Identification of circular RNA-Dcaf6 as a therapeutic target for optic nerve crush-induced RGC degeneration.

The death of retinal ganglion cells (RGCs) can cause irreversible injury in visual function. Clarifying the mechanism of RGC degeneration is critical for the development of therapeutic strategies. Circular RNAs (circRNAs) are important regulators in many biological and pathological processes. Herein, we performed circRNA microarrays to identify dysregulated circRNAs following optic nerve crush (ONC). The results showed that 221 circRNAs were differentially expressed between ONC retinas and normal retinas. Notably, the levels of circular RNA-Dcaf6 (cDcaf6) expression in aqueous humor of glaucoma patients were higher than that in cataract patients. cDcaf6 silencing could reduce oxidative stress-induced RGC apoptosis in vitro and alleviate retinal neurodegeneration in vivo as shown by increased neuronal nuclei antigen (NeuN, neuronal bodies) and beta-III-tubulin (TUBB3, neuronal filaments) staining and reduced glial fibrillary acidic protein (GFAP, activated glial cells) and vimentin (activated glial cells) staining. Collectively, this study identifies a promising target for treating retinal neurodegeneration.

Ocular A-to-I RNA editing signatures associated with SARS-CoV-2 infection.

Ophthalmic manifestations have recently been observed in acute and post-acute complications of COVID-19 caused by SARS-CoV-2 infection. Our precious study has shown that host RNA editing is linked to RNA viral infection, yet ocular adenosine to inosine (A-to-I) RNA editing during SARS-CoV-2 infection remains uninvestigated in COVID-19. Herein we used an epitranscriptomic pipeline to analyze 37 samples and investigate A-to-I editing associated with SARS-CoV-2 infection, in five ocular tissue types including the conjunctiva, limbus, cornea, sclera, and retinal organoids. Our results revealed dramatically altered A-to-I RNA editing across the five ocular tissues. Notably, the transcriptome-wide average level of RNA editing was increased in the cornea but generally decreased in the other four ocular tissues. Functional enrichment analysis showed that differential RNA editing (DRE) was mainly in genes related to ubiquitin-dependent protein catabolic process, transcriptional regulation, and RNA splicing. In addition to tissue-specific RNA editing found in each tissue, common RNA editing was observed across different tissues, especially in the innate antiviral immune gene MAVS and the E3 ubiquitin-protein ligase MDM2. Analysis in retinal organoids further revealed highly dynamic RNA editing alterations over time during SARS-CoV-2 infection. Our study thus suggested the potential role played by RNA editing in ophthalmic manifestations of COVID-19, and highlighted its potential transcriptome impact, especially on innate immunity.

Patients with ASPSCR1-TFE3 fusion achieve better response to ICI based combination therapy among TFE3-rearranged renal cell carcinoma.

BACKGROUND: TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is largely undefined. This study aims to evaluate and compare the efficacy of different systemic treatments and further explore the molecular correlates. METHODS: Thirty-eight patients with metastatic TFE3-rRCC were enrolled. Main outcomes included progression-free survival (PFS), overall survival, objective response rate (ORR) and disease control rate. RNA sequencing was performed on 32 tumors. RESULTS: Patients receiving first-line immune checkpoint inhibitor (ICI) based combination therapy achieved longer PFS than those treated without ICI (median PFS: 11.5 vs. 5.1 months, P = 0.098). After stratification of fusion partners, the superior efficacy of first-line ICI based combination therapy was predominantly observed in ASPSCR1-TFE3 rRCC (median PFS: not reached vs. 6.5 months, P = 0.01; ORR: 67.5% vs. 10.0%, P = 0.019), but almost not in non-ASPSCR1-TFE3 rRCC. Transcriptomic data revealed enrichment of ECM and collagen-related signaling in ASPSCR1-TFE3 rRCC, which might interfere with the potential efficacy of anti-angiogenic monotherapy. Whereas angiogenesis and immune activities were exclusively enriched in ASPSCR1-TFE3 rRCC and promised the better clinical outcomes with ICI plus tyrosine kinase inhibitor combination therapy. CONCLUSIONS: The current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC based on fusion partner stratification. ICI based combination therapy could serve as an effective first-line treatment option for metastatic ASPSCR1-TFE3 rRCC patients. Regarding with other fusion subtypes, further investigations should be performed to explore the molecular mechanisms to propose pointed therapeutic strategy accordingly.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone.

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.

Dysregulated RNA editing of EIF2AK2 in polycystic ovary syndrome: clinical relevance and functional implications.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear. METHODS: Ten RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression. RESULTS: RNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway. CONCLUSIONS: Our study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.

Single-cell profiling transcriptomic reveals cellular heterogeneity and cellular crosstalk in choroidal neovascularization model.

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD.

Co-exposure to fluoride and sulfur dioxide induces abnormal enamel mineralization in rats via the FGF9-mediated MAPK signaling pathway.

Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F-SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F-SO2 Co-exposure in rats. The results showed that F-SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F-SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F-SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F-SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.

Analysis of abnormal expression of signaling pathways in PQ-induced acute lung injury in SD rats based on RNA-seq technology.

Background: Paraquat (PQ) plays an important role in agricultural production due to its highly effective herbicidal effect. However, it has led to multiple organ failure in those who have been poisoned, with damage most notable in the lungs and ultimately leading to death. Because of little research has been performed at the genetic level, and therefore, the specific genetic changes caused by PQ exposure are unclear.Methods: Paraquat poisoning model was constructed in Sprague Dawley (SD) rats, and SD rats were randomly divided into Control group, paraquat (PQ) poisoning group and Anthrahydroquinone-2,6-disulfonate (AH2QDS) treatment group. Then, the data was screened and quality controlled, compared with reference genes, optimized gene structure, enriched at the gene expression level, and finally, signal pathways with significantly different gene enrichment were screened.Results: This review reports on lung tissues from paraquat-intoxicated Sprague Dawley (SD) rats that were subjected to RNA-seq, the differentially expressed genes were mainly enriched in PI3K-AKT, cGMP-PKG, MAPK, Focal adhesion and other signaling pathways.Conclusion: The signaling pathways enriched with these differentially expressed genes are summarized, and the important mechanisms mediated through these pathways in acute lung injury during paraquat poisoning are outlined to identify important targets for AH2QDS treatment of acute lung injury due to paraquat exposure, information that will be used to support a subsequent in-depth study on the mechanism of PQ action.

Knockdown of RTEL1 Alleviates Chronic Obstructive Pulmonary Disease by Modulating M1, M2 Macrophage Polarization and Inflammation.

Chronic obstructive pulmonary disease (COPD) is a common chronic disease characterized by airflow obstruction, which seriously threatens people's health. The COPD mouse model was established with cigarette smoke induction. Hematoxylin-eosin staining and Masson staining were carried out to observe the pathological changes of lung tissues in COPD mice. RTEL1 was silenced in COPD mice, and immunohistochemistry was used to detect RTEL1, ki67 and Caspase-3 expression. The role of RTEL1 in inflammation were evaluated by ELISA, and the impacts of RTEL1 on M1 and M2 macrophage markers (iNOS and CD206) were evaluated by qPCR and western blotting. In COPD model, there was an increase in the number of inflammatory cells, with slightly disorganized cell arrangement, unclear hierarchy, condensed and solidified nuclei, while knockdown of RTEL1 improved the inflammatory infiltration. Moreover, knockdown of RTEL1 reduced ki67-positive cells and increased Caspase-3 positive cells in COPD group. The increased inflammatory factors (IL-1ß, MMP-9, TNF-α, IL-4, IL-6, and IL-23) in COPD were suppressed by knockdown of RTEL1, while iNOS was raised and CD206 was inhibited. In conclusion, knockdown of RTEL1 promoted M1 and inhibited M2 macrophage polarization and inflammation to alleviate COPD.

Role of grapevine SEPALLATA-related MADS-box gene VvMADS39 in flower and ovule development.

Seedlessness is one of the most important breeding goals for table grapes; thus, understanding the molecular genetic regulation of seed development and abortion is critical for the development of seedless cultivars. In the present study, we characterized VvMADS39, a class E MADS-box gene of grapevine (Vitis vinifera) orthologous to Arabidopsis SEP2. Heterologous overexpression of VvMADS39 in tomato reduced the fruit and seed size and seed number. Targeted mutagenesis of the homologous SlMADS39 in tomato induced various floral and fruit defects. It could reasonable to suppose that active VvMADS39 expression in "Thompson Seedless" may restrict cellular expansion, resulting in the development of smaller fruits and seeds, VvMADS39 may play a role in the regulation of ovule development in grapevine and contributes to seedless fruit formation. In contrast, VvMADS39 suppression in "Red Globe" was associated with enhanced histone H3 lysine 27 trimethylation in the promoter region of VvMADS39, allowing normal ovule and fruit development; Meanwhile, VvMADS39 interacts with VvAGAMOUS, and the activity of the VvMADS39-VvAGAMOUS dimer to induce integument development requires the activation and maintenance of VvINO expression. The synergistic cooperation between VvMADS39 and related proteins plays an important role in maintaining floral meristem characteristics, and fruit and ovule development.

Avoiding Unnecessary Systematic Biopsy in Clinically Significant Prostate Cancer: Comparison Between MRI-Based Radiomics Model and PI-RADS Category.

BACKGROUND: MRI-targeted biopsy (MRTB) improves the clinically significant prostate cancer (csPCa) detection rate with fewer biopsy cores in men with suspected PCa. However, whether concurrent systematic biopsy (SB) can be avoided in patients undergoing MRTB remains unclear. PURPOSE: To evaluate the potential value of MRI-based radiomics models in avoiding unnecessary SB in biopsy-naïve patients. STUDY TYPE: Retrospective. POPULATION: A total of 226 patients (mean age 66.6 ± 9.02 years) with suspicion of PCa (PI-RADS score ≥ 3) and received combined cognitive MRTB with SB were retrospectively recruited and randomly divided into training (N = 180) and test (N = 46) cohorts at an 8:2 ratio. FIELD STRENGTH/SEQUENCE: A 3.0 T, biparametric MRI (bpMRI) including T2-weighted imaging (T2WI) and apparent diffusion coefficient (ADC) map. ASSESSMENT: The whole prostate gland (PG) and the index lesion (IL) were delineated. Three radiomics models of bpMRIPG , bpMRIIL , and bpMRIPG+IL were constructed, respectively, and the performance of each radiomics model was compared with that of PI-RADS assessment. STATISTICAL TESTS: The least absolute shrinkage and selection operator (LASSO) regression method was used to select texture features. The area under the curve (AUC) and decision curve analysis were used to estimate the models. RESULTS: The bpMRIPG+IL radiomics model exhibited good discrimination, calibration, and net benefits, which would reduce the SB biopsy in 71.2% and 71.4% of men with PI-RADS ≥ 5 lesions in the training and test cohorts, respectively. DATA CONCLUSION: A bpMRIPG+IL radiomics model may outperform PI-RADS category in help reducing unnecessary SB in biopsy-naïve patients. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 6.

Host ALDH2 deficiency aggravates nonalcoholic steatohepatitis through gut-liver axis.

Nonalcoholic steatohepatitis (NASH) is the major cause of liver dysfunction. Animal and population studies have shown that mitochondrial aldehyde dehydrogenase (ALDH2) is implicated in fatty liver disease. However, the role of ALDH2 in NASH and the underlying mechanisms remains unclear. To address this issue, ALDH2 knockout (ALDH2-/-) mice and wild-type littermate mice were fed a methionine-and choline-deficient (MCD) diet to induce a NASH model. Fecal, serum, and liver samples were collected and analyzed to investigate the impact of the gut microbiota and bile acids on this process. We found that MCD-fed ALDH2-/- mice exhibited increased serum pro-inflammation cytokines, hepatic inflammation and fat accumulation than their wild-type littermates. MCD-fed ALDH2-/- mice exhibited worsened MCD-induced intestinal inflammation and barrier damage, and gut microbiota disorder. Furthermore, mice receiving microbiota from MCD-fed ALDH2-/- mice had increased severity of NASH compared to those receiving microbiota from MCD-fed wild-type mice. Notably, the intestinal Lactobacillus was significantly reduced in MCD-fed ALDH2-/- mice, and gavage with Lactobacillus cocktail significantly improved MCD-induced NASH. Finally, we found that ALDH2-/- mice had reduced levels of bile salt hydrolase and specific bile acids, especially lithocholic acid (LCA), accompanied by downregulated expression of the intestinal FXR-FGF15 pathway. Supplementation of LCA in ALDH2-/- mice upregulated intestinal FXR-FGF15 pathway and alleviated NASH. In summary, ALDH2 plays a critical role in the development of NASH through modulation of gut microbiota and bile acid. The findings suggest that supplementing with Lactobacillus or LCA could be a promising therapeutic approach for treating NASH exacerbated by ALDH2 deficiency.

A Superhydrophobic Trifluoromethyl-Containing Covalent Organic Framework Membrane for Efficient Oil/Water Separation.

Oily water caused in the process of industry leads to not only the waste of resources, but also environmental pollution. Membrane separation, as a facile and efficient separation technology, has attracted widespread attention in the field of oil/water separation. The development of membrane materials with high separation performance is one of the key elements to improve separation efficiency. In this work, a superhydrophobic membrane composited with a trifluoromethyl-containing covalent organic framework (COF) is prepared, which exhibits excellent performance on separations of oil/water mixtures and water-in-oil emulsions. For different composition of oil/water mixtures, the highest flux of oil is up to 32 000 L m-2  h-1 and oil/water separation efficiency is above 99%. Moreover, the high oil/water separation efficiency remains unchanged after successive cycles. This work provides a feasible scheme for the design of high-efficiency oil/water separation membranes.

Efficacy and safety of high dose recombinant human thrombopoietin in the treatment of immune thrombocytopenia.

The conventional dose of recombinant human thrombopoietin (rhTPO) in the treatment of immune thrombocytopenia (ITP) is 300 U/kg per day, but the clinical reaction rate is not satisfactory. Accordingly, we explored the efficacy and safety of increasing rhTPO dose in the treatment of ITP. A retrospective study was conducted to collect the clinical data of 105 ITP patients who were divided into two groups, a low-dose group (15 000 U/day) and a high-dose group (30 000 U/day) according to the dose of rhTPO. The total effective rate of the low-dose group and the high-dose group was 31/44 (70.45%) vs. 56/61 (91.80%) (P = .049), and the average time of using rhTPO in the high-dose group was shorter than that in the low-dose group (7 days vs. 10 days, P = .001). On the 7th and 14th day of treatment, the efficacy of the high-dose group was better than that of the low-dose group [45/61 (73.77%) vs. 17/44 (38.64%), P < .001; 55/60 (91.67%) vs. 30/44 (68.18%), P < .05)]. The incidence of treatment related adverse events in the low-dose group and the high-dose group was 6/44 (13.64%) vs. 6/61 (9.84%) (P > .05), which were mild and transient in nature. In our study, high-dose rhTPO had good efficacy and high safety in the treatment of ITP with the efficacy better than low-dose rhTPO especially at day 7.

What is the context? Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by low platelet counts due to increased platelet destruction and impaired platelet production.The therapy direction of ITP involves promoting platelet generation, reducing excessive platelet destruction, immune regulation and so on.Recombinant human thrombopoietin (rhTPO), a promote platelet production drug, has pharmacological action similar to that of endogenous TPO. It can increase platelet count rapidly and effectively and has immunological regulation effect as well.It is found that rhTPO can rapidly and effectively increase platelet count, which has potential clinical application value in emergency situations.What is new? Traditionally, rhTPO has been recommended at 300 U/kg per day. Although it can greatly improve the treatment effect of ITP, the effect is not very satisfactory. In clinical practice, it has been observed that rhTPO dosage is often relatively insufficient and the therapeutic effect is poor. Therefore, we explored the efficacy and safety of increasing rhTPO dose in the treatment of ITP.Within the efficacy and safety of rhTPO 15 000 U/day and 30 000 U/day in the treatment of ITP, our analyses suggest that:The early response rate of the high-dose group was better than that of the low-dose group.In the high-dose group, the effective rate of rhTPO alone or combined with glucocorticoids was more than 90%.Treatment related adverse events occurred at a low rate and remained mild and transient.What is the impact? Comparing with conventional dose rhTPO, high-dose rhTPO may have better efficacy and safety in the treatment of immune thrombocytopenia and shorter administration time.

A Case of Adult Acute B-Cell Lymphoblastic Leukemia with Hypercalcemia and Osteolytic Bone Lesions.

BACKGROUND: We report a rare case of adult acute B-lymphoblastic leukemia (B-ALL) with hypercalcemia and osteolytic bone lesions in a 53-year-old man who died after chemotherapy. METHODS: The bone marrow examination was evaluated by Wright-Giemsa staining, tissue biopsy, immunohistochemical staining, and flow cytometry. Bone imaging was performed using positron emission tomography/computed tomography (PET/CT) technology. Total calcium levels were measured by biochemical analyzer. RESULTS: The result of PET/CT indicated the patient with B-ALL with severe osteolytic bone lesions. The serum total calcium level was as high as 4.09 mmol/L, and the cytokines interleukin-6 and 17A were significantly elevated. The patient was resistant to chemotherapy and had a poor prognosis. CONCLUSIONS: Hypercalcemia and osteolytic bone lesions are rare complications of adult B-ALL, and their co-occurrence may be an indicator of poor prognosis in patients with B-ALL.

The role of FGF9-mediated TGF-ß1/Smad signaling in enamel hypoplasia induced by exposure to fluoride and SO2 in rats.

Many geographical areas of the world are polluted by both fluoride and sulfur dioxide (SO2). However, the effects of simultaneous exposure to fluoride and SO2 on teeth are unknown. Fibroblast growth factor-9 (FGF9) and transforming growth factor-ß1 (TGF-ß1) are key signaling molecules in enamel development. The purpose of the study was to explore the effects of co-exposure to fluoride and sulfur dioxide on enamel and to investigate the role and mechanism of FGF9 and TGF-ß1. First, sodium fluoride (NaF) and SO2 derivatives were used to construct rat models and evaluate the enamel development of rats. Then, TGF-ß1 (cytokine) treatment, SIS3 (inhibitor) treatment and FGF9 gene knockdown were used to explore the mechanism of enamel damage in vitro. The results showed that enamel column crystals in the exposed group were characterized by enamel hypoplasia, as indicated by alterations such as disarrangement of enamel column crystals, space widening and breakage. Ameloblasts also showed pathological changes such as ribosome loss, mitochondrial swelling, nuclear fragmentation and chromatin aggregation. The protein expression of FGF9 was higher and the protein expression of AMBN, TGF-ß1 and p-Smad2/3 protein was lower in the groups treated with fluoride and SO2 individually or in combination compared with the control group. Further studies showed that TGF-ß1 significantly upregulated p-Smad2/3 and AMBN protein expression and reduced the inhibitory effects of fluoride and SO2; furthermore, SISI blocked the effect of TGF-ß1. In addition, knockdown of FGF9 upregulated TGF-ß1 protein expression, further activated Smad2/3 phosphorylation, eliminated the inhibitory effects of fluoride and SO2, and increased the protein expression of AMBN. In brief, the study confirms that co-exposure to fluoride and SO2 can result in enamel hypoplasia in rats and indicates that the underlying mechanism may be closely related to the effect of FGF9 on enamel matrix protein secretion through inhibition of the TGF-ß1/Smad signaling pathway.

Lead exposure disturbs ATP7B-mediated copper export from brain barrier cells by inhibiting XIAP-regulated COMMD1 protein degradation.

The brain barrier is an important structure for metal ion homeostasis. According to studies, lead (Pb) exposure disrupts the transportation of copper (Cu) through the brain barrier, which may cause impairment of the nervous system; however, the specific mechanism is unknown. The previous studies suggested the X-linked inhibitor of apoptosis (XIAP) is a sensor for cellular Cu level which mediate the degradation of the MURR1 domain-containing 1 (COMMD1) protein. XIAP/COMMD1 axis was thought to be an important regulator in Cu metabolism maintenance. In this study, the role of XIAP-regulated COMMD1 protein degradation in Pb-induced Cu disorders in brain barrier cells was investigated. Pb exposure significantly increased Cu levels in both cell types, according to atomic absorption technology testing. Western blotting and reverse transcription PCR (RT-PCR) showed that COMMD1 protein levels were significantly increased, whereas XIAP, ATP7A, and ATP7B protein levels were significantly decreased. However, there were no significant effects at the messenger RNA (mRNA) level (XIAP, ATP7A, and ATP7B). Pb-induced Cu accumulation and ATP7B expression were reduced when COMMD1 was knocked down by transient small interfering RNA (siRNA) transfection. In addition, transient plasmid transfection of XIAP before Pb exposure reduced Pb-induced Cu accumulation, increased COMMD1 protein levels, and decreased ATP7B levels. In conclusion, Pb exposure can reduce XIAP protein expression, increase COMMD1 protein levels, and specifically decrease ATP7B protein levels, resulting in Cu accumulation in brain barrier cells.

Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules.

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.

Bioaugmentation of intertidal sludge enhancing the development of salt-tolerant aerobic granular sludge.

Three parallel bioreactors were operated with different inoculation of activated sludge (R1), intertidal sludge (ItS) (R2), and ItS-added AS (R3), respectively, to explore the effects of ItS bioaugmentation on the formation of salt-tolerant aerobic granular sludge (SAGS) and the enhancement of COD removal performance. The results showed that compared to the control (R1-2), R3 promoted a more rapid development of SAGS with a cultivation time of 25 d. Following 110-day cultivation, R3 exhibited a higher granular diameter of 1.3 mm and a higher hydrophobic aromatic protein content than that in control. Compared to the control, the salt-tolerant performance in R3 was also enhanced with the COD removal efficiency of 96.4% due to the higher sludge specific activity of 14.4 g·gVSS-1·d-1 and the salinity inhibition constant of 49.3 gL-1. Read- and genome-resolved metagenomics together indicated that a higher level of tryptophan/tyrosine synthase gene (trpBD, tyrBC) and enrichment of the key gene hosts Rhodobacteraceae, Marinicella in R3, which was about 5.4-fold and 1.4-fold of that in control, could be the driving factors of rapid development of SAGS. Furthermore, the augmented salt-tolerant potential in R3 could result from that R1 was dominated by Rhodospirillaceae, Bacteroidales, which carried more trehalose synthase gene (otsB, treS), while the dominant members Rhodobacteraceae, Marinicella in R3 were main contributors to the glycine betaine synthase gene (ectC, betB, gbsA). This study could provide deeper insights into the rapid development and improved salt-tolerant potential of SAGS via bioaugmentation of intertidal sludge, which could promote the application of hypersaline wastewater treatment.

[Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province].

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.