Identification of crucial lncRNAs and mRNAs in liver regeneration after portal vein ligation through weighted gene correlation network analysis.

BACKGROUND: Portal vein ligation (PVL)-induced liver hypertrophy increases future liver remnant (FLR) volume and improves resectability of large hepatic carcinoma. However, the molecular mechanism by which PVL facilitates liver hypertrophy remains poorly understood. METHODS: To gain mechanistic insight, we established a rat PVL model and carried out a comprehensive transcriptome analyses of hepatic lobes preserving portal blood supply at 0, 1, 7, and 14-day after PVL. The differentially expressed (DE) long-non coding RNAs (lncRNAs) and mRNAs were applied to conduct weighted gene co-expression network analysis (WGCNA). LncRNA-mRNA co-expression network was constructed in the most significant module. The modules and genes associated with PVL-induced liver hypertrophy were assessed through quantitative real-time PCR. RESULTS: A total of 4213 DElncRNAs and 6809 DEmRNAs probesets, identified by transcriptome analyses, were used to carry out WGCNA, by which 10 modules were generated. The largest and most significant module (marked in black_M6) was selected for further analysis. Gene Ontology (GO) analysis of the module exhibited several key biological processes associated with liver regeneration such as complement activation, IL-6 production, Wnt signaling pathway, autophagy, etc. Sixteen mRNAs (Notch1, Grb2, IL-4, Cops4, Stxbp1, Khdrbs2, Hdac2, Gnb3, Gng10, Tlr2, Sod1, Gosr2, Rbbp5, Map3k3, Golga2, and Rev3l) and ten lncRNAs (BC092620, AB190508, EF076772, BC088302, BC158675, BC100646, BC089934, L20987, BC091187, and M23890) were identified as hub genes in accordance with gene significance value, module membership value, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network. Furthermore, the overexpression of 3 mRNAs (Notch1, Grb2 and IL-4) and 4 lncRNAs (BC089934, EF076772, BC092620, and BC088302) was validated in hypertrophic liver lobe tissues from PVL rats and patients undergoing hepatectomy after portal vein embolization (PVE). CONCLUSIONS: Microarray and WGCNA analysis revealed that the 3 mRNAs (Notch1, Grb2 and IL-4) and the 4 lncRNAs (BC089934, EF076772, BC092620 and BC088302) may be promising targets for accelerating liver regeneration before extensive hepatectomy.

Structural insight and engineering of a plastic degrading hydrolase Ple629.

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.

Exogenous melatonin improves cadmium tolerance in Solanum nigrum L. without affecting its remediation potential.

Although Solanum nigrum L. is a phytoremediator for different metals, its growth and physiology are still influenced by toxic levels of cadmium (Cd). Thus, the development of eco-friendly strategies to enhance its tolerance, maintaining remediation potential is of special interest. The present work aimed to evaluate the effects of exogenous application of melatonin (MT) in the physiological and biochemical responses of S. nigrum and remediation potential exposed to Cd. After 30 days of exposure, the results revealed that Cd-mediated inhibitory effects on biomass and photosynthetic pigment synthesis were efficiently mitigated upon application of melatonin, without affecting Cd accumulation. Higher levels of Cd were found in roots, regardless of the pretreatment with the melatonin. Foliar application of melatonin, however, induced distinctive effects, lowering malondialdehyde (MDA), relative electrical conductivity (REL), and proline levels in shoots. These changes contributed to improvements in the water status, photosynthetic pigment synthesis, and biomass production of S. nigrum under Cd stresses. Overall, our results indicate a protective effect of melatonin on S. nigrum response to excess Cd, contributing to a better tolerance and growth rate, without disturbing its phytoremediation potential.Novelty statementAlthough Solanum nigrum L. is a phytoremediator for different metals, its growth and physiology are still influenced by toxic levels of cadmium. This study evaluated the potential of melatonin to boost S. nigrum defence against Cd toward a better growth rate and remediation potential.

Transcriptome analysis of immune-related gene expression in Yellow River carp (Cyprinus carpio var.) after challenge with Flavobacterium columnare.

Yellow River carp (Cyprinus carpio) is an economically-important freshwater fish. It is the common host of the epizootic bacterium Flavobacterium columnare, a common fish pathogen that causes columnaris disease resulting in aquacultural losses. However, information on the functions and mechanisms of the immune system of Yellow River carp infected with F. columnare is limited. Therefore, the aim of this study is to evaluate the genetic and histopathological effects of an experimentally-induced F. columnare infection in Yellow River carp. Sixty fish were divided into control (CT group) and challenged groups. The gills were collected for histological and transcriptome analysis to understand the host immune response after challenge with F. columnare. The infected fish of the IF group presented typical columnaris disease symptoms and higher mortality, as well as histological changes. However, some challenged fish showed asymptomatic infection (IC group). Additionally, there were 1776 significant differentially-expressed genes (DEGs) between the IC and CT groups, 1853 DEGs between the IF and CT groups, and 1836 DEGs between the IF and IC groups, All the DEGs were classified into three gene ontology categories, which were allocated to 158 KEGG pathways. Moreover, immune-related genes were confirmed by qRT-PCR. we quantified the level of IL-1, IL-6, TNF-α and IL-8 by ELISA. The results showed the highest expression levels of inflammatory cytokines as well as stress proteins and the adhesion molecules in the lF group, which may contribute to severe infection, and a higher case fatality rate, while the high expression of chemokines, costimulatory molecules and the up regulation of antigen presentation function could help the carp resist F. columnare infection.

Thermal ablation with and without adjuvant systemic therapy: a nationwide multicenter observational cohort study of solitary colorectal liver metastases.

BACKGROUND: Thermal ablation is routinely used for solitary colorectal liver metastases (SCLM), but the added value of adjuvant systemic therapy in SCLM remains unclear. This study aimed to compare the long-term outcomes for SCLM treated by ablation alone (AB) versus ablation plus systemic therapy (AS). METHODS: This multicenter retrospective study using nationwide data from fourteen institutions between October 2010 and May 2023, 369 patients with initial SCLM smaller than 5 cm, no extrahepatic metastases, and colorectal cancer R0 resection treated by thermal ablation were included. The crude analysis was used to analyze eligible cases between the two groups. The propensity score matching (PSM) to control for potential confounders in each matched group. Subgroup analyses were performed to identify specific survival benefits. RESULTS: 61.2% (226/369) of eligible patients were treated with AS and 38.8% (143/369) with AB. During the median follow-up period of 8.8 years, 1-/3-/5-year DFS/OS rates did not differ between the two groups, when analyzed via PSM (P=0.52/0.08). Subgroup analysis revealed that AS was significantly associated with better OS than AB in patients with plasma CEA >5 ug/L (P=0.036), T (III-IV) category of primary cancer (P=0.034), or clinical risk score (1-2) (P=0.041). In each matched group, we did find a significant difference in drug-related adverse events (P<0.001) between AS group (24.1%, 28/116) and AB group (0.0%, 0/116). CONCLUSIONS: For patients with plasma CEA >5 ug/L, T (III-IV) category of primary cancer, or clinical risk score (1-2), thermal ablation plus systemic therapy appeared to be associated with improved overall survival. Thermal ablation was equally effective in disease-free survival for treating solitary colorectal liver metastasis, whether with or without adjuvant systemic therapy.

Microwave ablation versus liver resection for primary intrahepatic cholangiocarcinoma within Milan criteria: a long-term multicenter cohort study.

Background: Ablation has been recommended by worldwide guidelines as first-line treatment for hepatocellular carcinoma (HCC), while evidence regarding its efficacy for primary intrahepatic cholangiocarcinoma (iCCA) is lacking. We aimed to study the efficacy of ablation in treating iCCA by comparing its prognosis with surgery. Methods: In this real-world multicenter cohort study from January 2009 to June 2022, 10,441 iCCA patients from ten tertiary hospitals were identified. Patients who underwent curative-intent microwave ablation (MWA) or liver resection (LR) for tumors within Milan criteria were included. One-to-many propensity score matching (PSM) at variable ratios (1:n ≤4) was used to balance baseline characteristics. Mediation analysis was applied to identify potential mediators of the survival difference. Findings: 944 patients were finally enrolled in this study, with 221 undergoing MWA and 723 undergoing LR. After PSM, 203 patients in the MWA group were matched with 588 patients in the LR group. The median follow-up time was 4.7 years. Compared with LR, MWA demonstrated similar overall survival (5-year 44.8% versus 40.4%; HR 0.96, 95% CI 0.71-1.29, P = .761). There was an improvement in the 5-year disease-free survival rate for MWA from 17.1% during the period of 2009-2016 to 37.3% during 2017-2022, becoming comparable to the 40.8% of LR (P = .129). The proportion of ablative margins ≥5 mm increased from 25% to 61% over the two periods, while this proportion of surgical margins was 62% and 77%, respectively. 34.5% of DFS disparity can be explained by the mediation effect of margins (P < .0001). Similar DFS was observed when both ablative and surgical margins exceeded 5 mm (HR 0.83, 95% CI 0.52-1.32, P = .41). Interpretation: MWA may be considered as a viable alternative to LR for iCCA within Milan criteria when an adequate margin can be obtained. Funding: National Natural Science Foundation of China.

Surgical treatment of hilar cholangiocarcinoma: retrospective analysis.

BACKGROUND: Achieving a better prognosis for patients and reducing the risk of complications are primary considerations in surgical decisions for hilar cholangiocarcinoma. METHODS: A retrospective analysis of the authors' clinical practice outcomes in the surgical management of patients with hilar cholangiocarcinoma following the planned-hepatectomy surgical treatment programme between 2009 and 2018. RESULTS: Some 473 patients were included, of whom 127 (26.8 per cent) underwent bile duct tumour resection alone, 44 (9.3 per cent) underwent bile duct tumour resection combined with restrictive hepatectomy, and 302 (63.8 per cent) underwent bile duct tumour resection combined with extensive hepatectomy. R0 resection was achieved in 82.2 per cent and the postoperative complication rate was similar between the different operations. The 5-year survival rates after surgery were 37.0, 37.3, and 28.4 per cent in the bile duct tumour resection alone, restrictive hepatectomy, and extensive hepatectomy groups respectively, with no statistically significant differences. As TNM staging progressed, the 1-5-year cumulative survival rate for the patients in the three groups showed a significant downward trend. CONCLUSION: In the setting of a high-volume centre, a planned-hepatectomy surgical treatment programme helps to strike a better balance between achieving radical tumour resection for hilar cholangiocarcinoma and reasonable control of the extent of surgical damage.

[Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability].

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.

Insight into the Vacuolar Compartmentalization Process and the Effect Glutathione Regulation to This Process in the Hyperaccumulator Plant Solanum nigrum L.

Vacuole compartmentalization plays an important role in the storage of heavy metals in hyperaccumulators. Is the vacuolar compartmentation a simple shielding process or a dynamic process that continuously consumes cell sap resources? How does glutathione affect the process of vacuolar compartmentalization? These unknown questions are very important to understand the mechanism of vacuole compartmentalization and can provide a guide for the design of hyperaccumulator plants by genetic engineering. Therefore, this study explored the enzyme activities, total cadmium, Cd2+, glutathione, oxidized glutathione, and reactive oxygen species contents in protoplasts and vacuoles of leaf cells in Solanum nigrum L. through subcellular separation. The results showed that vacuolar compartmentalization was a dynamic process that actively induced the related substances produced by cell sap to enter the vacuole for detoxification. When regulating the decreased glutathione content with buthionine sulfoximine, the total cadmium and combined cadmium in protoplasm decreased significantly, but the vacuole still maintained a high proportion of cadmium content and stable ROS content, which indicated that various external resources were preferentially used to maintain cadmium storage and homeostasis in vacuole rather than outside vacuole. These findings could guide the use of genetic engineering to design hyperaccumulator plants.

Phytoremediation of cadmium-contaminated soils by Solanum nigrum L. enhanced with biodegradable chelating agents.

The application of biodegradable chelating agents to enhance phytoremediation is a low-cost and promising method to improve the remediation efficiency of heavy metal-contaminated soil. The effects of N, N-bis glutamic acid (GLDA) on the growth and heavy metal absorption of Solanum nigrum were studied by pot experiment. The addition of chelate on the 20th day after sowing can improve the bioavailability of cadmium (Cd) in the soil. The results showed that the addition of chelating agents effectively improved the migration rate of the target heavy metal Cd in the soil, and significantly increased the accumulation of heavy metal in the roots, stems, and leaves of plants. The results showed that compared with the control group, the chelating agent could increase the extraction rate of total Cd by 28.65-68.74%. The application of GLDA significantly increased the accumulation of Cd (20 mg kg-1 and 40 mg kg-1), reaching 24.28-40.30 and 25.71-33.16 µg of pot-1 DW, respectively. At the same time, GLDA increased Cd stress by decreasing plant biomass, inhibiting photosynthetic pigment synthesis and increasing MDA levels. These results indicated that GLDA could improve the absorption of Cd by S. nigrum, which provided a new idea for its practical application in the remediation of Cd-contaminated soil.

Cortical pain induced by optogenetic cortical spreading depression: from whole brain activity mapping.

BACKGROUND: Cortical spreading depression (CSD) is an electrophysiological event underlying migraine aura. Traditional CSD models are invasive and often cause injuries. The aim of the study was to establish a minimally invasive optogenetic CSD model and identify the active networks after CSD using whole-brain activity mapping. METHODS: CSD was induced in mice by light illumination, and their periorbital thresholds and behaviours in the open field, elevated plus-maze and light-aversion were recorded. Using c-fos, we mapped the brain activity after CSD. The whole brain was imaged, reconstructed and analyzed using the Volumetric Imaging with Synchronized on-the-fly-scan and Readout technique. To ensure the accuracy of the results, the immunofluorescence staining method was used to verify the imaging results. RESULTS: The optogenetic CSD model showed significantly decreased periorbital thresholds, increased facial grooming and freezing behaviours and prominent light-aversion behaviours. Brain activity mapping revealed that the somatosensory, primary sensory, olfactory, basal ganglia and default mode networks were activated. However, the thalamus and trigeminal nucleus caudalis were not activated. CONCLUSIONS: Optogenetic CSD model could mimic the behaviours of headache and photophobia. Moreover, the optogenetic CSD could activate multiple sensory cortical regions without the thalamus or trigeminal nucleus caudalis to induce cortical pain.

Molecular and Biochemical Differences of the Tandem and Cold-Adapted PET Hydrolases Ple628 and Ple629, Isolated From a Marine Microbial Consortium.

Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, α/ß hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase.

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

A Fast Linear Neighborhood Similarity-Based Network Link Inference Method to Predict MicroRNA-Disease Associations.

Increasing evidences revealed that microRNAs (miRNAs) play critical roles in important biological processes. The identification of disease-related miRNAs is critical to understand the molecular mechanisms of human diseases. Most existing computational methods require diverse features to predict miRNA-disease associations. However, diverse features are not available for all miRNAs or diseases. In addition, most methods can't predict links for miRNAs or diseases without association information. In this paper, we propose a fast linear neighborhood similarity-based network link inference method, named FLNSNLI, to predict miRNA-disease associations. First, known miRNA-disease associations are formulated as a bipartite network, and miRNAs (or diseases) are expressed as association profiles. Second, miRNA-miRNA similarity and disease-disease similarity are calculated by fast linear neighborhood similarity measure and association profiles. Third, the label propagation algorithm is respectively implemented on two sides to score candidate miRNA-disease associations. Finally, FLNSNLI adopts the weighted average strategy and makes predictions. Moreover, we develop a link complementing approach, and extend FLNSNLI to predict links for miRNAs (or diseases) without known associations. In computational experiments, FLNSNLI produces high-accuracy performances, and outperforms other state-of-the-art methods. More importantly, FLNSNLI requires less information but performs well. Case studies on three popular diseases show that FLNSNLI is useful for the microRNA-disease association prediction.

Visualization and quantification of cadmium accumulation, chelation and antioxidation during the process of vacuolar compartmentalization in the hyperaccumulator plant Solanum nigrum L.

Hyperaccumulators store metals in the vacuoles of leaf cells. To investigate the role of vacuolar compartmentalization in Cd accumulation, chelation and induced antioxidation, we quantified the amounts of total cadmium (Cd), Cd2+, glutathione (GSH) and reactive oxygen species (ROS) in leaf cells of Solanum nigrum L. The results confirmed that vacuoles were, indeed, the main storage compartments for Cd. We then found that with increased Cd treatment concentration, the proportion of vacuolar Cd in protoplasts showed its ultimate storage capacity (82.24 %-83.40 %), and the Cd concentration stored in the protoplast maintained at a certain level (73.81-77.46 mg L-1). Besides, studies on different forms of Cd showed that the chelation state was dominant in the protoplast. The large level appearance of Cd2+ outside the vacuole revealed the limitations of vacuolar Cd2+ sequestration. The relationships between the combined forms of Cd and GSH outside the vacuole (R2 = 0.9906) showed GSH was mainly distributed to important compartments for chelation, not to vacuoles. We also demonstrated the presence of ROS-induced oxidative stress and detoxification mediated by the antioxidant GSH in vacuoles, suggesting that sequestration into vacuoles is an active process accompanied by chelation and antioxidant-mediated detoxification.