Intron Retention of DDX39A Driven by SNRPD2 is a Crucial Splicing Axis for Oncogenic MYC/Spliceosome Program in Hepatocellular Carcinoma.

RNA splicing is a dynamic molecular process in response to environmental stimuli and is strictly regulated by the spliceosome. Sm proteins, constituents of the spliceosome, are key components that mediate splicing reactions; however, their potential role in hepatocellular carcinoma (HCC) is poorly understood. In the study, SNRPD2 (PD2) is found to be the most highly upregulated Sm protein in HCC and to act as an oncogene. PD2 modulates DDX39A intron retention together with HNRNPL to sustain the DDX39A short variant (39A_S) expression. Mechanistically, 39A_S can mediate MYC mRNA nuclear export to maintain high MYC protein expression, while MYC in turn potentiates PD2 transcription. Importantly, digitoxin can directly interact with PD2 and has a notable cancer-suppressive effect on HCC. The study reveals a novel mechanism by which DDX39A senses oncogenic MYC signaling and undergoes splicing via PD2 to form a positive feedback loop in HCC, which can be targeted by digitoxin.

TRIM71 mutations cause a neurodevelopmental syndrome featuring ventriculomegaly and hydrocephalus.

Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH.

The roles and mechanisms of SREBP1 in cancer development and drug response.

Cancer occurrence and development are closely related to increased lipid production and glucose consumption. Lipids are the basic component of the cell membrane and play a significant role in cancer cell processes such as cell-to-cell recognition, signal transduction, and energy supply, which are vital for cancer cell rapid proliferation, invasion, and metastasis. Sterol regulatory element-binding transcription factor 1 (SREBP1) is a key transcription factor regulating the expression of genes related to cholesterol biosynthesis, lipid homeostasis, and fatty acid synthesis. In addition, SREBP1 and its upstream or downstream target genes are implicated in various metabolic diseases, particularly cancer. However, no review of SREBP1 in cancer biology has yet been published. Herein, we summarized the roles and mechanisms of SREBP1 biological processes in cancer cells, including SREBP1 modification, lipid metabolism and reprogramming, glucose and mitochondrial metabolism, immunity, and tumor microenvironment, epithelial-mesenchymal transition, cell cycle, apoptosis, and ferroptosis. Additionally, we discussed the potential role of SREBP1 in cancer prognosis, drug response such as drug sensitivity to chemotherapy and radiotherapy, and the potential drugs targeting SREBP1 and its corresponding pathway, elucidating the potential clinical application based on SREBP1 and its corresponding signal pathway.

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin.

BACKGROUND: Isoquercitrin (quercetin-3-O-ß-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards. RESULTS: Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three ß-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor. CONCLUSION: The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.

E3 ubiquitin ligase RNF148 functions as an oncogene in colorectal cancer by ubiquitination-mediated degradation of CHAC2.

We previously reported that RNF148 was involved in the ubiquitination-mediated degradation of CHAC2. However, its molecular mechanism was not determined. In this study, we investigated the role and mechanism of RNF148 in the progression of colorectal cancer (CRC), especially in the process of ubiquitination-mediated degradation of CHAC2. Our results revealed that RNF148 was upregulated in most CRC tissues, and its expression significantly correlated with the 3-year overall survival rate and most clinicopathological parameters of CRC patients. Furthermore, RNF148 served as an independent prognostic biomarker of CRC and promoted CRC cell proliferation and migration while inhibiting cell apoptosis and sensitivity to 5-FU. Mechanistically, RNF148 used its protease-associated domain to bind to the CHAC domain of CHAC2 and target it for degradation. In addition, we identified two phosphorylation and three ubiquitination residues of CHAC2 and identified Y118 and K102 as the critical phosphorylation and ubiquitination residues, respectively. We also identified CHAC2's and RNF148's interacting proteins and discovered their potential interaction network. In conclusion, our current study unveiled the role of RNF148 in CRC and the mechanism of RNF148 in the ubiquitination-mediated degradation of CHAC2, which shed light on providing potential prognostic biomarkers and molecular targets for CRC patients.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

Collectin 11 has a pivotal role in host defense against kidney and bladder infection in mice.

The urinary tract is constantly exposed to microorganisms. Host defense mechanisms in protection from microbial colonization and development of urinary tract infections require better understanding to control kidney infection. Here we report that the lectin collectin 11 (CL-11), particularly kidney produced, has a pivotal role in host defense against uropathogen infection. CL-11 was found in mouse urine under normal and pathological conditions. Mice with global gene ablation of Colec11 had increased susceptibility to and severity of kidney and to an extent, bladder infection. Mice with kidney-specific Colec11 ablation exhibited a similar disease phenotype to that observed in global Colec11 deficient mice, indicating the importance of kidney produced CL-11 for protection against kidney and bladder infection. Conversely, intravesical or systemic administration of recombinant CL-11 reduced susceptibility to and severity of kidney and bladder infection. Mechanism analysis revealed that CL-11 can mediate several key innate defense mechanisms (agglutination, anti- adhesion, opsonophagocytosis), and limit local inflammatory responses to pathogens. Furthermore, CL-11-mediated innate defense mechanisms can act on clinically relevant microorganisms including multiple antibiotic resistant strains. CL-11 was detectable in eight of 24 urine samples from patients with urinary tract infections but not detectable in urine samples from ten healthy individuals. Thus, our findings demonstrate that CL-11 is a key factor of host defense mechanisms in kidney and bladder infection with therapeutic potential for human application.

Establishment and characterization of an immortalized bovine intestinal epithelial cell line.

Primary bovine intestinal epithelial cells (PBIECs) are an important model for studying the molecular and pathogenic mechanisms of diseases affecting the bovine intestine. It is difficult to obtain and grow PBIECs stably, and their short lifespan greatly limits their application. Therefore, the purpose of this study was to create a cell line for exploring the mechanisms of pathogen infection in bovine intestinal epithelial cells in vitro. We isolated and cultured PBIECs and established an immortalized BIEC line by transfecting PBIECs with the pCI-neo-hTERT (human telomerase reverse transcriptase) recombinant plasmid. The immortalized cell line (BIECs-21) retained structure and function similar to that of the PBIECs. The marker proteins characteristic of epithelial cells, cytokeratin 18, occludin, zonula occludens protein 1 (ZO-1), E-cadherin and enterokinase, were all positive in the immortalized cell line, and the cell structure, growth rate, karyotype, serum dependence and contact inhibition were normal. The hTERT gene was successfully transferred into BIECs-21 where it remained stable and was highly expressed. The transport of short-chain fatty acids and glucose uptake by the BIECs-21 was consistent with PBIECs, and we showed that they could be infected with the intestinal parasite, Neospora caninum. The immortalized BIECs-21, which have exceeded 80 passages, were structurally and functionally similar to the primary BIECs and thus provide a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro.

In dairy cattle, the intestine is essential for productivity as it contributes nearly 10% of the total metabolizable energy. The intestinal epithelium is at risk of infection from constant exposure to pathogenic microorganisms, which seriously endangers an animal's health, but no bovine intestinal epithelial cell line has been developed so far for research on intestine -related diseases. Thus, the goal of this study was to create an immortalized cell line from isolated primary bovine intestinal epithelial cells. The expression of an exogenous human telomerase reverse transcriptase (hTERT) gene can circumvent the Hayflick limit by maintaining telomere integrity and we used transfection with a plasmid expressing the hTERT gene to convert primary intestinal epithelial cells into an immortalized cell line, which we then characterized. The results showed that the immortalized cell line (BIECs-21) was structurally and functionally similar to the primary bovine intestinal epithelial cells (BIECs) and thus provided a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro.

The roles and mechanism of m6A RNA methylation regulators in cancer immunity.

N6-methyladenosine (m6A), the most common internal modification in RNA, can be regulated by three types of regulators, including methyltransferases (writers), demethylases (erasers), and m6A binding proteins (readers). Recently, immunotherapy represented by immune checkpoint blocking has increasingly become an effective cancer treatment, and increasing shreds of evidence show that m6A RNA methylation affects cancer immunity in various cancers. Until now, there have been few reviews about the role and mechanism of m6A modification in cancer immunity. Here, we first summarized the regulation of m6A regulators on the expression of target messenger RNAs (mRNA) and their corresponding roles in inflammation, immunity response, immune process and immunotherapy in various cancer cells. Meanwhile, we described the roles and mechanisms of m6A RNA modification in tumor microenvironment and immune response by affecting the stability of non-coding RNA (ncRNA). Moreover, we also discussed the m6A regulators or its target RNAs which might be used as predictor of cancer diagnosis and prognosis, and shed light on the potentiality of m6A methylation regulators as therapeutic targets in cancer immunity.

Multiomic analyses implicate a neurodevelopmental program in the pathogenesis of cerebral arachnoid cysts.

Cerebral arachnoid cysts (ACs) are one of the most common and poorly understood types of developmental brain lesion. To begin to elucidate AC pathogenesis, we performed an integrated analysis of 617 patient-parent (trio) exomes, 152,898 human brain and mouse meningeal single-cell RNA sequencing transcriptomes and natural language processing data of patient medical records. We found that damaging de novo variants (DNVs) were highly enriched in patients with ACs compared with healthy individuals (P = 1.57 × 10-33). Seven genes harbored an exome-wide significant DNV burden. AC-associated genes were enriched for chromatin modifiers and converged in midgestational transcription networks essential for neural and meningeal development. Unsupervised clustering of patient phenotypes identified four AC subtypes and clinical severity correlated with the presence of a damaging DNV. These data provide insights into the coordinated regulation of brain and meningeal development and implicate epigenomic dysregulation due to DNVs in AC pathogenesis. Our results provide a preliminary indication that, in the appropriate clinical context, ACs may be considered radiographic harbingers of neurodevelopmental pathology warranting genetic testing and neurobehavioral follow-up. These data highlight the utility of a systems-level, multiomics approach to elucidate sporadic structural brain disease.

Unveiling pathogenic mutations in BRCA1 and BRCA2 genes across head and neck squamous cell carcinoma patients via next generation sequencing.

Head and Neck Squamous Cell Carcinoma (HNSC) presents a formidable challenge in the field of oncology due to its aggressive nature and the limited therapeutic options available. In this study, our primary focus was on the Pakistani HNSC patient population, aiming to investigate germline oncogenic mutations within the BRCA1 and BRCA2 genes via Next Generation Sequencing (NGS) and explore their clinical implications. We sought to understand the functional consequences of these mutations via RT-qPCR and Immunohistochemistry (IHC) techniques. The key discovery of our research lies in the identification of three pathogenic mutations, including two within BRCA1 (p.Cys274Ter and p.Glu272Ter) and one within BRCA2 (p.Met1Val), among Pakistani HNSC patients. These mutations previously associated with an increased risk of various cancers. What sets our study apart is the uniqueness of these pathogenic mutations, absent in HNSC patients from other populations. This suggests a distinct genetic profile in Pakistani HNSC patients, possibly contributing to their susceptibility to this malignancy. Furthermore, our research revealed elevated expression levels of BRCA1 and BRCA2 genes in HNSC samples harboring pathogenic mutations, offering insights into mechanisms driving tumor progression in HNSC. Importantly, we identified significant enrichment of BRCA1/2 genes in pathways related to cancer development within the KEGG database. Finally, in our quest to explore therapeutic avenues, we systematically analyzed drugs targeting up-regulated and mutated BRCA1/2 genes, identifying promising candidates for tailored treatment modalities in HNSC. In conclusion, our study reveals the unique genetic profile of HNSC in Pakistani patients, featuring unique pathogenic mutations in BRCA1 and BRCA2 genes. These mutations offer promise as valuable diagnostic markers and potential therapeutic targets.

Protective role for C3aR in experimental chronic pyelonephritis.

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.

Mechanical instability generated by Myosin 19 contributes to mitochondria cristae architecture and OXPHOS.

The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.

The C5a/C5aR1 Axis Contributes to the Pathogenesis of Acute Cystitis Through Enhancement of Adhesion and Colonization of Uropathogenic E. coli.

Our previous work using a murine model of pyelonephritis demonstrated that the C5a/C5aR1 axis plays a pathogenic role in acute kidney infection. In this study, we report that the C5a/C5aR1 axis also plays a pathogenic role in acute bladder infection. C5aR1-deficient mice had reduced bladder bacterial load and attenuated bladder tissue injury, which is associated with reduced expression of terminal α-mannosyl residues (Man) (a potential ligand for type 1 fimbriae of E. coli) at the luminal surface of the bladder epithelium and reduced early bacterial colonization of the bladder. In vitro, C5a stimulation enhanced mannose expression in and facilitated bacterial adhesion/colonization to human bladder epithelial cells. C5a stimulation also upregulated the activation of ERK1/2 and NF-κB signaling and gene expression of proinflammatory cytokines (i.e., Il6, Il1b, Cxcl1, Ccl2) in the epithelial cells, which could drive pro-inflammatory responses leading to tissue injury. Administration of the C5aR1 antagonist effectively reduced bladder bacterial load and tissue injury. Thus, our findings demonstrate a previously unknown pathogenic role for the C5a/C5aR1 axis in bladder infection and suggest that the C5a/C5aR1 axis-mediated upregulation of Man expression, enhancement of bacterial adhesion/colonization, and excessive inflammatory responses contribute to acute bladder infection. These findings improve our understanding of the pathogenesis of bladder infection with therapeutic implications for UTI.

The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma.

Deregulation of alternative splicing is implicated as a relevant source of molecular heterogeneity in cancer. However, the targets and intrinsic mechanisms of splicing in hepatocarcinogenesis are largely unknown. Here, we report a functional impact of a Splicing Regulatory Glutamine/Lysine-Rich Protein 1 (SREK1) variant and its regulator, Serine/arginine-rich splicing factor 10 (SRSF10). HCC patients with poor prognosis express higher levels of exon 10-inclusive SREK1 (SREK1L). SREK1L can sustain BLOC1S5-TXNDC5 (B-T) expression, a targeted gene of nonsense-mediated mRNA decay through inhibiting exon-exon junction complex binding with B-T to exert its oncogenic role. B-T plays its competing endogenous RNA role by inhibiting miR-30c-5p and miR-30e-5p, and further promoting the expression of downstream oncogenic targets SRSF10 and TXNDC5. Interestingly, SRSF10 can act as a splicing regulator for SREK1L to promote hepatocarcinogenesis via the formation of a SRSF10-associated complex. In summary, we demonstrate a SRSF10/SREK1L/B-T signalling loop to accelerate the hepatocarcinogenesis.

Ginsenosides in Panax genus and their biosynthesis.

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.

A combination index and glycoproteomics-based approach revealed synergistic anticancer effects of curcuminoids of turmeric against prostate cancer PC3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicines (HMs) often exert integration effects, including synergistic, additive and antagonistic effects, in such ways that they act on multiple targets and multiple pathways on account of their multiple components. Turmeric, made from the rhizome of Curcuma longa L., is a well-known HM prescribed in the polyherbal formulas for cancer treatment in traditional Chinese medicines (TCMs). However, neither the multiple anticancer compounds of turmeric nor the integration effects of these components are fully known. AIM OF THE STUDY: This work aims to develop a systematic approach to reveal the integration effect mechanisms of multiple anticancer compounds in turmeric against prostate cancer PC3 cells. MATERIALS AND METHODS: Combination index and omics technologies were applied to profile the integration effect mechanisms of bioactive compounds in proportions naturally found in turmeric. PC3 cell line (a prostate cancer cell line) fishing and high resolution mass spectrometry were employed to screen and identify the anticancer compounds from turmeric. The combinations which contain different cell-bound compounds in natural proportions were prepared for further evaluation of anti-cancer activity by using cell viability assays, and assessment of cell apoptosis and cell cycle analysis. Combination index analysis was applied to study the integration effects of the anticancer compounds in their natural proportions. Finally, quantitative glycoproteomics/proteomics and Western blot were implemented to reveal the potential synergistic effect mechanisms of the anticancer compounds based on their natural proportions in turmeric. RESULTS: Three curcuminoids (curcumin, CUR; demethoxycurcumin, DMC; bisdemethoxycurcumin, BDMC) in turmeric were discovered and shown to possess significant synergistic anticancer activities. Combination index analysis revealed an additive effect of CUR combined with DMC or BDMC and a slight synergistic effect of DMC combined with BDMC in natural proportions in turmeric, while a combination of all three curcuminoids (CUR, DMC and BDMC) at a ratio of 1:1:1 yielded superior synergistic effects. Interestingly, the presence of BDMC and DMC are essential for synergistic effect. Glycoproteomics and proteomics demonstrated that different curcuminoids regulate various protein pathways, such as ribosome, glycolysis/gluconeogenesis, biosynthesis of amino acids, and combination of CUR + DMC + BDMC showed the most powerful effects on down-regulation of protein expression. CONCLUSIONS: Our analytical approach provides a systematic understanding of the holistic activity and integration effects of the anti-cancer compounds in turmeric and three curcuminoids of turmeric showed a synergistic effect on PC3 cells.

Genome-wide identification of R2R3-MYB family in wheat and functional characteristics of the abiotic stress responsive gene TaMYB344.

BACKGROUND: MYB superfamily is one of the most abundant families in plants, which plays important roles in plant growth, development, and productivity. However, to date, researches on MYBs in wheat (Triticum aestivum L.) are scattered mostly, not comprehensive. RESULTS: In this study, a total of 393 R2R3-MYBs and 12 R1R2R3-MYBs were identified and analyzed including gene structure, chromosomal distribution, synteny relationship, and evolutionary relationship. Then, 29 clusters tandem duplication and 8 clusters segmental duplication genes were discovered. The expression profile of the identified genes under abiotic and biotic stress was analyzed using RNA-seq data. Based on expression patterns analysis, we screened many candidate genes involved in plant response to abiotic and biotic stress. Among them, the functional characteristics of TaMYB344 were further studied. TaMYB344 was localized in the nucleus and functioned as a weak transcriptional activator. We demonstrated that TaMYB344-overexpressing transgenic tobacco plants had enhanced tolerance to drought, heat, and high salt stress. CONCLUSIONS: In this study, 393 R2R3-MYBs and 12 R1R2R3-MYBs in wheat were systemically identified and analyzed. Differential expression analysis indicated that many R2R3-MYBs were involved in abiotic and biotic stress response. We identified a potential candidate gene TaMYB344, overexpression of which in tobacco plants enhanced drought, heat, and salt stress tolerance. These results will provide abundant molecular data for breeding new varieties of wheat in the future.

Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.

smi-miR396b targeted SmGRFs, SmHDT1, and SmMYB37/4 synergistically regulates cell growth and active ingredient accumulation in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: MIR396b had been cloned and overexpressed in Salvia miltiorrhiza hairy roots. MiR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to regulate cell growth and secondary metabolism in S. miltiorrhiza hairy roots. Danshen (Salvia miltiorrhiza Bunge) is a valuable medicinal herb with two kinds of clinically used natural products, salvianolic acids and tanshinones. miR396 is a conserved microRNA and plays extensive roles in plants. However, it is still unclear how miR396 works in S. miltiorrhiza. In this study, an smi-MIR396b has been cloned from S. miltiorrhiza. Overexpression of miR396b in danshen hairy roots inhibited hairy root growth, reduced salvianolic acid concentration, but enhanced tanshinone accumulation, resulting in the biomass and total salvianolic acids respectively reduced to 55.5 and 72.1% of the control and total tanshinones increased up to 1.91-fold of the control. Applied degradome sequencing, 5'RLM-RACE, and qRT-PCR, 13 targets for miR396b were identified including seven conserved SmGRF1-7 and six novel ones. Comparative transcriptomics and microRNomics analysis together with qRT-PCR results confirmed that miR396b targets SmGRFs, SmHDT1, and SmMYB37/4 to mediate the phytohormone, especially gibberellin signaling pathways and consequentially resulted in the phenotype variation of miR396b-OE hairy roots. Furthermore, miR396b could be activated by methyl jasmonate, abscisic acid, gibberellin, salt, and drought stresses. The findings in this study indicated that smi-miR396b acts as an upstream and central regulator in cell growth and the biosynthesis of tanshinones and salvianolic acids, shedding light on the coordinated regulation of plant growth and biosynthesis of active ingredients in S. miltiorrhiza.