Microcystin-LR Induces Estrogenic Effects at Environmentally Relevant Concentration in Black-Spotted Pond Frogs (Pelophylax nigromaculatus): In Situ, In Vivo, In Vitro, and In Silico Investigations.

Harmful cyanobacterial blooms are frequent and intense worldwide, creating hazards for aquatic biodiversity. The potential estrogen-like effect of Microcystin-LR (MC-LR) is a growing concern. In this study, we assessed the estrogenic potency of MC-LR in black-spotted frogs through combined field and laboratory approaches. In 13 bloom areas of Zhejiang province, China, the MC-LR concentrations in water ranged from 0.87 to 8.77 µg/L and were correlated with sex hormone profiles in frogs, suggesting possible estrogenic activity of MC-LR. Tadpoles exposed to 1 µg/L, an environmentally relevant concentration, displayed a female-biased sex ratio relative to controls. Transcriptomic results revealed that MC-LR induces numerous and complex effects on gene expression across multiple endocrine axes. In addition, exposure of male adults significantly increased the estradiol (E2)/testosterone (T) ratio by 3.5-fold relative to controls. Downregulation of genes related to male reproductive endocrine function was also identified. We also showed how MC-LR enhances the expression of specific estrogen receptor (ER) proteins, which induce estrogenic effects by activating the ER pathway and hypothalamic-pituitary-gonadal (HPG) axis. In aggregate, our results reveal multiple lines of evidence demonstrating that, for amphibians, MC-LR is an estrogenic endocrine disruptor at environmentally relevant concentrations. The data presented here support the need for a shift in the MC-LR risk assessment. While hepatoxicity has historically been the focus of MC-LR risk assessments, our data clearly demonstrate that estrogenicity is a major mode of toxicity at environmental levels and that estrogenic effects should be considered for risk assessments on MC-LR going forward.

Timosaponin BII inhibits TGF-ß mediated epithelial-mesenchymal transition through Smad-dependent pathway during pulmonary fibrosis.

Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial-mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti-PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug-induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)-induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP-9/TIMP-1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF-ß1 and EMT-related marker proteins including E-cadherin, vimentin, and α-SMA. Besides, aberrant TGF-ß1 expression and phosphorylation of Smad2 and Smad3 in BLM-induced animal model and TGF-ß1-induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF-ß/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

Efficacy and safety of flumatinib in the treatment of newly diagnosed chronic myeloid leukemia in the chronic phase: A real-world single-center retrospective study, with a focus on premature drug discontinuation.

PURPOSE: To assess the real-world efficacy and safety of flumatinib as first-line and post-line treatments for chronic myeloid leukemia in the chronic phase (CML-CP). RESULTS: Among 141 patients receiving flumatinib as first-line and post-line treatment, the 12-month major molecular response (MMR) rates were 69.4% and 67.6%, respectively. The median time to response was 6 and 10.5 months, respectively. In post-line treatment, the early molecular response (EMR) of flumatinib as second-line is significantly superior to that of third-line treatment (3-month EMR rate: 79.2% vs. 39.3%, P<0.001; 3-month MMR rate: 45.8% vs. 21.4%, P=0.033). Contrastively, patients who switched to flumatinib due to intolerance had significantly higher MMR rates at 3, 6, and 12 months compared to patients who switched due to inadequate response (60.6% vs. 24.2%, P=0.003; 66.7% vs. 36.0%, P=0.027; 84.2% vs. 50.0%, P=0.038). Premature drug discontinuation was observed in 28.4% of the patients. Grades 3-4 hematologic adverse events (AEs) were identified as independent risk factors for premature drug discontinuation. Patients who discontinued treatment and those who previously received only imatinib therapy had a poorer molecular response and failure-free survival. CONCLUSIONS: Flumatinib demonstrates favorable efficacy and safety. Treatment discontinuation can result in a poorer molecular response and long-term prognosis.

Metformin as an Emerging Pollutant in the Aquatic Environment: Occurrence, Analysis, and Toxicity.

The use of human and veterinary drugs has led to the accumulation of pharmaceuticals in various aquatic environments at progressively increasing levels, exhibiting strong ecological risks. Metformin is widely used as a first-line prescription drug for the treatment of type 2 diabetes mellitus as well as a livestock drug. Unlike other drugs, metformin is not metabolized in the body, and almost all of its intake is excreted and released into the aquatic environment via urine and feces, causing adverse effects on aquatic ecosystems. This review provides an overview of the occurrence and detection of metformin in the aquatic environment and its toxic effects on different aquatic organisms (fish, daphnia, rotifers, chlorella). Metformin has been documented in a variety of aqueous environments such as wastewater, surface water, and groundwater as well as drinking water. The wide distribution of metformin in the aqueous environment calls for the development of more accurate detection methods. This paper reviews detection methods for metformin in the aqueous environment and evaluates their advantages and disadvantages. Toxicity studies have shown that metformin can cause adverse reactions in fish, such as oxidative stress, genotoxicity, disruption of intestinal flora, and morphological alterations; it also affects the growth and reproduction of small aquatic organisms. Knowledge gaps in the field of metformin research were assessed, and future research priorities were identified.

Smoking index and COPD duration as potential risk factors for development of osteoporosis in patients with non-small cell lung cancer - A retrospective case control study evaluated by CT Hounsfield unit.

Objective: To investigate the effect of smoking index (calculated as number of cigarettes per day × smoking years) and chronic obstructive pulmonary disease (COPD) duration on osteoporosis (OP)evaluated by opportunistic chest CT in patients with non-small cell lung cancer (NSCLC). Methods: A total of 101 patients diagnosed with NSCLC were included in our cohort study. Among them, 50 patients with a history of smoking and COPD were assigned to the experimental group, while 51 patients without a history of smoking and COPD were assigned to the control group. Hounsfield unit (HU) value was measured by conventional chest CT to investigate the bone mineral density; and the mean values of axial HU value in the upper, middle and lower parts of T4, T7, T10 and L1 vertebral bodies were measured as the study variables. Results: There were no significant differences in gender, age, body mass index, type of lung cancer, clinical stage of lung cancer and comorbidities between the two groups (P = 0.938，P = 0.158，P = 0.722,P = 0.596,P = 0.813,P = 0.655). The overall mean HU values of T4, T7, T10, L1 in the experimental group were 116.60 ± 30.67, 110.56 ± 30.03, 109.18 (96.85-122.95), 94.63 (85.20-104.12) and 106.86 ± 22.26, respectively, which were significantly lower than those in the control group (189.55 ± 34.57, 174.54 ± 35.30, 172.73 (156.33-199.50), 158.20 (141.60-179.40) and 177.50 ± 33.49) (P ＜0.05). And in the experimental group, smoking index and COPD duration were significantly and negatively correlated with HU values (r = -0.627, -0.542, P ＜0.05, respectively). Conclusion: Patients with NSCLC who have a history of smoking and COPD exhibit a notably lower HU value compared to the control groups. Additionally, it has been observed that the smoking index and duration of COPD may be influential factors affecting bone mineral density in NSCLC patients.

Genome-wide identification glutathione-S-transferase gene superfamily in Daphnia pulex and its transcriptional response to nanoplastics.

Glutathione S-transferases (GSTs) are key multifunctional phase II detoxification enzymes involved in the regulation of growth, development, and stress responses. However, the knowledge of GSTs in the model invertebrate organism Daphnia pulex at the genomic level remains limited. In the present study, 35 GST genes were identified in D. pulex (Dp-GST), belonging to eight subfamilies, with the sigma, mu, and delta/epsilon subfamilies constituting approximately 29 %, 20 %, and 20 % of the GST superfamily, respectively. Chromosome tandem duplication of genes within the same subfamily was observed, which may be the main force driving GST expansion in D. pulex. DpGST genes showed different expression patterns in response to nanoplastic exposure for 96 h and 21 days. Some homologous GST genes in D. pulex showed similar expression patterns in response to nanoplastic exposure, likely owing to their unique motifs. For example, motif 9 is found in all delta/epsilon GST genes, whereas motifs 1, 2, 3, 5, and 7 are highly conserved in sigma GST genes. The characterization of D. pulex GSTs extending the knowledge of GST-mediated environmental contaminants, especially nanoplastics.

Perfluorooctanoic acid and perfluorooctanesulfonic acid induce immunotoxicity through the NF-κB pathway in black-spotted frog (Rana nigromaculata).

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are widely detected in the environment and wild animals, thus posing a threat to wildlife and public health; however, knowledge about their immunotoxicity and the underlying mechanism remains limited. In the present study, male black-spotted frogs (Rana nigromaculata) were exposed to environmentally relevant concentrations (0, 1, and 10 µg/L) of PFOA or PFOS for 21 days; subsequently, biochemical analysis, molecular docking, and gene expression determination were conducted. The results indicated that exposure to 10 µg/L PFOA decreased the serum levels of immunoglobulin A. PFOS exposure significantly increased the hepatic levels of interleukin-1ß, interleukin-6, tumor necrosis factor-α, interferon-γ, and nitric oxide; but PFOA significantly increased the levels of only tumor necrosis factor-α. Furthermore, PFOA and PFOS exposure significantly decreased the activity of inducible nitric oxide synthase and total nitric oxide synthase. IBRv2 analysis indicated that PFOA and PFOS had a similar effect on these immune indicators, but PFOS was more toxic than PFOA. Molecular docking revealed that PFOA and PFOS can bind to nuclear factor-κB (NF-κB) by forming stable hydrogen bonds. PFOA and PFOS exposure upregulated the gene expression of NF-κB and its downstream genes. Significant correlations between the expression of genes involved in the NF-κB pathway and immune-related indicators suggests that PFOA- and PFOS-induced immunotoxicity was associated with the activation of NF-κB. Our findings provide novel insights into the potential role of NF-κB in immunotoxicity induced by PFOA and PFOS in frogs.

The complete mitochondrial genome of Hylarana latouchii (Anura: Ranidae) and its phylogenetic analysis.

We reported the complete mitochondrial genome (mitogenome) of broad-folded frog (Hylarana latouchii). This mitogenome is 17,007 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding sequence (D-loop). The total composition was 58.54% A + T and 41.46% G + C (T: 29.31%, C: 27.33%, A: 29.23%, and G: 14.13%). The phylogenetic analysis revealed that H. latouchii formed a clade with other two species of genus Hylarana. This mitogenomic sequence of H. latouchii provides useful data to study its population genetics and phylogeography.

[Clinical efficacy of arthroscopic simultaneous treatment for anterior cruciate ligament injury combined with meniscus bucket-handle tear].

Objective: To explore the clinical efficacy of arthroscopic simultaneous both anterior cruciate ligament (ACL) reconstruction and suture of the meniscus bucket-handle tear (BHT). Methods: Between January 2013 and April 2014, 22 patients (22 knees) with ACL injury and BHT, who accorded with the inclusion criteria, were studied. There were 14 males and 8 females with a mean age of 30.68 years (range, 15-44 years). The left side was involved in 10 cases and the right side in 12 cases. Injury located at the medial meniscus in 14 patients, and at the lateral meniscus in 8 patients. The median of interval from injury to operation was 40 days (range, 9 hours to 4 years). BHT was sutured, and then single bundle reconstruction of ACL was performed under arthroscopy. Results: All incisions healed by first intention, and there were no serious complications such as infection, vascular injury, and nerve injury. The patients were followed up for 26.7 months on average (range, 12-42 months). At 6 weeks after operation, one patient had limited motion of the knee, the function was recovered after release under anesthesia; and one patient had joint space tenderness, which was relieved after conservative treatment. The total effective rate was 90.9% (20/22). At last follow-up, the anterior drawer test, Lachman test, and McMurray test were negative in all the cases. The visual analogue scale (VAS), Tegner activity level score, and Lysholm score were significantly improved at 12 months after operation when compared with preoperative scores ( P<0.05). At 6-12 months after operation, complete healing was obtained in 7 cases, and partial healing in 11 cases, and nonunion in 4 cases based on MRI evaluation criteria by Crues et al. There was no rupture of reconstruc-tive ligament during follow-up. Conclusion: Arthroscopic simultaneous both ACL reconstruction and suture of BHT can improve the symptoms, reduce the risk of re-tear of sutured meniscus effectively, delay degeneration of articular cartilage, and maintain the stability of the knee joint.

Protease-Activated Receptor-2 Deficiency Attenuates Atherosclerotic Lesion Progression and Instability in Apolipoprotein E-Deficient Mice.

Inflammatory mechanisms are involved in the process of atherosclerotic plaque formation and rupture. Accumulating evidence suggests that protease-activated receptor (PAR)-2 contributes to the pathophysiology of chronic inflammation on the vasculature. To directly examine the role of PAR-2 in atherosclerosis, we generated apolipoprotein E/PAR-2 double-deficient mice. Mice were fed with high-fat diet for 12 weeks starting at ages of 6 weeks. PAR-2 deficiency attenuated atherosclerotic lesion progression with reduced total lesion area, reduced percentage of stenosis and reduced total necrotic core area. PAR-2 deficiency increased fibrous cap thickness and collagen content of plaque. Moreover, PAR-2 deficiency decreased smooth muscle cell content, macrophage accumulation, matrix metallopeptidase-9 expression and neovascularization in plaque. Relative quantitative PCR assay using thoracic aorta revealed that PAR-2 deficiency reduced mRNA expression of inflammatory molecules, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. In vitro experiment, we found that PAR-2 deficiency reduced mRNA expression of interferon-γ, interleukin-6, TNF-α and MCP-1 in macrophage under unstimulated and lipopolysaccharide-stimulated conditions. These results suggest that PAR-2 deficiency attenuates the progression and instability of atherosclerotic plaque.

Factor Xa induces pro-inflammatory cytokine expression in RAW 264.7 macrophages via protease-activated receptor-2 activation.

Coagulation proteases have been suggested to trigger a diversity of inflammatory responses in addition to their critical role in the coagulation cascade. It has been well established that the inflammatory and coagulation pathways are invariably linked. However, the mechanisms through which coagulation protease factor Xa (FXa) causes inflammation remain unclear. Thus, we assessed the pro-inflammatory effects of FXa in RAW 264.7 macrophages. We show that FXa elicits signal transduction in RAW 264.7 macrophages. FXa-induced signal transduction was dependent on the activation of protease-activated receptor 2 (PAR-2), PAR-2 desensitization but not PAR-1 desensitization abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to the expression of pro-inflammatory cytokines IL-6, IL-8, TNF-α and IFN-γ in RAW 264.7 macrophages. Furthermore, a specific inhibitor of the ERK1/2 pathway, U0126, decreased the FXa-induced pro-inflammatory cytokines expression significantly. Taken together, our data indicate that FXa induces PAR-2-dependent pro-inflammatory activity in RAW 264.7 macrophages through the ERK1/2 pathway.