The novel hydrolase IpcH initiates the degradation of isoprocarb in a newly isolated strain Rhodococcus sp. D-6.

Isoprocarb (IPC), a representative monocyclic carbamate insecticide, poses risks of environmental contamination and harm to non-target organisms. However, its degradation mechanism has not been reported. In this study, a newly IPC-degrading strain D-6 was isolated from the genus Rhodococcus, and its degradation characteristics and pathway of IPC were analyzed. A novel hydrolase IpcH, responsible for hydrolyzing IPC to 2-isopropylphenol (IPP), was identified. IpcH exhibited low similarity (< 27 %) with other reported hydrolases, including previously characterized carbamate insecticides hydrolases, indicating its novelty. The Km and kcat values of IpcH towards IPC were 69.99 ± 8.33 µM and 95.96 ± 4.02 s-1, respectively. Also, IpcH exhibited catalytic activity towards various types of carbamate insecticides, including monocyclic carbamates (IPC, fenobucarb and propoxur), bicyclic carbamates (carbaryl and carbofuran), and linear carbamates (oxamyl and aldicarb). The molecular docking and site-directed mutagenesis revealed that His254, His256, His329 and His376 were essential for IpcH activity. Strain D-6 can effectively reduce the toxicity of IPC and IPP towards sensitive organisms through its degradation ability. This study presents the initial report on IPC degradation pathway and molecular mechanism of IPC degradation, and provides a good potential strain for bioremediating IPC and IPP-contaminated environments.

Functional analysis, diversity, and distribution of the ean cluster responsible for 17ß-estradiol degradation in sphingomonads.

17ß-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2.IMPORTANCESteroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA, eanB1, eanC, and eanD) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.

The mechanism of lncRNA SNHG1 in osteogenic differentiation via miR-497-5p/ HIF1AN axis.

The pivotal role of lncRNAs in osteoporosis progression and development necessitates a comprehensive exploration of the functional and precise molecular mechanisms underlying lncRNA SNHG1's regulation of osteoblast differentiation and calcification. The study involved inducing BMSCs cells to differentiate into osteoblasts, followed by transfections of miR-497-5p inhibitors, pcDNA3.1-SNHG1, sh-HIF1AN, miR-497-5p mimics, and respective negative controls into BMSCs. Quantitative PCR (qPCR) was employed to assess the expression of SNHG1 and miR-497-5p. Western Blotting was conducted to measure the levels of short stature-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and HIF1AN. Alkaline phosphatase (ALP) activity was determined using appropriate assay kits. Calcium nodule staining was performed through Alizarin red staining. Dual luciferase reporter gene assays were executed to validate the interaction between SNHG1 and miR-497-5p, as well as HIF1AN. Throughout osteogenic differentiation, there was a down-regulation of SNHG1 and HIF1AN, in contrast to an elevation in miR-497-5p levels. Direct interactions between miR-497-5p and both SNHG1 and HIF1AN were observed. Notably, SNHG1 exhibited the ability to modulate HIF1AN by influencing miR-497-5p, thereby inhibiting osteogenic differentiation. Functioning as a competitive endogenous RNA, lncRNA SNHG1 exerts an inhibitory influence on osteogenic differentiation via the miR-497-5p/HIF1AN axis. This highlights the potential for lncRNA SNHG1 to emerge as a promising therapeutic target for osteoporosis. The study's findings pave the way for a novel target strategy in the future treatment of osteoporosis.

Long Non-coding RNA SNHG1 Suppresses the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Binding with HMGB1.

Osteoporosis (OP) has a significant detrimental impact on the health of the elder. Long-term clinical effectiveness of current drugs used for OP treatment is limited. Therefore, it is very important to explore novel treatment targets for OP. The expression of SNHG1, HMGB1, OCN and OPN in gene level was measured using RT-qPCR, and the protein expression was determined by Western blotting assay. The concentration of IL-1ß and IL-18 in supernatant of the bone marrow mesenchymal stem cells (BMSCs) was measured by ELISA. The interaction between SNHG1 and HMGB1 was confirmed by RNA pull down. Besides, alizarin red staining was performed to evaluate the differentiation of BMSCs into osteoblast. SNHG1 and HMGB1 were found to be upregulated in the serum of OP patients. During the osteogenic differentiation of BMSCs, the expression of osteoblastogenesis markers (OCN and OPN) and the activity of ALP were upregulated, while the expression levels of SNHG1 and HMGB1 were decreased in a time-dependent manner. In addition, the interaction between SNHG1 and HMGB1, expression of pyroptosis-associated factors (caspase-1 p20 and GSDMD-N), and secretion of IL-1ß and IL-18 were also decreased during osteogenic differentiation. Interestingly, increasing SNHG1 promoted HMGB1 expression, activated pyroptosis, but inhibited osteogenic differentiation. Silencing HMGB1 or inhibiting caspase-1 partially rescued the inhibitory effect of SNHG1 on osteogenic differentiation. Our findings indicate that SNHG1 suppresses the osteogenic differentiation of BMSCs by activating pyroptosis through interaction with HMGB1 and promotion of HMGB1 expression. Our work provides further evidence supporting SNHG1 acts as a potential target for OP treatment, and reveals for the first time that SNHG1 regulates osteogenic differentiation by affecting pyroptosis.

Cloning and expression of the phenazine-1-carboxamide hydrolysis gene pzcH and the identification of the key amino acids necessary for its activity.

Phenazine-1-carboxamide (PCN), a phenazine derivative, can cause toxicity risks to non target organisms. In this study, the Gram-positive bacteria Rhodococcus equi WH99 was found to have the ability to degrade PCN. PzcH, a novel amidase belonging to amidase signature (AS) family, responsible for hydrolyzing PCN to PCA was identified from strain WH99. PzcH shared no similarity with amidase PcnH which can also hydrolyze PCN and belong to the isochorismatase superfamily from Gram-negative bacteria Sphingomonas histidinilytica DS-9. PzcH also showed low similarity (˂ 39%) with other reported amidases. The optimal catalysis temperature and pH of PzcH was 30 °C and 9.0, respectively. The Km and kcat values of PzcH for PCN were 43.52 ± 4.82 µM and 17.028 ± 0.57 s-1, respectively. The molecular docking and point mutation experiment demonstrated that catalytic triad Lys80-Ser155-Ser179 are essential for PzcH to hydrolyze PCN. Strain WH99 can degrade PCN and PCA to reduce their toxicity against the sensitive organisms. This study enhances our understanding of the molecular mechanism of PCN degradation, presents the first report on the key amino acids in PzcH from the Gram-positive bacteria and provides an effective strain in the bioremediation PCN and PCA contaminated environments.

PcaR, a GntR/FadR Family Transcriptional Repressor Controls the Transcription of Phenazine-1-Carboxylic Acid 1,2-Dioxygenase Gene Cluster in Sphingomonas histidinilytica DS-9.

In our previous study, the phenazine-1-carboxylic acid (PCA) 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster) in Sphingomonas histidinilytica DS-9 was identified to be responsible for the conversion of PCA to 1,2-dihydroxyphenazine (Ren Y, Zhang M, Gao S, Zhu Q, et al. 2022. Appl Environ Microbiol 88:e00543-22). However, the regulatory mechanism of the pcaA1A2A3A4 cluster has not been elucidated yet. In this study, the pcaA1A2A3A4 cluster was found to be transcribed as two divergent operons: pcaA3-ORF5205 (named A3-5205 operon) and pcaA1A2-ORF5208-pcaA4-ORF5210 (named A1-5210 operon). The promoter regions of the two operons were overlapped. PcaR acts as a transcriptional repressor of the pcaA1A2A3A4 cluster, and it belongs to GntR/FadR family transcriptional regulator. Gene disruption of pcaR can shorten the lag phase of PCA degradation. The results of electrophoretic mobility shift assay and DNase I footprinting showed that PcaR binds to a 25-bp motif in the ORF5205-pcaA1 intergenic promoter region to regulate the expression of two operons. The 25-bp motif covers the -10 region of the promoter of A3-5205 operon and the -35 region and -10 region of the promoter of A1-5210 operon. The TNGT/ANCNA box within the motif was essential for PcaR binding to the two promoters. PCA acted as an effector of PcaR, preventing it from binding to the promoter region and repressing the transcription of the pcaA1A2A3A4 cluster. In addition, PcaR represses its own transcription, and this repression can be relieved by PCA. This study reveals the regulatory mechanism of PCA degradation in strain DS-9, and the identification of PcaR increases the variety of regulatory model of the GntR/FadR-type regulator. IMPORTANCE Sphingomonas histidinilytica DS-9 is a phenazine-1-carboxylic acid (PCA)-degrading strain. The 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster, encoding dioxygenase PcaA1A2, reductase PcaA3, and ferredoxin PcaA4) is responsible for the initial degradation step of PCA and widely distributed in Sphingomonads, but its regulatory mechanism has not been investigated yet. In this study, a GntR/FadR-type transcriptional regulator PcaR repressing the transcription of pcaA1A2A3A4 cluster and pcaR gene was identified and characterized. The binding site of PcaR in ORF5205-pcaA1 intergenic promoter region contains a TNGT/ANCNA box, which is important for the binding. These findings enhance our understanding of the molecular mechanism of PCA degradation.

[Study on correlation of JAK/STAT signal pathway with progression and prognosis in hepatocellular carcinoma].

AIM: To investigate the expression of JAK/STAT signal pathway in human hepatocellular carcinoma, and to evaluate its clinical significance in the progression and prognosis in hepatocellular carcinoma. METHODS: 196 patients with hepatocellular carcinoma were examined for the expression of JAK-1 protein and STAT-3 protein by SABC immunohistochemistry. RESULTS: The positive expression rates of JAK-1 protein and STAT-3 protein in patients with hepatocellular carcinoma were significantly higher than those in 20 cases of normal liver tissue (P=0.02 and 0.01, respectively). There was no significant relation between the positive expression rates of JAK-1 protein and STAT-3 protein and patients'sex, age, tumor size, and cirrhosis of the hepatocellular carcinoma tissues (all P>0.05). The JAK-1 protein and STAT-3 protein were expressed more frequently in hepatocellular carcinoma tissues with incomplete capsule (P=0.01 and 0.008, respectively), venous tumor emboli (both P=0.02), poorly differentiated (P=0.01 and 0.009, respectively) or clinical III-IV stage (P=0.02 and 0.008, respectively) than in those with complete capsule, no venous tumor emboli, maturely differentiated or clinical I-II stage. Cox proportional hazard regression model analysis indicated that the expression of JAK-1 protein and STAT-3 protein was significantly correlated with the prognosis of patients with hepatocellular carcinoma. CONCLUSION: The results suggest that the over-expression of JAK/STAT signal pathway may be an important feature of hepatocellular carcinoma.