TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

A validated high-performance liquid chromatography method for detecting geniposide, ellagic acid, piperine, costunolide and dehydrocostus lactone in Liuwei Muxiang capsules.

In this study, a sensitive high-performance liquid chromatography detector was established and validated for the simultaneous determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang Capsules. The analysis was achieved on CHANIN 100-5-C18-H column (5µm, 250 mm×4.6 mm) with the temperature of 30oC. Gradient elution was applied using 0.1% phosphoric acid solution-methanol-acetonitrile (50:50) as mobile phase at the flow rate of 1.0 mL/min. The determination was performed at the wavelength of 225 nm (detecting geniposide), 254 nm (detecting ellagic acid), 343 nm (detecting piperine) and 225 nm (detecting costunolide and dehydrocostuslactone) along with the sample volume of 10µL. The linear ranges of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone demonstrated good linear relationships within their respective determination ranges. The average recoveries were 100.04%, 99.86%, 99.79%, 100.17% and 100.41%, respectively. RSD% was 1.3%, 1.2%, 1.2%, 1.2%, 1.5%, respectively. The developed method was proved to be simple, accurate and sensitive, which can provide a quantitative analysis method for the content determination of geniposide, ellagic acid, piperine, costunolide and dehydrocostuslactone in Liuwei Muxiang capsules.

Molecular Correlates of Diapause in Aphidoletes aphidimyza.

The aphidophagous gall midge, Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae), a dominant natural enemy of aphids, has been used as a biological control agent in many countries to control aphids in greenhouses. To identify key factors that induce diapause in A. aphidimyza, we evaluated the effects of photoperiod and temperature on the incidence of diapause in A. aphidimyza under laboratory conditions. The results showed that temperature and photoperiod had significant impacts on development and diapause in A. aphidimyza. Low temperatures and a short photoperiod inhibited development, while high temperatures and a long photoperiod promoted development. Temperatures above 20 °C and a photoperiod greater than 14 h prevented diapause in A. aphidimyza. However, the highest diapause rate was recorded at under 15 °C and 10L:14D photoperiod conditions. At 15 °C, the first to third larvae were sensitive to a short photoperiod at any stage, and a short photoperiod had a cumulative effect on diapause induction. The longer the larvae received short light exposure, the higher the diapause rate appeared to be. Transcriptome sequencing analysis at different stages of diapause showed that differentially expressed genes were mainly enriched in the glucose metabolism pathway. Physiological and biochemical analyses showed that diapausing A. aphidimyza reduced water content; accumulated glycogen, trehalose, sorbitol, and triglycerides; and gradually reduced trehalose and triglyceride contents in the body with the extension of diapause time. Glycogen may be used as a source of energy, but sorbitol is usually used as a cryoprotectant. This study provided results on aspects of diapause in A. aphidimyza, providing data and theoretical support for promoting its commercial breeding and in-depth research on the molecular mechanisms underlying diapause regulation.

Bioaccumulation and transferreing for impacts on Cd and Pb by aphid consumption of the broad bean, Vicia faba L, in soil heavy metal pollution.

Heavy metal pollution threatens human and ecological health. Heavy metals can exist in the soil for a long time and migrate to organisms along the food chain. However, only a few studies have investigated the effects of a single stress on broad beans. Here, we aimed to characterize Cd and Pb bioaccumulation, at varying concentrations, in the broad bean, Vicia faba L. We also determined how the bioaccumulated metals are impacted by aphids that consume the plant. No significant difference was noted in the germination rates of broad beans at the early stage of planting (after 8 days), but eventually, the germination rates of broad beans at all time points first decreased and then increased, and the highest inhibition efficiency was observed in the T3 group (12.5 mg/L Cd2+ + 50 mg/L Pb2+). Fourteen days after planting, there was no significant difference in seedling height between the T5 (50 mg/L Cd2+ + 200 mg/L Pb2+) and control groups; however, that in the other groups decreased significantly and there was no dependence between stress concentration and inhibition efficiency. In addition, both Cd and Pb in the soil could be transferred to broad beans, and the concentration of Pb in the roots of broad beans was greater than that of Cd, whereas the opposite was observed in the stems and leaves. Notably, under mixed stress, aphids could significantly reduce the content of Cd in broad beans; similarly, the Pb content in the roots and stems of broad beans decreased significantly after being infested with aphids but increased significantly in the leaves. Further, the aphid infestation decreased the Pb content in the soil and the soil Cd content in the highest concentration group (T5 group) (50 mg/L Cd2+ + 200 mg/L Pb2+). These results highlight the necessity of focusing on the effect of insects on heavy metal remediation in plants and provide a new perspective for reducing plant Cd toxicity.

Endothelial NLRP3 inflammasome regulation in atherosclerosis.

AIM: The activation of Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. METHODS AND RESULTS: Our results demonstrated a significant upregulation of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli upregulated endothelial PFKFB3 expression via sterol regulatory element binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe-/- mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1ß (IL-1ß). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent upregulation of NLRP3, Caspase-1, and IL-1ß. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. CONCLUSIONS: Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

The Ability of Clinically Relevant Chemotherapeutics to Induce Immunogenic Cell Death in Squamous Cell Carcinoma.

BACKGROUND: Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. METHODS: We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. RESULTS: Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. CONCLUSION: The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

The features of multidrug-resistant organisms between 2016 and March 2023 and its change after the end of zero-COVID-19 policy in a teaching hospital in Shenzhen, China.

BACKGROUND: To investigate the clinical distribution and changing trend of antibiotic resistance profiles of multidrug-resistant organisms (MDROs), a retrospective study was undertaken. METHODS: The characteristics of MDROs isolated from 2016 to March 2023 were retrospectively analysed. The detection rate of these MDROs was compared prior to COVID-19 (Period 1, 2016-2019), during the COVID-19 pandemic with restrictions (Period 2, 2020-2022), and after the end of zero-policy (Period 3, Jan-March, 2023). Antibiotic-resistant genes were detected. RESULTS: The overall detection rates of CRPA, CRAB, CREC, CRKP, MRSA, and VREfm were 22.6%, 22.6%, 1.3%, 4.0%, 19.5%, and 3.1%, respectively. The detection rate of CRAB was significantly lower in Period 2 and 3 compared with Period 1 (P < 0.0001). The detection rate of CRPA and VREfm was significantly increased in Period 3 compared with Period 1 and 2 (P < 0.0001). The resistance rate to ticarcillin/clavulanic acid (TIM) and piperacillin/tazobactam (TZP) has gradually increased in CRPA since 2018. NDM and KPC were the most common carbapenemase genes identified in CREC (60.0%) and CRKP isolates (47.8%), respectively. All the 10 VREfm isolates carried the vanA gene. CONCLUSIONS: The detection rate of CRAB has decreased since 2018, but a significantly increased prevalence of CRPA and VREfm was seen after the end of zero-policy. An increasing resistance rate to TIM and TZP was seen in CRPA. NDM, KPC, and vanA were the common genes harboured by CREC, CRKP, and VREfm, respectively. Ongoing surveillance after the COVID-19 era is suggested.

[Clinicians' Practice and Opinions on Sedation Therapy in End-Stage Patients].

Objective To investigate clinicians' practice and opinions on sedation therapy in end-stage patients at Peking Union Medical College Hospital. Methods From August,2022 to April,2023,an online questionnaire survey was conducted among clinicians involved in end-stage patient management. Results A total of 205 questionnaires were distributed,with an effective response rate of 56.1%.Among the clinicians,55.7% of them had experience of applying sedation therapy in end-stage patients;85.2% of clinicians believed that sedation could relieve the suffering of terminal patients from physical refractory symptoms;75.7% of clinicians considered that sedation therapy could be used to relieve agony from psycho-existential distress.Most clinicians had concerns about sedation therapy due to the lack of legal support(86.1%)and the lack of understanding of patients or families(59.1%).The majority (90.4%) of clinicians were willing to receive training on palliative sedation. Conclusions A majority of clinicians agree that sedation therapy could relieve the physical distress and psycho-existential distress in end-stage patients.However,most clinicians have concerns about the application of sedation therapy due to the lack of legal support.It is necessary to enhance the training on palliative sedation.

Control effect and field application of four predatory Orius species on Megalurothrips usitatus (Thysanoptera: Thripidae).

Megalurothrips usitatus (Bagrall) is one of the most important pests of cowpea, Vigna unguiculata (Linn.) Walp in South China. Four Orius species, including Orius minutus (L.), Orius nagaii (Yasunaga), Orius sauteri (Poppius), and Orius strigicollis (Poppius), have been commercially produced and widely used as natural enemies of pests in China. In this study, we evaluated the control efficiency of these Orius species on M. usitatus in tropical Hainan Province, China, by recording the survival rates, developmental times, and predation effects in laboratory and semi-field conditions. Laboratory experiments showed that all these 4 Orius species preyed on M. usitatus under the experimental temperatures (25, 30, and 35 °C), and O. strigicollis exhibited the highest survival rate and predation effect. Semi-field cage experiments showed that the control effect of 4 Orius species on M. usitatus was significantly higher than that under normal chemical control, with O. strigicollis having the highest effect. Greenhouse experiments in Hainan Province, China, confirmed that O. strigicollis had a significant control effect on M. usitatus. Our study indicated that O. strigicollis has a significant potential for the control of M. usitatus in cowpea fields in southern China.

Effect of Guanylate Cyclase-22-like on Ovarian Development of Orius nagaii (Hemiptera: Anthocoridae).

This study identified and characterized the gene encoding recep tor-type guanylate cyclase-22-like (GCY-22; OnGCY) from the pirate bug Orius nagaii, an important biological control agent. The full-length cDNA of the GCY of O. nagaii was obtained by rapid amplification of cDNA ends (RACE); it had a total length of 4888 base pairs (bp), of which the open reading frame (ORF) was 3750 bp, encoding a polypeptide of 1249 amino acid residues. The physicochemical properties of OnGCY were predicted and analyzed by using relevant ExPASy software, revealing a molecular formula of C6502H10122N1698O1869S57, molecular weight of ~143,811.57 kDa, isoelectric point of 6.55, and fat index of 90.04. The resulting protein was also shown to have a signal peptide, two transmembrane regions, and a conserved tyrosine kinase (tyrkc). Silencing OnGCY by RNA interference significantly inhibited ovarian development and decreased fertility in female O. nagaii in the treated versus the control group. Additionally, OnGCY silencing significantly decreased the expression levels of other GCY and Vg genes. Thus, these results clarify the structure and biological function of OnGCY, which has an important role in insect fecundity. The results also provide a reference for agricultural pest control and future large-scale breeding of biological control agents.

Overexpression of NtDOGL4 improves cadmium tolerance through abscisic acid signaling pathway in tobacco.

The DELAY OF GERMINATION1-LIKE (DOGL) genes play an essential role in diverse biological processes in plants. However, their exact involvement in the response to cadmium (Cd) stress via the ABA pathway remains unclear. Here, we focused on NtDOGL4, a tobacco DOGL gene whose expression is highly induced upon exposure to Cd. Overexpression of NtDOGL4 in tobacco resulted in elevated endogenous ABA levels, reduced Cd accumulation, and increased tolerance to Cd. Moreover, NtDOGL4 overexpression led to decreased accumulation of reactive oxygen species (ROS) and improved ROS scavenging capacity under Cd stress. Further analyses revealed the direct binding of the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) to the NtDOGL4 promoter, positively regulating its expression in tobacco. Notably, NtDOGL4 overexpression promoted suberin formation and deposition, while suppressing the expression of Cd transporter genes in tobacco roots, as evidenced by histochemical staining, suberin fraction determination, and qRT-PCR assays. Collectively, our results demonstrate that NtDOGL4 overexpression reduces Cd accumulation, thereby improving Cd stress tolerance through the modulation of antioxidant system, transcription of Cd transporters, and suberin deposition. Notably, the NtABI5-NtDOGL4 module functions as a positive regulator in tobacco's Cd tolerance, underscoring its potential as a molecular target for developing low-Cd crops to ensure environmental safety.

LncRNA PSMB8-AS1 Instigates Vascular Inflammation to Aggravate Atherosclerosis.

BACKGROUND: Increasing evidence suggests that long noncoding RNAs play significant roles in vascular biology and disease development. One such long noncoding RNA, PSMB8-AS1, has been implicated in the development of tumors. Nevertheless, the precise role of PSMB8-AS1 in cardiovascular diseases, particularly atherosclerosis, has not been thoroughly elucidated. Thus, the primary aim of this investigation is to assess the influence of PSMB8-AS1 on vascular inflammation and the initiation of atherosclerosis. METHODS: We generated PSMB8-AS1 knockin and Apoe (Apolipoprotein E) knockout mice (Apoe-/-PSMB8-AS1KI) and global Apoe and proteasome subunit-ß type-9 (Psmb9) double knockout mice (Apoe-/-Psmb9-/-). To explore the roles of PSMB8-AS1 and Psmb9 in atherosclerosis, we fed the mice with a Western diet for 12 weeks. RESULTS: Long noncoding RNA PSMB8-AS1 is significantly elevated in human atherosclerotic plaques. Strikingly, Apoe-/-PSMB8-AS1KI mice exhibited increased atherosclerosis development, plaque vulnerability, and vascular inflammation compared with Apoe-/- mice. Moreover, the levels of VCAM1 (vascular adhesion molecule 1) and ICAM1 (intracellular adhesion molecule 1) were significantly upregulated in atherosclerotic lesions and serum of Apoe-/-PSMB8-AS1KI mice. Consistently, in vitro gain- and loss-of-function studies demonstrated that PSMB8-AS1 induced monocyte/macrophage adhesion to endothelial cells and increased VCAM1 and ICAM1 levels in a PSMB9-dependent manner. Mechanistic studies revealed that PSMB8-AS1 induced PSMB9 transcription by recruiting the transcription factor NONO (non-POU domain-containing octamer-binding protein) and binding to the PSMB9 promoter. PSMB9 (proteasome subunit-ß type-9) elevated VCAM1 and ICAM1 expression via the upregulation of ZEB1 (zinc finger E-box-binding homeobox 1). Psmb9 deficiency decreased atherosclerotic lesion size, plaque vulnerability, and vascular inflammation in Apoe-/- mice in vivo. Importantly, endothelial overexpression of PSMB8-AS1-increased atherosclerosis and vascular inflammation were attenuated by Psmb9 knockout. CONCLUSIONS: PSMB8-AS1 promotes vascular inflammation and atherosclerosis via the NONO/PSMB9/ZEB1 axis. Our findings support the development of new long noncoding RNA-based strategies to counteract atherosclerotic cardiovascular disease.

Par3L, a polarity protein, promotes M1 macrophage polarization and aggravates atherosclerosis in mice via p65 and ERK activation.

Proinflammatory M1 macrophages are critical for the progression of atherosclerosis. The Par3-like protein (Par3L) is a homolog of the Par3 family involved in cell polarity establishment. Par3L has been shown to maintain the stemness of mammary stem cells and promote the survival of colorectal cancer cells. In this study, we investigated the roles of the polar protein Par3L in M1 macrophage polarization and atherosclerosis. To induce atherosclerosis, Apoe-/- mice were fed with an atherosclerotic Western diet for 8 or 16 weeks. We showed that Par3L expression was significantly increased in human and mouse atherosclerotic plaques. In primary mouse macrophages, oxidized low-density lipoprotein (oxLDL, 50 µg/mL) time-dependently increased Par3L expression. In Apoe-/- mice, adenovirus-mediated Par3L overexpression aggravated atherosclerotic plaque formation accompanied by increased M1 macrophages in atherosclerotic plaques and bone marrow. In mouse bone marrow-derived macrophages (BMDMs) or peritoneal macrophages (PMs), we revealed that Par3L overexpression promoted LPS and IFNγ-induced M1 macrophage polarization by activating p65 and extracellular signal-regulated kinase (ERK) rather than p38 and JNK signaling. Our results uncover a previously unidentified role for the polarity protein Par3L in aggravating atherosclerosis and favoring M1 macrophage polarization, suggesting that Par3L may serve as a potential therapeutic target for atherosclerosis.

Hyperfractionated Accelerated Radiotherapy Versus Stereotactic Body Radiotherapy in the Treatment of Limited-Stage Small Cell Lung Cancer: A Matched-Pair Analysis.

BACKGROUND: Concurrent chemoradiotherapy based on hyperfractionated accelerated radiotherapy (HART) is the first-line recommended regimen for the treatment of small-cell lung cancer (SCLC). However, Stereotactic Body Radiotherapy (SBRT) is also regarded as an effective treatment for limited-stage (LS) SCLC, and the efficacy and safety of HART versus SBRT stay controversial. METHODS: In this study, 188 LS-SCLC patients were retrospectively divided into two groups receiving chemotherapy combined with either HART or SBRT. In HART group, patients received 4500 cGy in 30 fractions, administered twice daily for 3 weeks. Whereas in the SBRT group, a total radiation dose of 4000-4500 cGy was delivered in 10 fractions over 2 weeks. Thirty-three pairs of patients were finally included for next analysis. RESULTS: The estimated objective response rates were 63.6 % (21/33) and 78.8 % (26/33) in HART group and SBRT group, respectively (P = 0.269). Furthermore, there was no significant difference between HART and SBRT groups in overall survival (26 months vs. 29 months, P = 0.362) and progression free survival (11 months vs. 15 months, P = 0.223). As for the adverse events, toxicity of both groups is similar and slight that no grade 4 event was observed. Grade 3 pneumonitis cases were all occurred in the HART group (9.1%, 3/33, P = 0.238), and grade 3 esophagitis cases were all occurred in the SBRT group (6.1%, 2/33, P = 0.492). CONCLUSION: Compared with HART, SBRT could be another effective treatment with satisfactory safety for the concurrent chemoradiotherapy in patients with LS-SCLC.

DsFoxO knockout affects development and fecundity of Drosophila suzukii.

Forkhead box O (FoxO), a key transcription factor in many species, participates in numerous physiological and pathological processes of organisms through a variety of signaling pathways. In the present study, we established DsFoxO knockout (DsFoxO-KO) strain using CRISPR/Cas9, and the influence on development and fecundity of mutant strain were evaluated. To clarify the corresponding mechanism, a transcriptome analysis was conducted subsequently. The results showed that the survival rates of the DsFoxO-KO strain in larval, pupal, and adult stages were all significantly lower than those of control. The duration of the pupal stage was similar between the two strains; however, durations of egg, larva, adult preoviposition period (APOP), and total APOP (TPOP) in the DsFoxO-KO strain were all significantly longer compared to those of the control strain. The fecundity of the DsFoxO-KO strain was 20.31 eggs/female, which was significantly lower than that of the control strain (430.47 eggs/female). With the transcriptome analysis, 612 differentially expressed genes (DEGs) were identified. Following COG and GO analyses, we found that most of the DEGs were associated with the metabolic process. According to the KEGG database, the mTOR signaling, MAPK signaling, Wnt signaling, and Toll and Imd signaling pathways; insect hormone biosynthesis; autophagy; and apoptosis were altered in the DsFoxO-KO strain. These results demonstrated that knockout of DsFoxO in D. suzukii significantly influenced its development and fecundity, while transcriptome analysis provided insights to explore the corresponding molecular mechanism. These findings highlighted the critical role of FoxO in D. suzukii and might contribute to the development of novel management strategies for these flies in the future.

Glycolysis Promotes Angiotensin II-Induced Aortic Remodeling Through Regulating Endothelial-to-Mesenchymal Transition via the Corepressor C-Terminal Binding Protein 1.

BACKGROUND: Endothelial dysfunction plays a crucial role in aortic remodeling. Aerobic glycolysis and endothelial-to-mesenchymal transition (EndoMT) have, respectively, been suggested to contribute to endothelial dysfunction in many cardiovascular diseases. Here, we tested the hypothesis that glycolytic reprogramming is critical for EndoMT induction in aortic remodeling through an epigenetic mechanism mediated by a transcriptional corepressor CtBP1 (C-terminal binding protein 1), a sensor of glycolysis-derived NADH. METHODS: EndoMT program, aortic remodeling, and endothelial expression of the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) were evaluated in Ang (angiotensin) II-infused mice. Mice with endothelial-specific Pfkfb3 deficiency or CtBP1 inactivation, immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assay were employed to elucidate whether and how PFKFB3/CtBP1 epigenetically controls EndoMT. RESULTS: The EndoMT program and increased endothelial PFKFB3 expression were induced in remodeled thoracic aortas. In TGF-ß (transforming growth factor-ß)-treated human endothelial cells, activated SMAD2/3 (SMAD Family Member 2/3) transcriptionally upregulated PFKFB3 expression. In turn, the TGF-ß/SMAD signaling and EndoMT were compromised by silencing or inhibition of PFKFB3. Mechanistic studies revealed that PFKFB3-mediated glycolysis increased NADH content and activated the NADH-sensitive CtBP1. Through interaction with the transcription repressor E2F4 (E2F Transcription Factor 4), CtBP1 enhanced E2F4-mediated transcriptional repression of SMURF2 (SMAD ubiquitin regulatory factor 2), a negative regulator of TGF-ß/SMAD2 signaling. Additionally, EC-specific Pfkfb3 deficiency or CtBP1 inactivation in mice led to attenuated Ang II-induced aortic remodeling. CONCLUSIONS: Our results demonstrate a glycolysis-mediated positive feedback loop of the TGF-ß signaling to induce EndoMT and indicate that therapeutically targeting endothelial PFKFB3 or CtBP1 activity could provide a basis for treating EndoMT-linked aortic remodeling.

E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate ß-catenin signaling in glioblastoma.

Background: Elucidation of the mechanism of ubiquitation has led to novel ways to treat glioblastoma (GBM). A tripartite motif (TRIM) protein mediates a reversible, stringent ubiquitation which is closely related to glioma malignancy. This study intends to screen the most vital and abnormal regulating component of the tripartite motif protein and to explore its underlying mechanisms. Methods: TRIM21 is identified as an important oncogene that accelerates the progression of glioma cell through database in a multidimensional way and this is confirmed in human samples and cells. Tandem Mass Tags (TMT) and MS analysis are performed to discover the substrates of TRIM21.The underlying mechanisms are further investigated by CO-IP, luciferase reporter assays and gain and loss of function assays. In vivo treatment with siRNA is applied to evaluate the therapeutic significance of TRIM21. Result: We screened a panel of TRIM proteins and identified TRIM21, a E3 ubiquitin-protein ligase and autoantigen, as well as a prognostic biomarker for GBM. Functionally, high expression of wild-type TRIM21 accelerates tumor progression in vitro and in vivo, whereas TRIM21 mutants, including one with a critical RING-finger deletion, do not. Mechanistically, TRIM21 stimulates K63-linked ubiquitination and subcellular translocation of active ß-catenin from the cytoplasm to the nucleus. Moreover, TRIM21 forms a complex with the ß-catenin upstream regulator, TIF1γ, in the nucleus and accelerated its degradation by inducing K48-linked ubiquitination at K5 site, consequently increasing further nuclear ß-catenin presence. Endogenous TRIM21 levels are found to be inversely correlated with TIF1γ but positively correlated with ß-catenin in glioma tissue microarray experiments. Furthermore, direct injection of TRIM21 small interfering RNA (siRNA) into U87 cell-derived tumors (in vivo treatment with siRNA) is proved to inhibit tumor growth in nude mice. Conclusion: This work suggests that TRIM21/TIF1γ/ß-catenin axis is involved in the progression of human GBM. TRIM21 is a promising therapeutic and prognostic biomarker for glioma with hyperactive ß-catenin.

Client-counselor behavioral and inter-brain synchronization among dismissing and secure clients and its association with alliance quality and outcome.

Objective This study aimed to explore whether behavioral synchrony (BS) and inter-brain synchrony (IBS) could serve as potential biomarkers for alliance quality or outcomes among clients with different adult attachment styles. Method: We assessed the clients' self-report working alliance and clinical outcomes as well as simultaneously measured BS using motion energy analysis (MEA) and IBS with functional near-infrared spectroscopy (fNIRS) among 37 secure (N = 21) or dismissing (N = 16) clients with their counselors during the first psychological counseling meeting. Results: Dismissing dyads manifested significantly higher late-stage counselor-led and client-led IBS (p = .018) than secure dyads. Adult attachment style served as the moderators in the correlation of both whole-stage client-led BS with bond dimension of alliance (p = .015) as well as in the correlation of both whole-stage no-lag IBS with CORE-10 score changes (p = .022). Moreover, increases in the whole-stage client-led BS were significantly associated with decreases in early-stage, late-stage and whole-stage no-lag IBS (all ps ≤ 0.01). Conclusion: These findings revealed the potentially impeding role of interpersonal synchrony in alliance quality for dismissing clients, at least during the first psychological counseling meetings. They also might partially validate the relationship between different modalities of interpersonal synchrony.