HIF-1-mediated regulation of LDH gene unravels key insights into MCDV-1 pathogenesis in mud crabs Scylla paramamosain.

Hypoxia-inducible factors -1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α, targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α-silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction.

Enhanced Alkaline Oxygen Evolution Using Spin Polarization and Magnetic Heating Effects under an AC Magnetic Field.

Renewable electricity from splitting water to produce hydrogen is a favorable technology to achieve carbon neutrality, but slow anodic oxygen evolution reaction (OER) kinetics limits its large-scale commercialization. Electron spin polarization and increasing the reaction temperature are considered as potential ways to promote alkaline OER. Here, it is reported that in the alkaline OER process under an AC magnetic field, a ferromagnetic ordered electrocatalyst can simultaneously act as a heater and a spin polarizer to achieve significant OER enhancement at a low current density. Moreover, its effect obviously precedes antiferromagnetic, ferrimagnetic, and diamagnetic electrocatalysts. In particular, the noncorrected overpotential of the ferromagnetic electrocatalyst Co at 10 mA cm-2 is reduced by a maximum of 36.6% to 243 mV at 4.320 mT. It is found that the magnetic heating effect is immediate, and more importantly, it is localized and hardly affects the temperature of the entire electrolytic cell. In addition, the spin pinning effect established on the ferromagnetic/paramagnetic interface generated during the reconstruction of the ferromagnetic electrocatalyst expands the ferromagnetic order of the paramagnetic layer. Also, the introduction of an external magnetic field further increases the orderly arrangement of spins, thereby promoting OER. This work provides a reference for the design of high-performance OER electrocatalysts under a magnetic field.

Characterization of phosphofructokinase (PFK) from mud crab Scylla paramamosain and its role in mud crab dicistrovirus-1 proliferation.

Phosphofructokinase (PFK), the key enzyme of glycolysis, can catalyze the irreversible transphosphorylation of fructose-6-phosphate forming fructose-1, 6-biphosphate. In the present study, a PFK gene from the mud crab Scylla paramamosain, named SpPFK, was cloned and characterized. The full length of SpPFK contained a 5'untranslated region (UTR) of 249 bp, an open reading frame of 2,859 bp, and a 3'UTR of 1,248 bp. The mRNA of SpPFK was highly expressed in the gill, followed by the hemocytes and muscle. The expression of SpPFK was significantly up-regulated after mud crab dicistrovirus-1 (MCDV-1) infection. Knocking down SpPFK in vivo by RNA interference significantly reduced the expression of lactate dehydrogenase after MCDV-1 infection. Furthermore, silencing of SpPFK in vivo increased the survival rate of mud crabs and decreased the MCDV-1 copies in the gill and hepatopancreas after MCDV-1 infection. All these results suggested that SpPFK could play an important role in the process of MCDV-1 proliferation in mud crab.

[Prediction of Response to Neoadjuvant Chemotherapy for Breast Cancer Based on Histomorphology Analysis of Needle Biopsy Images].

OBJECTIVE: The deep learning method was used to automatically segment the tumor area and the cell nucleus based on needle biopsy images of breast cancer patients prior to receiving neoadjuvant chemotherapy (NAC), and then, the features of the cell clusters in the tumor area were identified to predict the level of pathological remission of breast cancer after NAC. METHODS: 68 breast cancer patients who were to receive NAC at Jiangsu Province Hospital were recruited and the hematoxylin-eosin (HE) stained preoperative biopsy sections of these patients were collected. Unet++ was used to establish a segmentation model and the tumor area and nucleus of the needle biopsy images were automatically segmented accordingly. Then, according to the nuclei in the automatically segmented tumor area, the features of the cells in the tumor were constructed. After that, effective features were selected through the feature selection method and the classifier model was constructed and trained with five-fold cross validation to predict the degree of post-NAC pathological remission. RESULTS: Predictions were made based on the needle biopsy images of the 68 patients. The model that combined the 10-dimensional features selected with the minimal redundancy-maximum-relevancy approach (mRMR) and training with the random forest (RF) classifier had the highest prediction accuracy, reaching 82.35%, and an area under curve ( AUC) value of 0.908 2. CONCLUSION: This model automatically segments tumor areas and cell nucleus on the biopsy images. The features of the cell clusters which are analyzed and identified in the tumor area can be used to predict the pathological response of the patient to NAC. The method is reliable and replicable. In addition, we found that the textural features of cells in the tumor area was a useful predictor of patient response to NAC, which further confirmed that cell cluster in the tumor area is of great significance to the prediction of treatment outcome.

[Characteristics of ANAMMOX Granular Sludge and Differences in Microbial Community Structure Under Different Culture Conditions].

Anaerobic ammonium oxidation (ANAMMOX) granular sludge was cultured during different operating conditions by an expanded granular sludge bed (EGSB) reactor and up-flow anaerobic sludge bed (UASB) reactors, and the characteristics of the granular sludge and microbial community were compared. The results showed that the flocculent ANAMMOX sludge can be granulated after being operated for 384 days by the EGSB and UASB reactors. The average particle size reached 1.17 mm and 1.21 mm, respectively. The particle size ratio of each range (<0.2, 0.2-1.5, 1.5-3, and>3 mm) was 6.06%, 60.05%, 25.25%, and 8.64% in the EGSB reactor, and 7.40%, 58.90%, 32.04%, and 1.66% in the UASB reactor, respectively. The results of scanning electron microscopy showed that the bacterial flora during different operating conditions were mainly Brevibacterium and Cocci aggregates. High-throughput sequencing results showed that the Shannon index of the EGSB reactor was 7.52, higher than the 7.18 of the UASB reactor on day 384; Proteobacteria was the main phylum of the sludge at each stage, and Planctomycetes increased from 3.30% to 12.30% in the EGSB reactor and 13.30% in the UASB reactor on day 384. The main ANAMMOX genera in the EGSB reactor were Candidatus Brocadia, accounting for 7.53%, followed by Candidatus Kuenenia accounting for 1.61%, whereas in the UASB reactor, Candidatus Kuenenia was the dominant anaerobic ammonia genus, accounting for 7.54%, followed by Candidatus Brocadia, which accounted for 3.69%. The proportion of dominant species was related to the change in environmental factors. The proportion of Candidatus Brocadia was positively correlated with the up-flow rate and nitrogen removal rate (NRR), but negatively correlated with hydraulic retention time (HRT). Candidatus Kuenenia was positively correlated with nitrogen removal efficiency (NRE), NRR, and HRT, but negatively correlated with the up-flow rate.

[Process Control and Operation Optimization of PN-SAD Coupling Process Based on SBR-ABR].

This study uses three different operating phases for a sequencing batch reactor (SBR) combined with an anaerobic baffled reactor (ABR) to determine the effect of deep nitrogen and carbon removal by the "partial nitrification-anaerobic ammonium oxidation combined denitrification" (termed PN-SAD) reaction. The effluent of the SBR (NO2--N/NH4+-N ratio range of 1-1.32) was accessed directly to the single compartment ABR anammox system in phase Ⅰ. The results showed that although the anammox reaction was stable, the combined process total nitrogen (TN) removal efficiency was<80%, and the TN concentration of effluent was~20 mg·L-1. In order to increase the denitrification function in the ABR, denitrifying sludge was added to the third compartment of the ABR in phase Ⅱ. We found that the TN removal efficiency of the coupling reaction was still low. An organic carbon source should be supplied in the latter stage of anammox if deep nitrogen removal is required. Therefore, in phase Ⅲ, the effluent of the SBR (NO2--N/NH4+-N ratio of ~5) was mixed with the partial raw water (mixed water NO2--N/NH4+-N ratio of ~1.4; C/N ratio of 2.5). The mixed water was connected to the single compartment of the ABR. The PN-SAD system not only achieved a good matrix ratio at the anammox stage, but also provided a good carbon source for denitrification. The chemical oxygen demand (COD) concentration of the effluent in the whole process was 50 mg·L-1, the TN concentration of the effluent was<6 mg·L-1, and the TN removal efficiency was 95%. We conclude that the stable operation of the combined PN-SAD reaction provides the basis for deep nitrogen and carbon removal using the combined SBR-ABR process.

Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

Ammonia toxicity in the mud crab (Scylla paramamosain): The mechanistic insight from physiology to transcriptome analysis.

Ammonia is a major aquatic environmental pollutants. However, the underlying molecular mechanism of ammonia-induced toxicity is not fully understood. In this study, we investigated the physiological response and molecular mechanism in mud crab (Scylla paramamosain) exposed to the acute total ammonia (30 mg L-1) for 48 h. The results shown that ammonia exposure induced oxidative stress, and subsequently led to cytological damage and DNA damage. Transcriptome analysis was applied to investigate the key genes and pathways involved in the responses to ammonia exposure. A total of 722 differentially expressed genes (DEGs) (526 up-regulated and 196 down-regulated) were identified. DEGs mainly involved in pathways including metabolism, cellular processes, signal transduction and immune functions. Additionally, transcriptome analysis revealed that ATM/p53-Caspase3 pathway involved in apoptosis induced by ammonia stress. These results provided a new insight into the mechanism of the potential toxic effects of ammonia on crustaceans.

4-Acetyl-1H-pyrrole-2-carbaldehyde.

The title compound, C(7)H(7)NO(2), was synthesized via a one-pot Vilsmeier-Haack and subsequent Friedel-Crafts reaction. The pyrazole ring makes dihedral angles of 4.50 (9) and 2.06 (8)°, respectively, with the aldehyde and acetyl groups. In the crystal, classical N-H⋯O hydrogen bonds and weak C-H⋯O inter-actions assemble the mol-ecules into a chain along the b axis.

[The effects of antisense oligonucleotides bcl-2/bcl-xl and bcl-2 on proliferation and apoptosis of breast cancer cells].

OBJECTIVE: To evaluate the effects of phosphorothioate antisense oligonucleotides (ASON) bcl-2/ bc-xl ASON and bcl-2 on the proliferation and apoptosis of breast cancer cells, MCF-7. METHODS: 1) bcl-2 ASON and bcl-2/bcl-xl ASON were transfected into MCF-7 cells with anionic long circulating liposomes (NA), cationic LCL (PA), respectively. 2) After incubation with bcl-2 ASON (FB1), bcl-2/bcl-xl ASON (FB2), NA loaded with bcl-2 ASON (NA-S) or bcl-2/bcl-xl ASON(NA-D), PA loaded with bcl-2 ASON(PA-S) or bcl-2/bc-xl ASON (PA-D) for 24 h, their inhibition on MCF-7 cells were evaluated by using HE staining, methythiazolyltetrazolium (MTT) and flow cytometry (FCM). RESULTS: The significant difference of nuclear condensation, chromatin fragmentation and apoptotic bodies in MCF-7 cells, typical of apoptotic cell death was observed in groups of bcl-2/ bcl-xl bispecific ASON by compared with that of bcl-2 ASON treatment. The fluorensence intensities of bcl-2 in groups of NA-D and NA-S, PA-S and PA-D, FB1 and FB2 were 1.92+/-0.08 and 2.83+/-0.16 (P=0.028); 4.20+/-0.18 and 2.85+/-0.57 (P=0.001); 5.70+/-1.16 and 4.35+/-0.11 (P=0.001), respectively. The cell survival rates at 24 h of NA-D and NA-S, PA-S and PA-D, FB1 and FB2 were (0.32+/-0.03)% and (0.58+/-0.07)% (P=0.014); (0.71+/-0.03)% and (0.45+/-0.04)% (P=0.014); (0.88+/-0.04)% and (0.57+/- 0.05)% (P=0.003), respectively. CONCLUSION: The bcl-2/bct-xl bispecific ASON could inhibit bcl-2 expression and induce apoptosis of breast cancer cells more efficiently than that treated with bcl-2 ASON alone.