IbNIEL-mediated degradation of IbNAC087 regulates jasmonic acid-dependent salt and drought tolerance in sweet potato.

Sweet potato (Ipomoea batatas [L.] Lam.) is a crucial staple and bioenergy crop. Its abiotic stress tolerance holds significant importance in fully utilizing marginal lands. Transcriptional processes regulate abiotic stress responses, yet the molecular regulatory mechanisms in sweet potato remain unclear. In this study, a NAC (NAM, ATAF1/2, and CUC2) transcription factor, IbNAC087, was identified, which is commonly upregulated in salt- and drought-tolerant germplasms. Overexpression of IbNAC087 increased salt and drought tolerance by increasing jasmonic acid (JA) accumulation and activating reactive oxygen species (ROS) scavenging, whereas silencing this gene resulted in opposite phenotypes. JA-rich IbNAC087-OE (overexpression) plants exhibited more stomatal closure than wild-type (WT) and IbNAC087-Ri plants under NaCl, polyethylene glycol, and methyl jasmonate treatments. IbNAC087 functions as a nuclear transcriptional activator and directly activates the expression of the key JA biosynthesis-related genes lipoxygenase (IbLOX) and allene oxide synthase (IbAOS). Moreover, IbNAC087 physically interacted with a RING-type E3 ubiquitin ligase NAC087-INTERACTING E3 LIGASE (IbNIEL), negatively regulating salt and drought tolerance in sweet potato. IbNIEL ubiquitinated IbNAC087 to promote 26S proteasome degradation, which weakened its activation on IbLOX and IbAOS. The findings provide insights into the mechanism underlying the IbNIEL-IbNAC087 module regulation of JA-dependent salt and drought response in sweet potato and provide candidate genes for improving abiotic stress tolerance in crops.

The tuber-specific StbHLH93 gene regulates proplastid-to-amyloplast development during stolon swelling in potato.

In potato, stolon swelling is a complex and highly regulated process, and much more work is needed to fully understand the underlying mechanisms. We identified a novel tuber-specific basic helix-loop-helix (bHLH) transcription factor, StbHLH93, based on the high-resolution transcriptome of potato tuber development. StbHLH93 is predominantly expressed in the subapical and perimedullary region of the stolon and developing tubers. Knockdown of StbHLH93 significantly decreased tuber number and size, resulting from suppression of stolon swelling. Furthermore, we found that StbHLH93 directly binds to the plastid protein import system gene TIC56 promoter, activates its expression, and is involved in proplastid-to-amyloplast development during the stolon-to-tuber transition. Knockdown of the target TIC56 gene resulted in similarly problematic amyloplast biogenesis and tuberization. Taken together, StbHLH93 functions in the differentiation of proplastids to regulate stolon swelling. This study highlights the critical role of proplastid-to-amyloplast interconversion during potato tuberization.

Genome-Wide Analysis of Family-1 UDP-Glycosyltransferases in Potato (Solanum tuberosum L.): Identification, Phylogenetic Analysis and Determination of Response to Osmotic Stress.

Family-1 UDP-glycosyltransferases (UGTs) are the most common and functional glycosyltransferases in the plant world. UGT is closely related to plant growth and the response to abiotic stress. However, despite systematic research, our understanding of potato UGT genes is still unclear. In this study, we identified 174 potato UGT proteins based on their conserved plant secondary product glycosyltransferase (PSPG) motifs. Phylogenetic analyses were used to compare these proteins with Arabidopsis UGTs and other plant UGTs, and it was found that they could be clustered into 18 distinct groups. Patterns of intron gain/loss and intron phases within potato UGTs revealed highly conserved intron insertion events. The promoter cis-elements of these 174 UGT genes were systematically investigated. The promoter regions of these UGT genes are known to contain various classes of cis-acting compounds. These include elements that are light-responsive, phytohormone-responsive, and stress-responsive. Transcriptome data analysis established that 25, 10, 6, and 4 of these 174 UGT genes were specifically expressed in leaves, roots, stolons, and young tubers, respectively. The mannitol-treated transcriptomic data showed thirty-eight UGT genes were significantly upregulated. The quantitative real-time PCR results showed that the four genes were all responsive to osmotic stress under a 10% PEG6000 treatment. The results of our study provide a basis for clarifying the molecular mechanism of potato osmotic stress resistance and better understanding its function in the future.

Based on Bioinformatics to Explore the Mechanism of "Tangzhiqing" Decoction Alleviating Type 2 Diabetes-associated Cognitive Dysfunction in Mice by Regulating Hippocampal Neuron Apoptosis and Autophagy.

BACKGROUND: Diabetic cognitive dysfunction (DCD) is emerging as a chronic complication of diabetes that is gaining increasing international recognition. The traditional Chinese medicine (TCM) formulation, Tangzhiqing decoction (TZQ), has shown the capacity to modulate the memory function of mice with DCD by ameliorating insulin resistance. Nevertheless, the precise mechanism underlying the effects of TZQ remains elusive. METHODS: The chemical constituents of TZQ were screened using TCMSP databases, and DCDassociated disease targets were retrieved from various databases. Subsequently, core targets were identified through network topology analysis. The core targets underwent analysis using Gene Ontology (GO) functional annotations and enrichment in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Models were established through high-fat and high-glucose diet feeding along with intraperitoneal injection of streptozotocin (STZ). TZQ and metformin were administered at varying doses over 8 weeks. The Morris water maze was employed to evaluate the cognitive capabilities of each rat group, while indicators of oxidative stress and insulin were assessed in mice. Neuronal apoptosis in distinct groups of mice's hippocampi was detected using TdT-mediated dUTP Nick-End Labeling (TUNEL), and western blot (WB) analysis was conducted to assess the expression of apoptosis- and autophagy-related proteins, including Bax, Bcl2, Caspase3, Caspase8, Beclin1, ATG7, LC3, p62, and Lamp2, within the hippocampus. RESULTS: TZQ exhibited the capacity to modulate neuronal autophagy, ameliorate endoplasmic reticulum stress, apoptosis, inflammation, and oxidative stress, as well as to regulate synaptic plasticity and conduction. TZQ mitigated cognitive dysfunction in mice, while also regulating hippocampal inflammation and apoptosis. Additionally, it influenced the protein expression of autophagy-related factors such as Bax, Bcl2, Caspase3, Caspase8, Beclin1, ATG7, and LC3. Notably, this modulation significantly reduced neuronal apoptosis in the hippocampus and curbed excessive autophagy. CONCLUSION: TZQ demonstrated a substantial reduction in neuronal apoptosis within the hippocampus and effectively suppressed excessive autophagy.

SARS-CoV-2 N protein induced acute kidney injury in diabetic db/db mice is associated with a Mincle-dependent M1 macrophage activation.

"Cytokine storm" is common in critically ill COVID-19 patients, however, mechanisms remain largely unknown. Here, we reported that overexpression of SARS-CoV-2 N protein in diabetic db/db mice significantly increased tubular death and the release of HMGB1, one of the damage-associated molecular patterns (DAMPs), to trigger M1 proinflammatory macrophage activation and production of IL-6, TNF-α, and MCP-1 via a Mincle-Syk/NF-κB-dependent mechanism. This was further confirmed in vitro that overexpression of SARS-CoV-2 N protein caused the release of HMGB1 from injured tubular cells under high AGE conditions, which resulted in M1 macrophage activation and production of proinflammatory cytokines via a Mincle-Syk/NF-κB-dependent mechanism. This was further evidenced by specifically silencing macrophage Mincle to block HMGB1-induced M1 macrophage activation and production of IL-6, TNF-α, and MCP-1 in vitro. Importantly, we also uncovered that treatment with quercetin largely improved SARS-CoV-2 N protein-induced AKI in db/db mice. Mechanistically, we found that quercetin treatment significantly inhibited the release of a DAMP molecule HMGB1 and inactivated M1 pro-inflammatory macrophage while promoting reparative M2 macrophage responses by suppressing Mincle-Syk/NF-κB signaling in vivo and in vitro. In conclusion, SARS-CoV-2 N protein-induced AKI in db/db mice is associated with Mincle-dependent M1 macrophage activation. Inhibition of this pathway may be a mechanism through which quercetin inhibits COVID-19-associated AKI.

Decline in Morel Production upon Continuous Cropping Is Related to Changes in Soil Mycobiome.

The black morel Morchella sextelata (Morchellaceae, Pezizales) is a valuable edible mushroom that can be cultivated on a large scale, but a severe yield decline is observed during continuous cropping. The effect of long-term cropping on soil-borne diseases and the dysbiosis of the microbiome and how these biotic factors affect the morel yield are not well understood. To help fill this knowledge gap, we designed an indoor experiment to investigate the influence of black morel cropping regimes on soil physicochemical properties, richness and distribution of fungal community, and morel primordial production. In this study, we employed rDNA metabarcoding and microbial network analysis to evaluate the effect of non-continuous and continuous cropping regimes on the fungal community during three developmental stages of black morel production, namely, the bare soil mycelium, mushroom conidial, and primordial stages. The results showed that during the first year, M. sextelata mycelium overwhelmed the resident soil fungal community by reducing the alpha diversity and niche breadth of soil fungal patterns by a greater amount compared to the continuous cropping regime, leading to high crop yield of 12.39 ± 6.09/quadrat but less complex soil mycobiome. To sustain continuous cropping, exogenous nutrition bags and morel mycelial spawn were consecutively added to the soil. The additional nutrient input stimulated the growth of fungal saprotrophic decomposers. The degrading activity of soil saprotrophs, including M.sextelata, caused a significant increase in soil nutrient content. This led to an inhibitory effect on the formation of morel primordia, resulting in a sharp decline to 0.29 ± 0.25/quadrat and 0.17 ± 0.24/quadrat, respectively, in the final morel cropping. Our findings provided a dynamic overview of the soil fungal community during morel mushroom production, allowing us to identify beneficial and detrimental fungal taxa in the soil mycobiome involved in morel cultivation. The information acquired from this study can be applied to mitigate the adverse impact of continuous cropping on the yield of black morel.

Sweet potato NAC transcription factor NAC43 negatively regulates plant growth by causing leaf curling and reducing photosynthetic efficiency.

Leaves comprise one of the most important organs for plant growth and development. Although there have been some reports on leaf development and the establishment of leaf polarity, their regulatory mechanisms are not very clear. In this study, we isolated a NAC (NAM, ATAF, and CUC) transcription factor (TF), i.e., IbNAC43, from Ipomoea trifida, which is a wild ancestor of sweet potato. This TF was highly expressed in the leaves and encoded a nuclear localization protein. The overexpression of IbNAC43 caused leaf curling and inhibited the growth and development of transgenic sweet potato plants. The chlorophyll content and photosynthetic rate in transgenic sweet potato plants were significantly lower than those in wild-type (WT) plants. Scanning electron microscopy (SEM) and paraffin sections showed that the ratio of cells in the upper and lower epidermis of the transgenic plant leaves was unbalanced; moreover, the abaxial epidermal cells were irregular and uneven in transgenic plants. In addition, the xylem of transgenic plants was more developed than that of WT plants, while their lignin and cellulose contents were significantly higher than those of WT. Quantitative real-time PCR (qRT-PCR) analysis showed that the overexpression of IbNAC43 upregulated the genes involved in leaf polarity development and lignin biosynthesis in transgenic plants. Moreover, it was found that IbNAC43 could directly activate the expression of the leaf adaxial polarity-related genes IbREV and IbAS1 by binding to their promoters. These results indicate that IbNAC43 might play a critical role in plant growth by affecting the establishment of leaf adaxial polarity. This study provides new insights regarding leaf development.

Genome-Wide Characterization of the PIFs Family in Sweet Potato and Functional Identification of IbPIF3.1 under Drought and Fusarium Wilt Stresses.

Phytochrome-interacting factors (PIFs) are essential for plant growth, development, and defense responses. However, research on the PIFs in sweet potato has been insufficient to date. In this study, we identified PIF genes in the cultivated hexaploid sweet potato (Ipomoea batatas) and its two wild relatives, Ipomoea triloba, and Ipomoea trifida. Phylogenetic analysis revealed that IbPIFs could be divided into four groups, showing the closest relationship with tomato and potato. Subsequently, the PIFs protein properties, chromosome location, gene structure, and protein interaction network were systematically analyzed. RNA-Seq and qRT-PCR analyses showed that IbPIFs were mainly expressed in stem, as well as had different gene expression patterns in response to various stresses. Among them, the expression of IbPIF3.1 was strongly induced by salt, drought, H2O2, cold, heat, Fusarium oxysporum f. sp. batatas (Fob), and stem nematodes, indicating that IbPIF3.1 might play an important role in response to abiotic and biotic stresses in sweet potato. Further research revealed that overexpression of IbPIF3.1 significantly enhanced drought and Fusarium wilt tolerance in transgenic tobacco plants. This study provides new insights for understanding PIF-mediated stress responses and lays a foundation for future investigation of sweet potato PIFs.

Yishen-Qingli-Huoxue formula attenuates renal fibrosis by inhibiting indoxyl sulfate via AhR/snai1 signaling.

BACKGROUND: Chronic kidney disease (CKD) is challenging to reverse and its treatment options are limited. Yishen-Qingli-Huoxue Formula (YQHF) is an effective treatment Chinese formula for CKD, as verified by clinical randomized controlled trial. However, the correlative YQHF therapeutic mechanisms are still unknown. PURPOSE: The current study aimed to investigate the potential anti-renal fibrosis effects of YQHF as well as the underlying mechanism. METHODS: After affirming the curative effects of YQHF on adenine-induced CKD rats, Masson staining, immunohistochemistry, and ELISA were used to assess the effects of YQHF on renal fibrosis. Subsequently, metabolomics and transcriptomics analyses were conducted to clarify the potential mechanisms. Furthermore, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), molecular docking analysis and in vitro experiments were used to verify final mechanism of anti-fibrosis. RESULTS: Our results demonstrated that YQHF could improve renal morphology, decrease blood urea nitrogen (BUN), serum creatinine (Scr), and increase body weight gain of model rats. Masson staining, immunohistochemistry of collagen I, fibronectin (FN), α-smooth muscle actin (α-SMA), vimentin and E-cadherin showed that YQHF delayed CKD progression by alleviating renal fibrosis, and the expression of fibrotic factors smoc2 and cdh11 were obviously suppressed by YQHF. Metabolomic and transcriptomic measures discovered that indoxyl sulfate might be a crucial factor inducing renal fibrosis, and the antagonistic effect of YQHF on renal fibrosis may be exerted via AhR/snai1 signaling. Subsequently, western blot and immunohistochemical experiments revealed YQHF indeed inhibited AhR/snai1 signaling in adenine-induced renal fibrosis of CKD rat, which confirmed previous results. In addition, molecular docking and in vitro experiments further supported this conclusion, in which astilbin, the main compound identified YQHF, was certified to exert a significant effect on AhR. CONCLUSION: Our findings showed that YQHF can effectively treat CKD by antagonizing renal fibrosis, the potential mechanisms were relating with the regulation on AhR/snai1 signaling.

A Novel WRKY Transcription Factor from Ipomoea trifida, ItfWRKY70, Confers Drought Tolerance in Sweet Potato.

WRKY transcription factors are one of the important families in plants, and have important roles in plant growth, abiotic stress responses, and defense regulation. In this study, we isolated a WRKY gene, ItfWRKY70, from the wild relative of sweet potato Ipomoea trifida (H.B.K.) G. Don. This gene was highly expressed in leaf tissue and strongly induced by 20% PEG6000 and 100 µM abscisic acid (ABA). Subcellar localization analyses indicated that ItfWRKY70 was localized in the nucleus. Overexpression of ItfWRKY70 significantly increased drought tolerance in transgenic sweet potato plants. The content of ABA and proline, and the activity of SOD and POD were significantly increased, whereas the content of malondialdehyde (MDA) and H2O2 were decreased in transgenic plants under drought stress. Overexpression of ItfWRKY70 up-regulated the genes involved in ABA biosynthesis, stress-response, ROS-scavenging system, and stomatal aperture in transgenic plants under drought stress. Taken together, these results demonstrated that ItfWRKY70 plays a positive role in drought tolerance by accumulating the content of ABA, regulating stomatal aperture and activating the ROS scavenging system in sweet potato.

Cognition in chronic kidney disease patients: Evaluation with the Beijing version of the Montreal Cognitive Assessment.

Patients with chronic kidney disease (CKD) may undergo cognitive impairment. We aimed to explore the cognition of patients with cognitive impairment (CI) and no cognitive impairment (NCI) respectively and the effect of demographics, estimated glomerular filtration rate (eGFR), number of comorbidities (NCD), and hemoglobin on CI in Chinese patients with CKD at stage 3-5 treated by nondialysis by using the Beijing version of the Montreal Cognitive Assessment (MoCA-BJ). A total of 120 patients with CKD were recruited from the Department of Nephrology at the Affiliated Hospital of Nanjing University of Chinese Medicine at in-patient and out-patient follow up. A logistic regression model was performed to assess the effect of these variables on CI of CKD patients. The results indicated that the CI group was mainly in the decline of visuospatial and executive function, abstraction, and memory, compared with the NCI group. In addition, years of education, eGFR and NCD were found as predictors of CI of CKD patients at stage 3-5. Specifically, lower eGFR, less years of education and more comorbidities were risk predictors of CI.

Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis.

BACKGROUND: Curcumin has a potential therapeutic role in ovarian cancer. However, whether curcumin plays anti-cancer role in ovarian cancer by mediating the circular RNA (circRNA)/microRNA (miRNA)/mRNA network is still unclear. METHODS: The expression of circ-PLEKHM3, miR-320a, and suppressor of morphogenesis in genitalia 1 (SMG1) was detected via qRT-PCR. Cell viability, colony-formation ability and apoptosis were analyzed via cell counting kit-8 assay, colony formation analysis, and flow cytometry. Protein expression was measured using western blot. The in vivo experiments were performed using a xenograft model. Target association was evaluated via dual-luciferase reporter analysis and RIP assay. RESULTS: Curcumin suppressed ovarian cancer cell proliferation and promoted apoptosis. Circ-PLEKHM3 was downregulated in ovarian cancer, and its expression could be promoted by curcumin treatment. Circ-PLEKHM3 overexpression exacerbated the effect of curcumin on ovarian cancer cell proliferation and apoptosis, as well as anti-tumor effect. MiR-320a was targeted by circ-PLEKHM3. The inhibition effect of circ-PLEKHM3 overexpression on cell proliferation and the enhancing effect on cell apoptosis could be reversed by miR-320a mimic. SMG1 was targeted by miR-320a, and its knockdown also reversed the regulation of miR-320a inhibitor on the proliferation and apoptosis of ovarian cancer cells. In addition, circ-PLEKHM3 could upregulate SMG1 expression via sponging miR-320a. CONCLUSION: Curcumin restrained proliferation and facilitated apoptosis in ovarian cancer by regulating the circ-PLEKHM3/miR-320a/SMG1 axis.

Kir6.2 is essential to maintain neurite features by modulating PM20D1-reduced mitochondrial ATP generation.

Kir6.2, a pore-forming subunit of the ATP-sensitive potassium (KATP) channels, regulates the functions of metabolically active tissues and acts as an ideal therapeutic target for multiple diseases. Previous studies have been conducted on peripheral kir6.2, but its precise physiological roles in the central nervous system (CNS) have rarely been revealed. In the current study, we evaluated the neurophenotypes and neuroethology of kir6.2 knockout (kir6.2-/-) mice. We demonstrated the beneficial effects of kir6.2 on maintaining the morphology of mesencephalic neurons and controlling the motor coordination of mice. The mechanisms underlying the abnormal neurological features of kir6.2 deficiency were analyzed by RNA sequencing (RNA-seq). Pm20d1, a gene encoding PM20D1 secretase that promotes the generation of endogenous mitochondria uncouplers in vivo, was dramatically upregulated in the midbrain of kir6.2-/- mice. Further investigations verified that PM20D1-induced increase of N-acyl amino acids (N-AAAs) from circulating fatty acids and amino acids promoted mitochondrial impairments and cut down the ATP generation, which mediated the morphological defects of the mesencephalic neurons and thus led to the behavioral impairments of kir6.2 knockout mice. This study is the first evidence to demonstrate the roles of kir6.2 in the morphological maintenance of neurite and motor coordination control of mice, which extends our understanding of kir6.2/KATP channels in regulating the neurophysiological function.

Isolation and Characterization of a Virulent Bacteriophage for Controlling Salmonella Enteritidis Growth in Ready-to-Eat Mixed-Ingredient Salads.

ABSTRACT: Ready-to-eat vegetable salads have gained popularity worldwide. However, the microbial safety of these salads is a health concern, primarily due to Salmonella Enteritidis contamination during the growing, harvesting, processing, and handling of produce. In this study, a bacteriophage-based strategy was developed to control Salmonella Enteritidis growth in mixed-ingredient salads. The lytic Salmonella-specific phage SapYZU01 was isolated from a soil sample from a suburban vegetable field in Yangzhou (People's Republic of China). SapYZU01 has a short latent period, a large burst size, and a lytic effect against 13 Salmonella Enteritidis strains isolated from various sources (human samples, pork, deli foods, chickens, and chicken meat). The SapYZU01 genome did not contain virulence or antibiotic resistance genes. SapYZU01 significantly decreased the viability of Salmonella Enteritidis cells in iceberg lettuce, chicken meat, and mixed-ingredient (lettuce plus chicken) salads at 37 and 25°C. Bacterial levels in the salad decreased significantly (by 4.0 log CFU/g) at 25°C after treatment of contaminated lettuce before salad preparation with SapYZU01 at a multiplicity of infection (MOI) of 100. Bacterial levels were decreased by 3.8 log CFU/g at 25°C in lettuce plus chicken salads treated after the salad preparation with SapYZU01 at an MOI of 100. In contrast, treating cooked chicken meat with SapYZU01 at an MOI of 100 before mixing it with contaminated lettuce decreased the bacterial level of the salad by 1.2 log CFU/g at 25°C. These findings indicate the potential application of SapYZU01 as a natural biocontrol agent against Salmonella Enteritidis in mixed-ingredient salads. However, both the treatment method and the bacteriophage MOI must be considered when using this lytic bacteriophage in mixed-ingredient salads.

A novel sweetpotato RING-H2 type E3 ubiquitin ligase gene IbATL38 enhances salt tolerance in transgenic Arabidopsis.

Arabidopsis Toxicos en Levadura (ATL) proteins compose a subfamily of E3 ubiquitin ligases and play major roles in regulating plant growth, cold, drought, oxidative stresses response and pathogen defense in plants. However, the role in enhancing salt tolerance has not been reported to date. Here, we cloned a novel RING-H2 type E3 ubiquitin ligase gene, named IbATL38, from sweetpotato cultivar Lushu 3. This gene was highly expressed in the leaves of sweetpotato and strongly induced by NaCl and abscisic acid (ABA). This IbATL38 was localized to nucleus and plasm membrane and possessed E3 ubiquitin ligase activity. Overexpression of IbATL38 in Arabidopsis significantly enhanced salt tolerance, along with inducible expression of a series of stress-responsive genes and prominently decrease of H2O2 content. These results suggest that IbATL38 as a novel E3 ubiquitin ligase may play an important role in salt stress response.

Repetitive δ-integration of a cellulase-encoding gene into the chromosome of an industrial Angel Yeast-derived strain by URA3 recycling.

Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167 bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77 g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.

Small molecule-driven NLRP3 inflammation inhibition via interplay between ubiquitination and autophagy: implications for Parkinson disease.

Aging-related, nonresolving inflammation in both the central nervous system (CNS) and periphery predisposes individuals to the development of neurodegenerative disorders (NDDs). Inflammasomes are thought to be especially relevant to immune homeostasis, and their dysregulation contributes to inflammation and NDDs. However, few agents have been clinically shown to reduce NDD incidence by targeting inflammasomes. Our study indicated that NLRP3 (NLR family, pyrin domain containing 3) inflammasome is involved in Parkinson disease (PD) progression in patients and various murine models. In addition, the small molecule kaempferol (Ka) protected mice against LPS- and SNCA-induced neurodegeneration by inhibiting NLRP3 inflammasome activation as evidenced by the fact that Ka reduced cleaved CASP1 expression and disrupted NLRP3-PYCARD-CASP1 complex assembly with concomitant decreased IL1B secretion. Mechanically, Ka promoted macroautophagy/autophagy in microglia, leading to reduced NLRP3 protein expression, which in turn deactivated the NLRP3 inflammasome. Intriguingly, ubiquitination was involved in Ka-induced autophagic NLRP3 degradation. These findings were further confirmed in vivo as knockdown of Atg5 expression or autophagy inhibitor treatment significantly inhibited the Ka-mediated NLRP3 inflammasome inhibition and neurodegeneration amelioration. Thus, we demonstrated that Ka promotes neuroinflammatory inhibition via the cooperation of ubiquitination and autophagy, suggesting that Ka is a promising therapeutic strategy for the treatment of NDDs. Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTB: actin, beta; AIF1/IBA1: allograft inflammatory factor 1; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; CASP1: caspase 1; CNS: central nervous system; CQ: chloroquine; DA neurons: dopaminergic neurons; DAMPS: damage-associated molecular patterns; DAPI: 4',6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFAP: glial fibrillary acidic protein; IP: immunoprecipitation; i.p.: intraperitoneally; Ka: kaempferol; KD: knockdown; KO: knockout; LPS: lipopolysaccharide; IL1B: interleukin 1 beta; IL6: interleukin 6; Ly: lysate; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NC: negative control; NDD: neurodegenerative diseases; NLRP3: NLR family, pyrin domain containing 3; OE: overexpression; PD: Parkinson disease; poly-Ub: poly-ubiquitin; PTM: post-translational modification; PYCARD/ASC: PYD and CARD domain containing; Rapa: rapamycin; RFP: red fluorescent protein; SN: supernatant; SNCA: synuclein alpha; SNpc: substantia nigra pars compacta; SQSTM1: sequestosome 1; TH: tyrosine hydroxylase; TNF/TNF-alpha: tumor necrosis factor; Ub: ubiquitin; WT: wild type.

MicroRNA-212-5p Prevents Dopaminergic Neuron Death by Inhibiting SIRT2 in MPTP-Induced Mouse Model of Parkinson's Disease.

Recently, emerging evidences show that sirtuins (SIRTs) modulate aging progress and affect neurodegenerative diseases. For example, inhibition of SIRT2 has been recognized to exert neuroprotective effects in Parkinson's disease (PD). However, current SIRT2 inhibitors are lack of selective property distinguished from its homolog. In this study, we found that SIRT2 protein level was highly increased in PD model, which was negatively regulated by miR-212-5p. In detail, miR-212-5p transfection reduced SIRT2 expression and inhibited SIRT2 activity. In vivo study, miR-212-5p treatment prevented dopaminergic neuron loss and DAT reduction by targeting SIRT2, which means miR-212-5p shows neuroprotective effect in PD. Mechanismly, we found nuclear acetylated p53 was up-regulation according to p53 is a major deacetylation substrate of SIRT2. Furthermore, decreased cytoplasmic p53 promoted autophagy in PD model, which was showed as autophagosomes, autophagic flux, LC3 B and p62 expression. Meanwhile, we also found miR-212-5p treatment somehow alleviated apoptosis in PD model, which might have some underlying mechanisms. In conclusions, our study provides a direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD. These findings will give us an insight into the development of highly specifically SIRT2 inhibitor of opening up novel therapeutic avenues for PD.

Deletion of Kir6.2/SUR1 potassium channels rescues diminishing of DA neurons via decreasing iron accumulation in PD.

ATP-sensitive potassium (K-ATP) channels express in the central nervous system extensively which coupling cell metabolism and cellular electrical activity. K-ATP channels in mature substantia nigra (SN) dopaminergic (DA) neurons are composed of inwardly rectifying potassium channel (Kir) subunit 6.2 and sulfonylurea receptor 1 (SUR1). Our previous study revealed that regulating K-ATP channel exerts the protective effect on DA neurons in a mouse model of Parkinson's disease (PD). However, the detailed mechanism underlying the role of Kir6.2/K-ATP remains unclear. In the present study, we found the deletion of Kir6.2 dramatically alleviated PD-like motor dysfunction of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model. We further found that Kir6.2 knockout selectively restored the reduction of both DA neuronal number and dopamine transmitter level in the nigrostriatal of MPTP-treated PD mice. To gain some understanding on the molecular basis of this effect, we focused on the regulation of Kir6.2 deletion on iron metabolism which is tightly associated with DA neuron damage. We found that Kir6.2 knockout suppressed the excessive iron accumulation in MPTP-treated mouse midbrain and inhibited the upregulation of ferritin light chain (FTL), which is a main intracellular iron storage protein. We probed further and found out that the deletion of Kir6.2 inhibited the excessive production of FTL via IRP-IRE regulatory system, and thereby protecting SN DA neurons against MPTP challenge. Our findings suggest that Kir6.2 plays a crucial role in the pathogenesis of PD and regulating Kir6.2/K-ATP channel may be a promising strategy for PD treatment.

Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson's Disease.

Epidemiological studies have shown that both lipid metabolism disorder and mitochondrial dysfunction are correlated with the pathogenesis of neurodegenerative diseases (NDDs), including Parkinson's disease (PD). Emerging evidence suggests that deposition of intracellular lipid droplets (LDs) participates in lipotoxicity and precedes neurodegeneration. Perilipin family members were recognized to facilitate LD movement and cellular signaling interactions. However, the direct interaction between Perilipin-regulated LD deposition and mitochondrial dysfunction in dopaminergic (DA) neurons remains obscure. Here, we demonstrate a novel type of lipid dysregulation involved in PD progression as evidenced by upregulated expression of Plin4 (a coating protein and regulator of LDs), and increased intracellular LD deposition that correlated with the loss of TH-ir (Tyrosine hydroxylase-immunoreactive) neurons in the MPTP/p-induced PD model mouse mesencephalon. Further, in vitro experiments showed that inhibition of LD storage by downregulating Plin4 promoted survival of SH-SY5Y cells. Mechanistically, reduced LD storage restored autophagy, leading to alleviation of mitochondrial damage, which in turn promoted cell survival. Moreover, the parkin-poly-Ub-p62 pathway was involved in this Plin4/LD-induced inhibition of mitophagy. These findings were further confirmed in primary cultures of DA-nergic neurons, in which autophagy inhibitor treatment significantly countermanded the ameliorations conferred by Plin4 silencing. Collectively, these experiments demonstrate that a dysfunctional Plin4/LD/mitophagy axis is involved in PD pathology and suggest Plin4-LDs as a potential biomarker as well as therapeutic strategy for PD.