS5 in calli improves salinity tolerance by maintaining active oxygen balance and reducing Na+ toxicity in Juglans sigillata.

The escalating global climate change significantly threatens plant growth, development, and production through salinity stress. Flavonoids, a crucial category of secondary metabolites, have been extensively studied for their role in modulating plant growth and development mechanisms in the face of biological and abiotic stress. The flavonol synthetase (FLS) gene plays a key role in the biosynthesis and accumulation of flavonoids. To investigate the correlation between salt tolerance and flavonol synthesis, JsFLS5 was overexpressed in the callus of Juglans sigillata cv. "Qianhe-7." This study shows that the upregulation of JsFLS5 significantly elevates the overall flavonoid content by modulating the expression of genes associated with flavonoid synthesis under salinity stress conditions. Additionally, the overexpressing callus exhibited enhanced resistance to salt stress compared to the wild-type callus, as evidenced by reduced levels of reactive oxygen species accumulation, electrolyte leakage, and malondialdehyde content in the overexpressing callus relative to the wild type (WT). Moreover, the overexpressing callus showed higher antioxidant enzyme activity and a more efficient ascorbic acid-glutathione cycle. Furthermore, the concentration of Na+ in the overexpressing callus was lower than WT, resulting in a decreased Na+ /K+ ratio. These findings suggest that JsFLS5 overexpression in calli effectively mitigates the oxidative damage induced by osmotic stress and reduces Na+ toxicity by enhancing flavonoid synthesis under salt stress conditions. Consequently, this study offers a novel perspective for comprehending the role of JsFLS5 in the response to abiotic stress in J. sigillata.

Systematical Characterization of the AT-Hook Gene Family in Juglans regia L. and the Functional Analysis of the JrAHL2 in Flower Induction and Hypocotyl Elongation.

AT-hook motif nuclear localization (AHL) proteins play essential roles in various plant biological processes. Yet, a comprehensive understanding of AHL transcription factors in walnut (Juglans regia L.) is missing. In this study, 37 AHL gene family members were first identified in the walnut genome. Based on the evolutionary analysis, JrAHL genes were grouped into two clades, and their expansion may occur due to segmental duplication. The stress-responsive nature and driving of developmental activities of JrAHL genes were revealed by cis-acting elements and transcriptomic data, respectively. Tissue-specific expression analysis showed that JrAHLs had a profound transcription in flower and shoot tip, JrAHL2 in particular. Subcellular localization showed that JrAHL2 is anchored to the nucleus. Overexpression of JrAHL2 in Arabidopsis adversely affected hypocotyl elongation and delayed flowering. Our study, for the first time, presented a detailed analysis of JrAHL genes in walnut and provided theoretical knowledge for future genetic breeding programs.

African walnut (Tetracarpidium conophorum) extract upregulates glucocerebrosidase activity and circumvents Parkinsonian changes in the Hippocampus via the activation of heatshock proteins.

BACKGROUND: Neurodegenerative illnesses like Parkinson's and Alzheimer's are largely caused by the accumulation of aggregated proteins. Heat shock proteins (HSPs), which are molecular chaperons, have been linked with the modulation of ß-glucocerebrosidase (GCase) function encoded by GBA1 and Synucleinopathies. Herein, the chaperonic properties of African walnut ethanolic extract (WNE) in manganese-induced Parkinsonian neuropathology in the hippocampus was examined. METHODOLOGY: 48 adult male rats weighing 185 g ± 10 g were randomly assigned into 6 (A - F) groups (n = 8) and treated orally as follows: A-PBS (1 ml daily for 28 days), B-WNE (200 mg/kg daily for 28 days), C- WNE (400 mg/kg daily for 28 days), D-Mn (100 mg/kg daily for 28 days), E-Mn plus WNE (100 mg/kg Mn + 200 mg/kg WNE daily concomitantly for 28 days), F-Mn plus WNE (100 mg/kg Mn + 400 mg/kg WNE daily concomitantly for 28 days). RESULTS: Rats treated with WNE showed increased levels of HSP70 and HSP90 in comparison with the Mn-intoxicated group. GCase activity also increased significantly in animals treated with WNE. Our results further revealed the therapeutic tendencies of WNE against Mn toxicity by modulating oligomeric α-synuclein levels, redox activity, and glucose bioenergetics. Furthermore, immunohistochemical evaluation revealed reduced expression of neurofibrillary tangles, and reactive astrogliosis following WNE treatment. CONCLUSION: The ethanolic extract of African Walnut induced the activation of HSPs and increased the expression of GBA1 gene in the hippocampus. Activated heat shock proteins suppressed neurodegenerative changes due to Manganese toxicity. WNE was also shown to modulate neuroinflammatory, bioenergetics and neural redox balance in Parkinson-like neuropathology. This study was limited to the use of crude walnut extract and the evaluation of non-motor cascades of Parkinson's disease.

Effects of dietary walnut (Juglans regia) leaves extract on immunity, gene expression responses, and disease resistance in Oreochromis niloticus.

The dietary effects of walnut leaf extract (WLE) on the growth, immunity, and resistance of Oreochromis niloticus to bacterial infection have been investigated. Five diets were prepared with various WLE doses of 0, 250, 500, 750, and 1000 mg/kg, termed as Con (control), WLE250, WLE500, WLE750, and WLE1000, respectively. Fish (11.67 ± 0.21 g) were fed these diets for 60 days and then challenged with Plesiomonas shigelloides. Before the challenge, it was observed that dietary WLE did not significantly affect the growth, blood proteins (globulin, albumin, and total protein), and liver function enzymes (ALT and AST) activities. The WLE250 group significantly increased serum SOD and CAT activities more than other groups. The serum immunological indices (lysozyme and myeloperoxidase activities) and hematological parameters (phagocytic activity %, phagocytic index, respiratory burst activity, and potential activity) were significantly increased in the WLE groups compared with the Con group. The expression of IgM heavy chain, IL-1ß, and IL-8 genes were significantly upregulated in all WLE-supplemented groups in comparison with the Con group. The fish survival rates (SR; %) post challenge in the Con, WLE250, WLE500, WLE750 and WLE1000 groups were 40.0%, 49.3%, 86.7%, 73.3%, and 70.7%, respectively. The Kaplan-Meier survivorship curves illustrated that the highest SR% was found in the WLE500 group (86.7%) amongst the other groups. Accordingly, we can suggest that feeding O. niloticus with a diet supplied with WLE at a dose rate of 500 mg/kg over 60 days could enrich haemato-immune responses and increase the fish survival against the challenge with P. shigelloides. These results recommend using WLE as a herbal dietary supplement to substitute antibiotic use in aquafeed.

Lipidomics characterized TAG biosynthesis of developing kernels in three walnut cultivars in Xinjiang region.

Walnut oil with very high proportion of polyunsaturated fatty acids exhibits many health beneficial effects. We hypothesized that the oil composition is led by a special pattern/mechanism for triacylglycerol (TAG) biosynthesis as well as accumulation in walnut kernel during embryo development. To test this hypothesis, shotgun lipidomics was performed for class-targeted lipid analysis (including TAG, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and lysophosphatidylcholine species) in walnut kernels from three cultivar collected at three critical stages of embryo development. The results indicated that TAG synthesis in the kernel happened before 84 days after flowering (DAF) and was significantly enhanced between 84 and 98 DAF. Moreover, TAG profile was changing along with DAFs due to the increased composition of 18:1 FA in TAG pool. Moreover, lipidomics also demonstrated that the enhanced acyl editing was responsible for the flux of FA through phosphatidylcholine for eventual TAG synthesis. Therefore, TAG biosynthesis in walnut kernel was characterized directly from lipid metabolism.