Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts.

DNA-demethylating agents have shown clinical anti-tumor efficacy via an unknown mechanism of action. Using a combination of experimental and bioinformatics analyses in colorectal cancer cells, we demonstrate that low-dose 5-AZA-CdR targets colorectal cancer-initiating cells (CICs) by inducing viral mimicry. This is associated with induction of dsRNAs derived at least in part from endogenous retroviral elements, activation of the MDA5/MAVS RNA recognition pathway, and downstream activation of IRF7. Indeed, disruption of virus recognition pathways, by individually knocking down MDA5, MAVS, or IRF7, inhibits the ability of 5-AZA-CdR to target colorectal CICs and significantly decreases 5-AZA-CdR long-term growth effects. Moreover, transfection of dsRNA into CICs can mimic the effects of 5-AZA-CdR. Together, our results represent a major shift in understanding the anti-tumor mechanisms of DNA-demethylating agents and highlight the MDA5/MAVS/IRF7 pathway as a potentially druggable target against CICs.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Milestones in understanding transport, sensing, and signaling of the plant nutrient phosphorus.

As an essential nutrient element, phosphorus (P) is primarily acquired and translocated as inorganic phosphate (Pi) by plant roots. Pi is often sequestered in the soil and becomes limited for plant growth. Plants have developed a sophisticated array of adaptive responses, termed P starvation responses, to cope with P deficiency by improving its external acquisition and internal utilization. Over the past 2 to 3 decades, remarkable progress has been made toward understanding how plants sense and respond to changing environmental P. This review provides an overview of the molecular mechanisms that regulate or coordinate P starvation responses, emphasizing P transport, sensing, and signaling. We present the major players and regulators responsible for Pi uptake and translocation. We then introduce how P is perceived at the root tip, how systemic P signaling is operated, and the mechanisms by which the intracellular P status is sensed and conveyed. Additionally, the recent exciting findings about the influence of P on plant-microbe interactions are highlighted. Finally, the challenges and prospects concerning the interplay between P and other nutrients and strategies to enhance P utilization efficiency are discussed. Insights obtained from this knowledge may guide future research endeavors in sustainable agriculture.

Identification of KU-55933 as an anti-atherosclerosis compound by using a hemodynamic-based high-throughput drug screening platform.

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 µM), Apicidin (10 µM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.

Genome-wide association study identifies multiple HLA loci for sarcoidosis susceptibility.

Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.

Nuclear factors NF-YC3 and NF-YBs positively regulate arbuscular mycorrhizal symbiosis in tomato.

The involvement of nuclear factor Y (NF-Y) in transcriptional reprogramming during arbuscular mycorrhizal symbiosis has been demonstrated in several plant species. However, a comprehensive picture is lacking. We showed that the spatial expression of NF-YC3 was observed in cortical cells containing arbuscules via the cis-regulatory element GCC boxes. Moreover, the NF-YC3 promoter was transactivated by the combination of CYCLOPS and autoactive calcium and calmodulin-dependent kinase (CCaMK) via GCC boxes. Knockdown of NF-YC3 significantly reduced the abundance of all intraradical fungal structures and affected arbuscule size. BCP1, SbtM1, and WRI5a, whose expression associated with NF-YC3 levels, might be downstream of NF-YC3. NF-YC3 interacted with NF-YB3a, NF-YB5c, or NF-YB3b, in yeast (Saccharomyces cerevisiae) and in planta, and interacted with NF-YA3a in yeast. Spatial expression of three NF-YBs was observed in all cell layers of roots under both mock and mycorrhizal conditions. Simultaneous knockdown of three NF-YBs, but not individually, reduced the fungal colonization level, suggesting that there might be functional redundancy of NF-YBs to regulate AM symbiosis. Collectively, our data suggest that NF-YC3 and NF-YBs positively regulate AM symbiosis in tomato, and arbuscule-related NF-YC3 may be an important downstream gene of the common symbiosis signaling pathway.

Excessive load promotes temporomandibular joint chondrocyte apoptosis via Piezo1/endoplasmic reticulum stress pathway.

Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The specific mechanism through which excessive load induces TMJ osteoarthritis is not fully understood; however, mechanically-activated (MA) ion channels play a crucial role. Among these channels, Piezo1 has been identified as a mediator of chondrocyte catabolic responses and is markedly increased in osteoarthritis. Our observations indicate that, under excessive load conditions, endoplasmic reticulum stress in chondrocytes results in apoptosis of the TMJ chondrocytes. Importantly, using the Piezo1 inhibitor GsMTx4 demonstrates its potential to alleviate this condition. Furthermore, Piezo1 mediates endoplasmic reticulum stress in chondrocytes by inducing calcium ion influx. Our research substantiates the role of Piezo1 as a pivotal ion channel in mediating chondrocyte overload. It elucidates the link between excessive load, cell apoptosis, and calcium ion influx through Piezo1. The findings underscore Piezo1 as a key player in the pathogenesis of TMJ osteoarthritis, shedding light on potential therapeutic interventions for this condition.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

The costs and benefits of symbiotic interactions: variable effects of rhizobia and arbuscular mycorrhizae on Vigna radiata accessions.

BACKGROUND: The symbiosis among plants, rhizobia, and arbuscular mycorrhizal fungi (AMF) is one of the most well-known symbiotic relationships in nature. However, it is still unclear how bilateral/tripartite symbiosis works under resource-limited conditions and the diverse genetic backgrounds of the host. RESULTS: Using a full factorial design, we manipulated mungbean accessions/subspecies, rhizobia, and AMF to test their effects on each other. Rhizobia functions as a typical facilitator by increasing plant nitrogen content, plant weight, chlorophyll content, and AMF colonization. In contrast, AMF resulted in a tradeoff in plants (reducing biomass for phosphorus acquisition) and behaved as a competitor in reducing rhizobia fitness (nodule weight). Plant genotype did not have a significant effect on AMF fitness, but different mungbean accessions had distinct rhizobia affinities. In contrast to previous studies, the positive relationship between plant and rhizobia fitness was attenuated in the presence of AMF, with wild mungbean being more responsive to the beneficial effect of rhizobia and attenuation by AMF. CONCLUSIONS: We showed that this complex tripartite relationship does not unconditionally benefit all parties. Moreover, rhizobia species and host genetic background affect the symbiotic relationship significantly. This study provides a new opportunity to re-evaluate the relationships between legume plants and their symbiotic partners.

Recent Progress in Phage-Based Nanoplatforms for Tumor Therapy.

Nanodrug delivery systems have demonstrated a great potential for tumor therapy with the development of nanotechnology. Nonetheless, traditional drug delivery systems are faced with issues such as complex synthetic procedures, low reproducibility, nonspecific distribution, impenetrability of biological barrier, systemic toxicity, etc. In recent years, phage-based nanoplatforms have attracted increasing attention in tumor treatment for their regular structure, fantastic carrying property, high transduction efficiency and biosafety. Notably, therapeutic or targeting peptides can be expressed on the surface of the phages through phage display technology, enabling the phage vectors to possess multifunctions. As a result, the drug delivery efficiency on tumor will be vastly improved, thereby enhancing the therapeutic efficacy while reducing the side effects on normal tissues. Moreover, phages can overcome the hindrance of biofilm barrier to elicit antitumor effects, which exhibit great advantages compared with traditional synthetic drug delivery systems. Herein, this review not only summarizes the structure and biology of the phages, but also presents their potential as prominent nanoplatforms against tumor in different pathways to inspire the development of effective nanomedicine.

Multi-omic signatures of sarcoidosis and progression in bronchoalveolar lavage cells.

BACKGROUND: Sarcoidosis is a heterogeneous granulomatous disease with no accurate biomarkers of disease progression. Therefore, we profiled and integrated the DNA methylome, mRNAs, and microRNAs to identify molecular changes associated with sarcoidosis and disease progression that might illuminate underlying mechanisms of disease and potential biomarkers. METHODS: Bronchoalveolar lavage cells from 64 sarcoidosis subjects and 16 healthy controls were used. DNA methylation was profiled on Illumina HumanMethylationEPIC arrays, mRNA by RNA-sequencing, and miRNAs by small RNA-sequencing. Linear models were fit to test for effect of sarcoidosis diagnosis and progression phenotype, adjusting for age, sex, smoking, and principal components of the data. We built a supervised multi-omics model using a subset of features from each dataset. RESULTS: We identified 1,459 CpGs, 64 mRNAs, and five miRNAs associated with sarcoidosis versus controls and four mRNAs associated with disease progression. Our integrated model emphasized the prominence of the PI3K/AKT1 pathway, which is important in T cell and mTOR function. Novel immune related genes and miRNAs including LYST, RGS14, SLFN12L, and hsa-miR-199b-5p, distinguished sarcoidosis from controls. Our integrated model also demonstrated differential expression/methylation of IL20RB, ABCC11, SFSWAP, AGBL4, miR-146a-3p, and miR-378b between non-progressive and progressive sarcoidosis. CONCLUSIONS: Leveraging the DNA methylome, transcriptome, and miRNA-sequencing in sarcoidosis BAL cells, we detected widespread molecular changes associated with disease, many which are involved in immune response. These molecules may serve as diagnostic/prognostic biomarkers and/or drug targets, although future testing is required for confirmation.

Fractional exhaled nitric oxide, a potential biomarker for evaluating glucocorticoids treatment and prognosis in allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by enhanced TH2 inflammatory response. Fractional exhaled nitric oxide (FeNO) measurement has been used as a valuable tool in predicting the development and management of asthma, another typical TH2 inflammation. However, the clinical significance of FeNO in ABPA remains unclear. OBJECTIVE: To investigate the association between FeNO and the prognosis of patients with ABPA to provide a basis for the use of FeNO in evaluating the efficacy of glucocorticoids in ABPA treatment. METHODS: This study comprised 2 parts; 58 patients were enrolled in the retrospective study. Clinical indexes in patients with different prognoses were compared, and receiver operating characteristic curve analysis was used to determine the threshold value. The prospective observational study involved 61 patients who were regularly followed up at 4 to 6 weeks and 6 months since the initial treatment. Patients were grouped on the basis of baseline FeNO values; correlation analysis was performed in the clinical data. RESULTS: Different prognoses were observed between patients with high and low baseline FeNO values, with a threshold value of 57 parts per billion. The percentage of Aspergillus fumigatus-specific IgE, percentage of positive A fumigatus-specific IgG, and relapse/exacerbation rate differed significantly between the high and low FeNO groups. Patients with higher FeNO needed longer treatment duration and showed shorter interval between glucocorticoid withdrawal and the next relapse/exacerbation. CONCLUSION: Our findings indicate that the level of FeNO is associated with the prognosis of ABPA. It can serve as an independent and valuable biomarker for evaluating the effectiveness of glucocorticoid treatment.

Atrophy of the cholinergic regions advances from early to late mild cognitive impairment.

PURPOSE: We investigated the volumetric changes in the components of the cholinergic pathway for patients with early mild cognitive impairment (EMCI) and those with late mild cognitive impairment (LMCI). The effect of patients' apolipoprotein 4 (APOE-ε4) allele status on the structural changes were analyzed. METHODS: Structural magnetic resonance imaging data were collected. Patients' demographic information, plasma data, and validated global cognitive composite scores were included. Relevant features were extracted for constructing machine learning models to differentiate between EMCI (n = 312) and LMCI (n = 541) and predict patients' neurocognitive function. The data were analyzed primarily through one-way analysis of variance and two-way analysis of covariance. RESULTS: Considerable differences were observed in cholinergic structural changes between patients with EMCI and LMCI. Cholinergic atrophy was more prominent in the LMCI cohort than in the EMCI cohort (P < 0.05 family-wise error corrected). APOE-ε4 differentially affected cholinergic atrophy in the LMCI and EMCI cohorts. For LMCI cohort, APOE-ε4 carriers exhibited increased brain atrophy (left amygdala: P = 0.001; right amygdala: P = 0.006, and right Ch123, P = 0.032). EMCI and LCMI patients showed distinctive associations of gray matter volumes in cholinergic regions with executive (R2 = 0.063 and 0.030 for EMCI and LMCI, respectively) and language (R2 = 0.095 and 0.042 for EMCI and LMCI, respectively) function. CONCLUSIONS: Our data confirmed significant cholinergic atrophy differences between early and late stages of mild cognitive impairment. The impact of the APOE-ε4 allele on cholinergic atrophy varied between the LMCI and EMCI groups.

Discovery of Uncommon Tryptophan-Containing Diketopiperazines from Aspergillus homomorphus CBS 101889 Using an Aspergillus nidulans Heterologous Expression System.

Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute's MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans. The expression of this refactored BGC in A. nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F (2, 4-8). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l-allo-isoleucine.

Sensitive tumour detection and classification using plasma cell-free DNA methylomes.

The use of liquid biopsies for cancer detection and management is rapidly gaining prominence1. Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations2-5. By contrast, large-scale epigenetic alterations-which are tissue- and cancer-type specific-are not similarly constrained6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns.

α4 nicotinic receptors on GABAergic neurons mediate a cholinergic analgesic circuit in the substantia nigra pars reticulata.

Nicotinic acetylcholine receptors (nAChRs) regulate pain pathways with various outcomes depending on receptor subtypes, neuron types, and locations. But it remains unknown whether α4ß2 nAChRs abundantly expressed in the substantia nigra pars reticulata (SNr) have potential to mitigate hyperalgesia in pain states. We observed that injection of nAChR antagonists into the SNr reduced pain thresholds in naïve mice, whereas injection of nAChR agonists into the SNr relieved hyperalgesia in mice, subjected to capsaicin injection into the lower hind leg, spinal nerve injury, chronic constriction injury, or chronic nicotine exposure. The analgesic effects of nAChR agonists were mimicked by optogenetic stimulation of cholinergic inputs from the pedunculopontine nucleus (PPN) to the SNr, but attenuated upon downregulation of α4 nAChRs on SNr GABAergic neurons and injection of dihydro-ß-erythroidine into the SNr. Chronic nicotine-induced hyperalgesia depended on α4 nAChRs in SNr GABAergic neurons and was associated with the reduction of ACh release in the SNr. Either activation of α4 nAChRs in the SNr or optogenetic stimulation of the PPN-SNr cholinergic projection mitigated chronic nicotine-induced hyperalgesia. Interestingly, mechanical stimulation-induced ACh release was significantly attenuated in mice subjected to either capsaicin injection into the lower hind leg or SNI. These results suggest that α4 nAChRs on GABAergic neurons mediate a cholinergic analgesic circuit in the SNr, and these receptors may be effective therapeutic targets to relieve hyperalgesia in acute and chronic pain, and chronic nicotine exposure.

A novel small-molecule PCSK9 inhibitor E28362 ameliorates hyperlipidemia and atherosclerosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor precursor homologous domain A (EGF-A) of low-density lipoprotein receptor (LDLR) in the liver and triggers the degradation of LDLR via the lysosomal pathway, consequently leading to an elevation in plasma LDL-C levels. Inhibiting PCSK9 prolongs the lifespan of LDLR and maintains cholesterol homeostasis in the body. Thus, PCSK9 is an innovative pharmacological target for treating hypercholesterolemia and atherosclerosis. In this study, we discovered that E28362 was a novel small-molecule PCSK9 inhibitor by conducting a virtual screening of a library containing 40,000 compounds. E28362 (5, 10, 20 µM) dose-dependently increased the protein levels of LDLR in both total protein and the membrane fraction in both HepG2 and AML12 cells, and enhanced the uptake of DiI-LDL in AML12 cells. MTT assay showed that E28362 up to 80 µM had no obvious toxicity in HepG2, AML12, and HEK293a cells. The effects of E28362 on hyperlipidemia and atherosclerosis were evaluated in three different animal models. In high-fat diet-fed golden hamsters, administration of E28362 (6.7, 20, 60 mg·kg-1·d-1, i.g.) for 4 weeks significantly reduced plasma total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and PCSK9 levels, and reduced liver TC and TG contents. In Western diet-fed ApoE-/- mice (20, 60 mg·kg-1·d-1, i.g.) and human PCSK9 D374Y overexpression mice (60 mg·kg-1·d-1, i.g.), administration of E28362 for 12 weeks significantly decreased plasma LDL-C levels and the area of atherosclerotic lesions in en face aortas and aortic roots. Moreover, E28362 significantly increased the protein expression level of LDLR in the liver. We revealed that E28362 selectively bound to PCSK9 in HepG2 and AML12 cells, blocked the interaction between LDLR and PCSK9, and induced the degradation of PCSK9 through the ubiquitin-proteasome pathway, which finally resulted in increased LDLR protein levels. In conclusion, E28362 can block the interaction between PCSK9 and LDLR, induce the degradation of PCSK9, increase LDLR protein levels, and alleviate hyperlipidemia and atherosclerosis in three distinct animal models, suggesting that E28362 is a promising lead compound for the treatment of hyperlipidemia and atherosclerosis.

Efficacy and long-term outcomes of single-balloon enteroscopy-assisted treatment for biliary obstruction after choledochojejunostomy.

BACKGROUND: To evaluate the long-term efficacy of single-balloon enteroscopy endoscopic retrograde cholangiography (SBE-ERC) for the treatment of biliary obstruction and to analyze the factors affecting the recurrence of benign bilioenteric anastomotic stricture after SBE-ERC treatment. METHODS: The clinical data of patients with biliary diseases treated with SBE-ERC after choledochojejunostomy in our hospital from January 2015 to December 2021 were analyzed retrospectively for the success rates of diagnosis and treatment and the incidence of complications. Patients who were diagnosed with benign bilioenteric anastomotic stricture were followed up. The independent factors affecting recurrence were obtained by univariate and multivariate analyses using the Kaplan‒Meier method and Cox proportional hazard regression model. RESULTS: A total of 289 SBE-ERCs were performed in 165 patients. The overall success rate was 83.0% (240/289). The incidence of postoperative complications was 5.2% (15/289). The 108 successfully treated patients diagnosed with benign bilioenteric anastomotic stricture were followed up. Twenty-six percent (29/108) of patients had recurrent stricture after SBE-ERC. The biliary patency rates at 1 year, 2 years and 5 years after SBE-ERC were 90.1%, 69.3%, and 53.9%, respectively. Single-factor analysis revealed the absence of intrahepatic biliary gas imaging during endoscopy ( χ 2 =5.366, P = 0.021), a diameter of balloon dilatation during the last endoscopic treatment less than 0.8 cm ( χ 2 =4.552, P = 0.033), and the presence of a thread in the anastomosis ( χ 2 =8.921, P = 0.003) as risk factors for recurrence. A non-indwelling biliary plastic stent ( χ 2 =14.868, P < 0.001) and undergoing only one ERCP treatment ( χ 2 =13.313, P = 0.001) were risk factors for the recurrence of benign stricture after SBE-ERC resection. Multivariate analysis revealed that the absence of a stent (HR = 0.15, 95% CI 0.06-0.40, P = 0.001), absence of intrahepatic biliary gas imaging during endoscopy (HR = 0.39, 95% CI 0.17-0.91, P = 0.03) and the presence of a thread in the anastomosis (HR = 3.69, 95% CI 1.59-8.57, P = 0.002) were independent risk factors for stricture recurrence. CONCLUSIONS: Treating biliary disease after choledochojejunostomy with SBE-ERC is safe and effective, with a good immediate technical success rate and an acceptable incidence of complications. SBE-ERC has long-term efficacy in the treatment of benign bilioenteric anastomotic stricture. The absence of intrahepatic biliary gas imaging during endoscopy, non-indwelling biliary stents and the existence of anastomotic threads are independent risk factors for the recurrence of benign bilioenteric anastomotic stricture.

New mitogenomes from the genus Cricotopus (Diptera: Chironomidae, Orthocladiinae): Characterization and phylogenetic implications.

Cricotopus is a large and diverse genus of non-biting midges composed of several subgenera. Complete mitogenome sequences are available for very few Cricotopus species. The subgenus Pseudocricotopus unites species with unusual morphological structures in adult male and pupal stages, however, molecular methods are needed to verify the placement of this subgenus within Cricotopus. We obtained mitogenomes of C. (Pseudocricotopus) cf. montanus and nine other Cricotopus species for phylogenetic analysis, coupled with two Rheocricotopus species and one Synorthocladius species as outgroups. The structure of the mitogenome was similar among these Cricotopus species, exhibiting A+T bias and retaining ancestral gene order. Mutation rate, estimated as Ka/Ks, varied among genes, and was highest for ATP8 and lowest for COI. The phylogenetic relationships among species of Cricotopus, Rheocricotopus and Synorthocladius was reconstructed using Bayesian inference and maximum likelihood estimation. The phylogenetic trees confirmed placement of subgenus Pseudocricotopus, represented by Cricotopus cf. montanus, within Cricotopus. Our study increases the library of chironomid mitogenomes and provides insight into the properties of their constituent genes.