Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

Three-Dimensional-Cultured MSC-Derived Exosome-Hydrogel Hybrid Microneedle Array Patch for Spinal Cord Repair.

Exosomes derived from mesenchymal stem cells (MSCs) have been proven to exhibit great potentials in spinal cord injury (SCI) therapy. However, conventional two-dimensional (2D) culture will inevitably lead to the loss of stemness of MSCs, which substantially limits the therapeutic potency of MSCs exosomes (2D-Exo). Exosomes derived from three-dimensional culture (3D-Exo) possess higher therapeutic efficiency which have wide applications in spinal cord therapy. Typically, conventional exosome therapy that relies on local repeated injection results in secondary injury and low efficiency. It is urgent to develop a more reliable, convenient, and effective exosome delivery method to achieve constant in situ exosomes release. Herein, we proposed a controlled 3D-exohydrogel hybrid microneedle array patch to achieve SCI repair in situ. Our studies suggested that MSCs with 3D-culturing could maintain their stemness, and consequently, 3D-Exo effectively reduced SCI-induced inflammation and glial scarring. Thus, it is a promising therapeutic strategy for the treatment of SCI.

Unique Features of the m6A Methylome and Its Response to Salt Stress in the Roots of Sugar Beet (Beta vulgaris).

Salt is one of the most important environmental factors in crop growth and development. N6-methyladenosine (m6A) is an epigenetic modification that regulates plant-environment interaction at transcriptional and translational levels. Sugar beet is a salt-tolerant sugar-yielding crop, but how m6A modification affects its response to salt stress remains unknown. In this study, m6A-seq was used to explore the role of m6A modification in response to salt stress in sugar beet (Beta vulgaris). Transcriptome-wide m6A methylation profiles and physiological responses to high salinity were investigated in beet roots. After treatment with 300 mM NaCl, the activities of peroxidase and catalase, the root activity, and the contents of Na+, K+, and Ca2+ in the roots were significantly affected by salt stress. Compared with the control plants, 6904 differentially expressed genes (DEGs) and 566 differentially methylated peaks (DMPs) were identified. Association analysis revealed that 243 DEGs contained DMP, and 80% of these DEGs had expression patterns that were negatively correlated with the extent of m6A modification. Further analysis verified that m6A methylation may regulate the expression of some genes by controlling their mRNA stability. Functional analysis revealed that m6A modifications primarily affect the expression of genes involved in energy metabolism, transport, signal transduction, transcription factors, and cell wall organization. This study provides evidence that a post-transcriptional regulatory mechanism mediates gene expression during salt stress by affecting the stability of mRNA in the root.

Rezvilutamide versus bicalutamide in combination with androgen-deprivation therapy in patients with high-volume, metastatic, hormone-sensitive prostate cancer (CHART): a randomised, open-label, phase 3 trial.

BACKGROUND: Rezvilutamide, a novel androgen-receptor inhibitor with low blood-brain barrier penetration, has shown potent antitumour activity against metastatic castration-resistant prostate cancer. In this study, we aimed to evaluate the efficacy and safety of rezvilutamide versus bicalutamide in combination with androgen-deprivation therapy (ADT) for high-volume, metastatic, hormone-sensitive prostate cancer. METHODS: CHART is a randomised, open-label, phase 3 study done at 72 hospitals in China, Poland, Czech Republic, and Bulgaria. Eligible patients were aged 18 years or older, had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and had high-volume metastatic, hormone-sensitive prostate cancer. Previous chemotherapy or other localised treatment for prostate cancer were not allowed. Patients were randomly assigned (1:1) to receive ADT plus either rezvilutamide (240 mg) or bicalutamide (50 mg) orally once daily. Randomisation was done via an interactive response technology system (block size of four) and stratified according to ECOG performance status and presence of visceral metastasis (excluding lymph nodes). Herein, we present the results of the preplanned interim analyses for the two co-primary endpoints of radiographic progression-free survival assessed by a blinded independent review committee and overall survival in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study medication. This study is ongoing, but is closed to recruitment. This trial is registered with ClinicalTrials.gov, NCT03520478. FINDINGS: Between June 28, 2018, and Aug 6, 2020, 792 patients were screened and 654 patients were randomly assigned to receive rezvilutamide plus ADT (n=326) or bicalutamide plus ADT (n=328). At the preplanned interim analysis for radiographic progression-free survival (data cutoff May 16, 2021), the median follow-up duration was 21·2 months (IQR 16·6-25·8). Rezvilutamide significantly improved radiographic progression-free survival compared with bicalutamide (median radiographic progression-free survival not reached [95% CI not reached-not reached] vs 25·1 months [95% CI 15·7-not reached]; hazard ratio [HR] 0·44 [95% CI 0·33-0·58]; p<0·0001). At the preplanned interim analysis for overall survival (data cutoff Feb 28, 2022), the median follow-up duration was 29·3 months (IQR 21·0-33·3). Rezvilutamide significantly improved overall survival compared with bicalutamide (HR 0·58 [95% CI 0·44-0·77]; p=0·0001; median overall survival was not reached [95% CI not reached-not reached] vs not reached [36·2-not reached]). The most common grade 3 or worse adverse events of any cause in the safety population were hypertension (26 [8%] of 323 patients in the rezvilutamide group vs 24 [7%] of 324 patients in the bicalutamide group), hypertriglyceridaemia (24 [7%] vs seven [2%]), increased weight (20 [6%] vs 12 [4%]), anaemia (12 [4%] vs 16 [5%]), and hypokalaemia (11 [3%] vs four [1%]). Serious adverse events were reported in 90 (28%) of 323 patients in the rezvilutamide group and 69 (21%) of 324 patients in the bicalutamide group. No treatment-related deaths occurred in patients in the rezvilutamide group; one treatment-related death of unknown specific cause (<1%) occurred in the bicalutamide group. INTERPRETATION: In the two interim analyses, rezvilutamide plus ADT significantly improved radiographic progression-free survival and overall survival compared with bicalutamide plus ADT in patients with high-volume, metastatic, hormone-sensitive prostate cancer, with a tolerable safety profile. FUNDING: Jiangsu Hengrui Pharmaceuticals.

Graphene Infrared Radiation Management Targeting Photothermal Conversion for Electric-Energy-Free Crude Oil Collection.

Graphene has been widely used as a solar absorber for its broad-band absorption. However, targeting a higher photothermal efficiency, the intrinsic infrared radiation loss of graphene requires to be further reduced. Herein, band structure engineering is performed to modulate graphene infrared radiation. Nitrogen-doped vertical graphene is grown on quartz foam (NVGQF) by the plasma-enhanced chemical vapor deposition method. Under the premise of keeping high solar absorption (250-2500 nm), graphitic nitrogen doping effectively modulates the infrared emissivity (2.5-25 µm) of NVGQF from 0.96 to 0.68, reducing the radiation loss by ∼31%. Based on the excellent photothermal properties of NVGQF, a temperature-gradient-driven crude oil collecting raft is designed, where the crude oil flows along the collecting path driven by the viscosity gradient without any external electric energy input. Compared with a nondoped vertical graphene quartz foam raft, the NVGQF raft with a superior photothermal efficiency shows a significantly enhanced crude oil collecting efficiency by three times. The advances in this work suggest broad radiation-managed application platforms for graphene materials, such as seawater desalination and personal or building thermal management.

Adaptive immune responses and cytokine immune profiles in humans following prime and boost vaccination with the SARS-CoV-2 CoronaVac vaccine.

BACKGROUND: Adaptive immune response has been thought to play a key role in SARS-CoV-2 infection. The role of B cells, CD4+T, and CD8+T cells are different in vaccine-induced immune response, thus it is imperative to explore the functions and kinetics of adaptive immune response. We collected blood samples from unvaccinated and vaccinated individuals. To assess the mechanisms contributing to protective immunity of CoronaVac vaccines, we mapped the kinetics and durability of humoral and cellular immune responses after primary and boost vaccination with CoronaVac vaccine in different timepoints. MATERIALS AND METHODS: We separate PBMC and plasma from blood samples. The differentiation and function of RBD-spcific CD4+T and CD8+T cells were analyzed by flow cytometry and ELISA. Antibodies response was analyzed by ELISA. ELISPOT analysis was perfomed to detected the RBD-spcific memory B cells. CBA analysis was performed to detected the cytokine immune profiles. Graphpad prism 8 and Origin 2021 were used for statistical analysis. RESULTS: Vaccine-induced CD4+T cell responses to RBD were more prominent than CD8+T cell responses, and characterized by a predominant Th1 and weak Th17 helper response. CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination. Robust antigen-specific memory B cells were detected (p < 0.0001) following booster vaccination and maintained at 6 months (p < 0.0001) following primary vaccination. Vaccine-induced CD4+T cells correlated with CD8+T cells (r = 0.7147, 0.3258, p < 0.0001, p = 0.04), memory B cell responses (r = 0.7083, p < 0.0001), and IgG and IgA (r = 0.6168, 0.5519, p = 0.0006, 0.003) after vaccination. In addition, vaccine induced a broader and complex cytokine pattern in plasma at early stage. CONCLUSION: Taken together, these results highlight the potential role of B cell and T cell responses in vaccine-induced long-term immunity.

Seed priming with protein hydrolysate promotes seed germination via reserve mobilization, osmolyte accumulation and antioxidant systems under PEG-induced drought stress.

KEY MESSAGE: Seed priming with pig blood protein hydrolysate improves tomato seed germination and seedling growth via regulation of reserve mobilization, osmotic adjustment, and antioxidant mechanism under drought conditions. Protein hydrolysates obtained from agro-industrial byproducts are widely recognized because of their positive roles in regulating plant responses to environmental stresses. However, little is known regarding the roles of animal protein hydrolysates in mediating seed drought tolerance and its underlying mechanisms. This study investigated the potential effects of seed priming on tomato seed germination and seedling growth under PEG-induced drought stress using protein hydrolysates derived from pig blood (PP). PP priming effectively alleviated the drought-induced reduction in seed germination traits, resulting in improved tomato seedling growth. PP priming enhanced the gene expressions and activities of amylase and sucrose synthase and soluble sugar, soluble protein, and free amino acid levels, thereby promoting reserve mobilization in seeds. PP priming also reduced osmotic toxicity through increased accumulations of proline, soluble protein, and soluble sugar. Drought stress substantially enhanced reactive oxygen species production and the subsequent increases in malondialdehyde levels and Evans blue solution uptake, which were substantially alleviated after PP priming via the improved activities of enzymatic and non-enzymatic antioxidants. Moreover, the increased DPPH free radical scavenging capacity and ferric reducing antioxidant power indicated that PP-treated tomato seedings had high antioxidant activities under drought stress. Therefore, PP priming is a novel, promising, and practicable method for improving tomato seed germination and seedling growth under drought stress.

Transcriptome and Metabolome Analyses Revealed the Response Mechanism of Sugar Beet to Salt Stress of Different Durations.

Salinity is one of the most serious threats to agriculture worldwide. Sugar beet is an important sugar-yielding crop and has a certain tolerance to salt; however, the genome-wide dynamic response to salt stress remains largely unknown in sugar beet. In the present study, physiological and transcriptome analyses of sugar beet leaves and roots were compared under salt stress at five time points. The results showed that different salt stresses influenced phenotypic characteristics, leaf relative water content and root activity in sugar beet. The contents of chlorophyll, malondialdehyde (MDA), the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were also affected by different salt stresses. Compared with control plants, there were 7391 and 8729 differentially expressed genes (DEGs) in leaves and roots under salt stress, respectively. A total of 41 hub genes related to salt stress were identified by weighted gene co-expression network analysis (WGCNA) from DEGs, and a transcriptional regulatory network based on these genes was constructed. The expression pattern of hub genes under salt stress was confirmed by qRT-PCR. In addition, the metabolite of sugar beet was compared under salt stress for 24 h. A total of 157 and 157 differentially accumulated metabolites (DAMs) were identified in leaves and roots, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that DEGs and DAMs act on the starch and sucrose metabolism, alpha-linolenic acid metabolism, phenylpropanoid biosynthesis and plant hormone signal transduction pathway. In this study, RNA-seq, WGCNA analysis and untargeted metabolomics were combined to investigate the transcriptional and metabolic changes of sugar beet during salt stress. The results provided new insights into the molecular mechanism of sugar beet response to salt stress, and also provided candidate genes for sugar beet improvement.

A screened PirB antagonist peptide antagonizes Aß42-mediated inhibition of neurite outgrowth in vitro.

Alzheimer's disease (AD) is a type of progressive neurodegenerative disease, and amyloid ß-protein 42 (Aß42) serves an important role in the pathological process of development of AD. Paired immunoglobulin-like receptor B (PirB) is a functional receptor for myelin inhibitors of neuron regeneration in the CNS, and it has also been identified to function as a high-affinity receptor for Aß. Here, we used a phage display to identify a specific PirB antagonist peptide 11(PAP11, PFRLQLS), which could reverse Aß42-induced neurotoxicity and promote neurite outgrowth in vitro. Immunofluorescence analysis showed that PAP11 colocalized with PirB on the membrane of cortical neurons. Horseradish peroxidase-streptavidin-biotin assay further proved that PAP11 directly binds to PirB and the dissociation constant (Kd) was 0.128µM. PAP11 functionally antagonized the neurite outgrowth inhibitory effect induced by Aß42 in cortical neurons, and the underlying mechanism was associated with a PirB-ROCK2/CRMP2 signaling pathway. The novel PirB antagonist peptide PAP11 may be a promising candidate therapeutic agent for the treatment of AD and other neurodegenerative diseases. KEY POINTS: • PAP11 was the first PirB antagonist peptide screened by phage display technology. • PAP11 could protect neurons by blocking the binding of Aß42 and PirB. • PAP11 reverse inhibitory effect of neurite outgrowth through ROCK2/CRMP2 pathway.

Comparison of Posterior Fixation Strategies for Thoracolumbar Burst Fracture: A Finite Element Study.

The management of thoracolumbar (TL) burst fractures remained challenging. Due to the complex nature of the fractured vertebrae and the lack of clinical and biomechanical evidence, currently, there was still no guideline to select the optimal posterior fixation strategy for TL burst fracture. We utilized a T10-L3 TL finite element model to simulate L1 burst fracture and four surgical constructs with one- or two-level suprajacent and infrajacent instrumentation (U1L1, U1L2, U2L1, and U2L2). This study was aimed to compare the biomechanical properties and find an optimal fixation strategy for TL burst fracture in order to minimize motion in the fractured level without exerting significant burden in the construct. Our result showed that two-level infrajacent fixation (U1L2 and U2L2) resulted in greater global motion reduction ranging from 66.0 to 87.3% compared to 32.0 to 47.3% in one-level infrajacent fixation (U1L1 and U2L1). Flexion produced the largest pathological motion in the fractured level but the differences between the constructs were small, all within 0.26 deg. Comparisons in implant stress showed that U2L1 and U2L2 had an average 25.3 and 24.8% less von Mises stress in the pedicle screws compared to U1L1 and U1L2, respectively. The construct of U2L1 had better preservation of the physiological spinal motion while providing sufficient range of motion reduction at the fractured level. We suggested that U2L1 is a good alternative to the standard long-segment fixation with better preservation of physiological motion and without an increased risk of implant failure.

Exploring dynamics and associations of dominant lignocellulose degraders in tomato stalk composting.

The plant residues of tomato bring pressures to the environment and composting provides a feasible method to treat such agricultural waste. However, little is known about the succession and associations of the dominant lignocellulose degraders in the compost system. To further accelerate the process by inoculating key functional microorganisms, a compost pile composed of tomato stalk with maize straw addition was constructed, and the whole community structure and functions of the dominant were investigated by applying the integrated mata-omics. Results showed that Actinobacteria, Firmicutes, and Ascomycota dominated and drove the assembly of the co-occurrence network. In the thermophilic stage, Thermobifida was the exclusive degrader of cellulose, and Thermobifida fusca was the most important cellulolytic actinomycete. Saccharomonospora viridis, Planifilum fulgidum, Thermobacillus sp. and the dominant ascomycota of Aspergillus sclerotialis participated in hemicellulose decomposing. In the cooling phase, functional microorganisms became more diverse, with Nocardiopsis flavescens, Glycomyces artemisiae, Glycomyces sambucus, Streptomyces rubrolavendulae and Streptomyces vietnamensis joining the cellulose-degrading rank, and Chaetomium thermophilum emerging as the main hemicellulose degrader. More than two thirds of the bacteria-bacteria interactions and all the fungi-fungi associations were positive, while, both competition (for the same substrate of hemicellulose) and synergy (preference for cellulose and hemicellulose) coexisted in the bacteria-fungi interactions. In conclusion, these findings provide useful information for understanding the biodegradation of tomato plant residues better, and effects of the functional agents identified on composting process should be further studied.

Integrative genomic analyses identify WDR12 as a novel oncogene involved in glioblastoma.

Glioblastoma (GBM) is the most malignant primary brain tumor in adults. Due to its invasive nature, it cannot be thoroughly eliminated. WD repeat domain 12 (WDR12) processes the 32S precursor rRNA but cannot affect the synthesis of the 45S/47S primary transcript. In this study, we found that WDR12 is highly expressed in GBM according to the analysis results of mRNA expression by The Cancer Genome Atlas database. The high expression level of WDR12 is dramatically related to shorter overall survival and reduced disease-free survival. Next, we knocked down WDR12 and found that knockdown of WDR12 promoted the apoptosis and inhibited the proliferation by cell biology experiments. Differential expression genes in gene-chip revealed that WDR12 knockdown mainly inhibited cell cycle. Finally, we also found that WDR12 is associated with PLK1 and EZH2 in cell proliferation of GBM. Resumptively, this report showed a possible evidence that WDR12 drove malignant behavior of GBM, whose expression may present a neoteric independent prognostic biomarker in GBM.

iTRAQ protein profile analysis of sugar beet under salt stress: different coping mechanisms in leaves and roots.

BACKGROUND: Salinity is one of the most serious threats to world agriculture. An important sugar-yielding crop sugar beet, which shows some tolerance to salt via a mechanism that is poorly understood. Proteomics data can provide important clues that can contribute to finally understand this mechanism. RESULTS: Differentially abundant proteins (DAPs) in sugar beet under salt stress treatment were identified in leaves (70 DAPs) and roots (76 DAPs). Functions of these DAPs were predicted, and included metabolism and cellular, environmental information and genetic information processing. We hypothesize that these processes work in concert to maintain cellular homeostasis. Some DAPs are closely related to salt resistance, such as choline monooxygenase, betaine aldehyde dehydrogenase, glutathione S-transferase (GST) and F-type H+-transporting ATPase. The expression pattern of ten DAPs encoding genes was consistent with the iTRAQ data. CONCLUSIONS: During sugar beet adaptation to salt stress, leaves and roots cope using distinct mechanisms of molecular metabolism regulation. This study provides significant insights into the molecular mechanism underlying the response of higher plants to salt stress, and identified some candidate proteins involved in salt stress countermeasures.

Long Non-Coding RNA (lncRNA) BMP/OP-Responsive Gene (BORG) Promotes Development of Chemoresistance of Colorectal Cancer Cells to Carboplatin.

BACKGROUND The function of long non-coding RNA (lncRNA) BMP/OP-responsive gene (BORG) has only been studied in breast cancer. We analyzed the role of BORG in colorectal cancer (CRC). MATERIAL AND METHODS BORG in CRC tissues and non-cancer tissues from 66 CRC patients was detected by performing quantitative reverse-transcription PCR (RT-qPCR). BORG in plasma of CRC patients was detected at 3 times-points: before treatment and at 3 and 6 months after treatment. p53 expression in tumor tissues was also detected by RT-qPCR. QPCR was performed to confirm the overexpression of p53 in cells of both CRC cell lines. RESULTS We found that BORG expression was upregulated in CRC tissues and was inversely correlated with p53. With application of carboplatin-based treatment, the expression level of BORG was further upregulated. In CRC cells, carboplatin upregulated the expression of BORG and BORG negatively regulated p53. Under carboplatin treatment, BORG positively regulated the viability of CRC cells. In addition, p53 overexpression attenuated the effects of BORG overexpression. CONCLUSIONS BORG promotes the development of chemoresistance of CRC cells to carboplatin.

Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin.

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The Km value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

Whole-Transcriptome RNA Sequencing Reveals the Global Molecular Responses and CeRNA Regulatory Network of mRNAs, lncRNAs, miRNAs and circRNAs in Response to Salt Stress in Sugar Beet (Beta vulgaris).

Sugar beet is an important sugar-yielding crop with some tolerance to salt, but the mechanistic basis of this tolerance is not known. In the present study, we have used whole-transcriptome RNA-seq and degradome sequencing in response to salt stress to uncover differentially expressed (DE) mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in both leaves and roots. A competitive endogenous RNA (ceRNA) network was constructed with the predicted DE pairs, which revealed regulatory roles under salt stress. A functional analysis suggests that ceRNAs are implicated in copper redistribution, plasma membrane permeability, glycometabolism and energy metabolism, NAC transcription factor and the phosphoinositol signaling system. Overall, we conducted for the first time a full transcriptomic analysis of sugar beet under salt stress that involves a potential ceRNA network, thus providing a basis to study the potential functions of lncRNAs/circRNAs.

Role of sucrose in modulating the low-nitrogen-induced accumulation of phenolic compounds in lettuce (Lactuca sativa L.).

BACKGROUND: Phenolic compounds are phytochemicals present in vegetables which contribute to human health. Although nitrogen deficiency and sucrose (Suc) are linked to phenolic production in vegetables, the relationship between them in the regulation of phenolic biosynthesis remains unknown. This study investigated the potential role of Suc in regulating phenolic biosynthesis of lettuce under low-nitrogen (LN) conditions. RESULTS: Our results showed that LN treatment significantly increased Suc content in lettuce by inducing rapid increases in activities of sucrose synthesis-related enzymes. Exogenous Suc further stimulated LN-induced phenolic accumulation in lettuce by upregulating the expression of genes (PAL, CHS, F3H, DFR, F35H and UFGT) involved in phenolic biosynthesis. The opposite effects were true for exogenous 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) application. No changes were observed in chlorophyll content in LN-treated lettuce, in either the presence or absence of Suc application. Notably, exogenous DCMU resulted in decreases of maximum quantum efficiency of photosystem II (PSII) photochemistry, actual efficiency of PSII and electron transport rate in PSII and increase of quantum yield of non-regulated energy dissipation in PSII in lettuce under LN conditions, whereas these effects were reversed on Suc application. Exogenous Suc also increased glutamine synthetase and glutamate synthase activities in LN-treated lettuce. CONCLUSIONS: These results suggest that Suc is involved in LN-induced phenolic production in lettuce by enhancing photosynthetic and nitrogen assimilation efficiency to increase the supply of carbon resources and precursors for phenolic biosynthesis. © 2020 Society of Chemical Industry.

Tissue-Specific Monoallelic Expression of Bovine AXL is Associated with DNA Methylation of Promoter DMR.

The AXL protein is a receptor tyrosine kinase and is often implicated in proliferation, migration and therapy resistance in various cancers. The AXL gene in humans is maternally expressed and paternally imprinted with differentially methylated regions (DMR) surrounding the promoter region. However, the imprinting status and epigenetic regulation of AXL gene in cattle remain unclear. Therefore, we explored the molecular structure along with the patterns of allelic expression and DNA methylation of the bovine AXL gene. First, the complete cDNA sequence of bovine AXL was gathered by Sanger method, from transcripts obtained from RT-PCR, 5' and 3' -RACE. In silico BLAST alignments showed that the longest mRNA sequence of bovine AXL consists of 19 exons and encodes a protein of 887 amino acids. We further analyzed the allelic expression of bovine AXL by employing single-nucleotide polymorphism (SNP)-based sequencing method. A SNP site (GenBank Accession no: rs210020651) found in exon 7 allowed us to distinguish the two parental alleles. Monoallelic expression of AXL was observed in four adult bovine tissues (heart, liver, spleen and fat), while biallelic expression was found in the other adult tissues such as the lung, kidney, muscle, brain and placenta. To determine whether the DNA methylation played a role in the tissue-specific imprinting of bovine AXL, we performed bisulfite sequencing of two regions: region 1 was a CpG island (CGI) in AXL promoter, mapping to 643 bp upstream of the transcription start site of AXL 5'-v1 transcripts, while region two was homologous to the region of human AXL DMR, with 10 CpG sites overlapping the first translation start site (TSS1) of bovine AXL. In region 2, DNA from both monoallelic and biallelic expressed tissues were mostly found to be completely unmethylated. However, tissue-specific differential methylation patterns were found in monoallelic expressed tissues such as the heart and liver while hypomethylation was noted in the promoter CpG island in biallelic expressed tissues such as the lung. These observations demonstrated that the tissue-specific monoallelic expression of bovine AXL is dependent on the DNA methylation of its promoter region.

Sumoylation Negatively Regulates CSR1-Dependent Prostate Cancer Cell Death.

BACKGROUND/AIMS: SUMOylation is a dynamic process and reversed by the activity of SUMO-specific proteases (SENPs) family. SENP1, a member of this family, is highly expressed and plays oncogenic roles in diverse cancers including prostate cancer. However, the SENP1-transgenic mice exhibit aberrant transformation of the mouse prostate gland but do not develop cancer. Cellular Stress Response 1 (CSR1) is a tumor suppressor gene and frequently deleted in prostate cancers. Overexpression of CSR1 in prostate cancer cells inhibits colony formation, anchorage-independent growth and induces cell death. METHODS: The relationship between CSR1 and SENP1 were determined by immunoprecipitation-based proteomics screen and verified by GST-pull down assay. In vivo SUMOylation assay was used to detect the direct effect of SENP1 in the regulation of CSR1. Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing was used to generate Senp1-/- and CSR1-/- PC3 cells. FACS assay was used to determine the apoptosis ratio of cells after transfection. RESULTS: CSR1 is SUMOylated at K582 and rapid ubiquitinated and degradated in prostate cancer cells. SENP1 interacts with and deSUMOylates CSR1 to prevent its degradation and enhances CSR1-dependent prostate cancer cell death. CONCLUSION: Thus, our data indicates that CSR1 is a critical SUMOylated substrate of SENP1 that might partially explain the controversial roles of SENP1 in prostate cancer development.

Polymorphic Imprinting of SLC38A4 Gene in Bovine Placenta.

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (< 3%) of the DNA was observed in all the three detected placentas and their corresponding paternal sperms. The absence of DMR in bovine SLC38A4 promoter region implied that DNA methylation of these three CGIs does not directly or indirectly affect the polymorphic imprinting of SLC38A4 in bovine placenta. This suggested other epigenetic features other than DNA methylation are needed in regulating the imprinting of bovine SLC38A4, which is different from that of mouse with respect to a DMR existence at the mouse's slc38a4 promoter region. Although further work is needed, this first characterization of polymorphic imprinting status of SLC38A4 in cattle placenta provides valuable information on investigating the genomic imprinting phenomenon itself.