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Abstract Roles of liver-specific genes (LSGs) in tumor initiation and progression are rarely explored in hepatocellular carcinoma (HCC). Here we show that LSGs are generally downregulated in HCC tumor tissues compared to non-HCC liver tissues, and low-LSG HCCs show poor prognosis and the activated c-Myc pathway. Among the c-Myc- and patient prognosis-associated LSGs, PGRMC1 significantly blocks c-Myc-induced orthotopic HCC formation. The role of PGRMC1 depends on its localization to the endoplasmic reticulum (ER) membrane, where PGRMC1 interacts with PERK through their ER luminal domains. This interaction in turn activates PERK in an ER stress-independent manner, which phosphorylates eIF2α and consequently inhibits c-Myc protein translation. In HCC patients, PGRMC1 level is significantly reduced in tumor tissues and negatively associated with the c-Myc signature. Patients with low-PGRMC1 in their tumors have poor prognosis. Collectively, deregulated LSGs in HCC are associated with the c-Myc pathway activation and PGRMC1 blocks c-Myc-induced hepatic carcinogenesis through promoting ER stress-independent PERK activation. more...

Abstract Gallbladder cancer (GBC) presents as an aggressive malignancy with poor patient outcome. Like other epithelial cancers, the mechanisms of GBC cancer progression remain vague and efforts in finding targeted therapies fall below expectations. This study combined proteomic analysis of formalin-fixed paraffin-embedded (FFPE) GBC samples, functional and molecular characterization of potential oncogenes and identification of potential therapeutic strategies for GBC. We identified Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) as one of the significantly most upregulated proteins in GBC. CEACAM6 overexpression has been observed in other cancer entities but the molecular function remains unclear. Our functional analyses in vitro and in vivo mouse models revealed that CEACAM6 supported the initial steps of cancer progression and metastasis by decreasing cell adhesion and promoting migration and invasion of GBC cells. Conversely, CEACAM6 knockdown abolished GBC aggressiveness by increasing cell adhesion while reducing cell migration, cell proliferation, and colony formation. BirA-BioID followed by mass-spectrometry revealed Integrin Beta-1 (ITGB1) and Protein Kinase C Delta (PRKCD) as direct molecular and functional partners of CEACAM6 supporting GBC cell migration. ERK and AKT signaling and their downstream target genes were regulated by CEACAM6 and thus the treatment with AKT inhibitor capivasertib or ERK inhibitor ulixertinib mitigated the CEACAM6-induced migration. These findings demonstrate that CEACAM6 is crucially involved in gallbladder cancer progression by promoting migration and inhibiting cell adhesion through ERK and AKT signaling providing specific options for treatment of CEACAM6-positive cancers. more...

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is one of the most lethal malignancies and highly heterogeneous. We thus aimed to identify and characterize iCCA cell subpopulations with severe malignant features. Methods Transcriptomic datasets from three independent iCCA cohorts (iCCA cohorts 1–3, n = 382) and formalin-fixed and paraffin-embedded tissues from iCCA cohort 4 (n = 31) were used. An unbiased global screening strategy was established, including the transcriptome analysis with the activated malignancy/stemness (MS) signature in iCCA cohorts 1–3 and the mass spectrometry analysis of the sorted stemness reporter-positive iCCA cells. A group of cellular assays and subcutaneous tumor xenograft assay were performed to investigate functional roles of the candidate. Immunohistochemistry was performed in iCCA cohort 4 to examine the expression and localization of the candidate. Molecular and biochemical assays were used to evaluate the membrane localization and functional protein domains of the candidate. Cell sorting was performed and the corresponding cellular molecular assays were utilized to examine cancer stem cell features of the sorted cells. Results The unbiased global screening identified RRM2 as the top candidate, with a significantly higher level in iCCA patients with the MS signature activation and in iCCA cells positive for the stemness reporter. Consistently, silencing RRM2 significantly suppressed iCCA malignancy phenotypes both in vitro and in vivo. Moreover, immunohistochemistry in tumor tissues of iCCA patients revealed an unreported cell membrane localization of RRM2, in contrast to its usual cytoplasmic localization. RRM2 cell membrane localization was then confirmed in iCCA cells via immunofluorescence with or without cell membrane permeabilization, cell fractionation assay and cell surface biotinylation assay. Meanwhile, an unclassical signal peptide and a transmembrane domain of RRM2 were revealed experimentally. They were essential for RRM2 trafficking to cell membrane via the conventional endoplasmic reticulum (ER)–Golgi secretory pathway. Furthermore, the membrane RRM2-positive iCCA cells were successfully sorted. These cells possessed significant cancer stem cell malignant features including cell differentiation ability, self-renewal ability, tumor initiation ability, and stemness/malignancy gene signatures. Patients with membrane RRM2-positive iCCA cells had poor prognosis. Conclusions RRM2 had an alternative cell membrane localization. The membrane RRM2-positive iCCA cells represented a malignant subpopulation with cancer stem cell features. more...

Abstract RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy. more...

Summary: Stress granules (SGs) are membrane-less cellular compartments which are dynamically assembled via biomolecular condensation mechanism when eukaryotic cells encounter environmental stresses. SGs are important for gene expression and cell fate regulation. Dysregulation of SG homeostasis has been linked to human neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we report that the HRD1-SEL1L ubiquitin ligase complex specifically regulates the homeostasis of heat shock-induced SGs through the ubiquitin-proteasome system (UPS) and the UPS-associated ATPase p97. Mechanistically, the HRD1-SEL1L complex mediates SG homeostasis through the BiP-coupled PERK-eIF2α signaling axis of endoplasmic reticulum (ER) stress, thereby coordinating the unfolded protein response (UPR) with SG dynamics. Furthermore, we show that the distinctive branches of ER stress play differential roles in SG homeostasis. Our study indicates that the UPS and the UPR together via the HRD1-SEL1L ubiquitin ligase to maintain SG homeostasis in a stressor-dependent manner. more...

Abstract Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal biliary epithelial cancer in the liver. Here, Laminin subunit gamma‐2 (LAMC2) with important oncogenic roles in iCCA is discovered. In a total of 231 cholangiocarcinoma patients (82% of iCCA patients) across four independent cohorts, LAMC2 is significantly more abundant in iCCA tumor tissue compared to normal bile duct and non‐tumor liver. Among 26.3% of iCCA patients, LAMC2 gene is amplified, contributing to its over‐expression. Functionally, silencing LAMC2 significantly blocks tumor formation in orthotopic iCCA mouse models. Mechanistically, it promotes EGFR protein translation via interacting with nascent unglycosylated EGFR in the endoplasmic reticulum (ER), resulting in activated EGFR signaling. LAMC2‐mediated EGFR translation also depends on its interaction with the ER chaperone BiP via their C‐terminus. Together LAMC2 and BiP generate a binding “pocket” of nascent EGFR and facilitate EGFR translation. Consistently, LAMC2‐high iCCA patients have poor prognosis in two iCCA cohorts. LAMC2‐high iCCA cells are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs) treatment both in vitro and in vivo. Together, these data demonstrate LAMC2 as an oncogenic player in iCCA by promoting EGFR translation and an indicator to identify iCCA patients who may benefit from available EGFR‐targeted TKIs therapies. more...

The dysfunction of the ubiquitin-proteasome system (UPS) facilitates the malignant progression of hepatocellular carcinoma (HCC). While targeting the UPS for HCC therapy has been proposed, identifying effective targets has been challenging. In this study, we conducted a focused screen of siRNA libraries targeting UPS-related WD40 repeat (WDR) proteins and found that silencing WDR20, a deubiquitinating enzyme activating factor, selectively inhibited the proliferation of HCC cells without affecting normal hepatocytes. Moreover, the downregulation of WDR20 expression induced HCC cellular senescence and suppressed tumor progression in xenograft, sleeping beauty transposon/transposase, and hydrodynamic tail vein injection-induced HCC models, and Alb-Cre+/MYC+ HCC transgenic mouse models. Mechanistically, we found that WDR20 silencing disturbed the protein stability of c-Myc, orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc, thereby promoting the transcriptional activation of CDKN1A. Further investigation revealed a positive coexpression of WDR20 and c-Myc in a tissue microarray with 88 HCC clinical samples. By employing three patient-derived organoids from individuals with HCC, we have validated the decrease in c-Myc expression and the significant induction of senescence and growth inhibition following silencing of WDR20. This study not only uncovers the biological function of WDR20 and elucidates the molecular mechanism underlying its negative regulation of HCC cellular senescence but also highlight the potential of WDR20 as a promising target for HCC therapy. [ABSTRACT FROM AUTHOR] more...

Feeding sheep with high-concentrate diet (HCD) to shorten production cycle is a well-developed feeding strategy to increase lamb production. Here, metabolomics were performed to explore the mechanism that HCD changes lamb nutrition composition. Differential metabolites were enriched in primary bile acid biosynthesis. Significantly higher content of bile acids including taurodeoxycholic acid sodium salt (TDCA), taurochenodeoxycholic acid sodium salt (TCDCA) and taurocholic acid (TCA) was observed in lamb of HCD, while the content of lithocholic acid (LCA), cholic acid (CA), chenodeoxycholic acid (CDCA) and Chenodeoxycholic acid-3-beta-D-glucuronide (CDCA-3Gln) were higher in the controls. Furthermore, a significantly decreased content of fatty acids was observed in lamb of HCD group. Finally, primary skeletal cells treated with CA or TCA showed a significant decrease in contents of fatty acids, while TCA showed a stronger effect in decreasing fatty acid contents. Collectively, we suggest that HCD decreases lamb fatty acid contents by regulating bile acid composition. more...

N6-Methyladenosine (m6A) RNA modification plays an essential role in many biological processes. To investigate the regulatory role of m6A on the skeletal muscle development in Hu sheep, this study took newborn Hu sheep (b_B Group) and six-month-old Hu sheep (s_B Group) as the objects. MeRIP-seq and RNA-Seq analysis techniques were used to detect differentially methylated genes (DMGs) and differentially expressed genes (DEGs) in the longissimus dorsi muscle of Hu sheep at different months of age. Then, conjoint analysis was further employed to screen for key genes involved in skeletal muscle development that are modified by m6A and expressed by mRNA. According to the results of the MeRIP-seq analysis, there were 285 m6A differentially methylated peaks (DMPs) in total between b_B Group and s_B Group, with 192 significant upregulated peaks and 93 significant downregulated peaks. GO and KEGG analysis revealed that DMGs are mainly enriched in actin-binding, cellular transport, and metabolic pathways. According to the results of the RNA-seq analysis, there were 4,349 DEGs in total between b_B Group and s_B Group, with 2010 upregulated genes and 2,339 downregulated genes. DEGs are found to be mainly enriched in the regulation of actin cytoskeleton tissue, AMPK and FoxO signaling pathways, etc. The conjoint analysis demonstrated that 283 genes were both modified by m6A and expressed by mRNA. Among them, three genes relevant to muscle growth (RGMB, MAPK8IP3, and RSPO3) were selected as candidates for quantitative validation, and the results were in line with the sequencing results. The results mentioned above all suggest that m6A plays a certain role in the skeletal muscle development in Hu sheep. more...

Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/μL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency. more...

Lamb meat has become very popular with consumers in recent years due to its nutritional benefits. As a lean red meat, lamb is an important natural source of polyunsaturated and saturated fatty acids, which can be modified by adjustments in livestock feed. This study used proteomic and metabolic analyses to compare a basal ration supplemented with either mulberry silage or corn silage. Supplementation with mulberry silage led to a reduction in subcutaneous carcass fatness compared with corn silage. Additionally, changes in the proteome associated with fatty acid metabolism and oxidation resulted in decreased levels of saturated and trans fatty acids, while significantly increasing the levels of α-linolenic acid (ALA) and oleic acid and reducing linoleic acid content. more...

The urban ecological environment is crucial to the quality of life of residents and the sustainable development of the region, and the assessment and prediction of the ecological environment quality can provide a scientific guidance for ecological environment management and improvement. We proposed a novel approach to assess and simulate the urban ecological environment quality using the Geographic Information System Ecological Index (GISEI). First, we calculated the remote sensing ecological index (RSEI) for Xi’an in 2020. Second, we selected land use data, mean annual temperature, and mean annual relative humidity as ecological indicators. We regressed these indicators on the RSEI to obtain the GISEI of Xi’an in 2020. Finally, we simulated the GISEI of Xi’an in 2030 by predicting the ecological indicators and analyzed the changes in the ecological environment quality. The results of the study show that the ecological environment quality in Xi’an in 2020 is better overall. By 2030, most of the ecological environment quality in Xi’an will be worse, and the proportion of the excellent area will decrease from 42.8% to 3.8%. The more serious ecological degradation is mainly located in the regions bordering the Qinling Mountains and the Guanzhong Plain, and the ecological environment quality in most areas of the Qinling Mountains will deteriorate from excellent to good. more...

Background: Rumen epithelium plays a central role in absorbing, transporting, and metabolizing of short-chain fatty acids. For dairy calves, the growth of rumen papillae greatly enhances the rumen surface area to absorb nutrients. However, the molecular mechanism underlying dairy calves rumen postnatal development remains rarely understood.Results: Here, we firstly describe the histological change of rumen epithelium from birth to day 90 of age. Then, a shotgun approach and bioinformatics analyses were used to investigate and compare proteomic profiles of Holstein calve rumen epithelium on day 0, 30, 60 and 90 of age. A total of 4372 proteins were identified, in which we found 852, 342, 164 and 95 differentially expressed proteins between D0 and D30, between D30 and D60, between D60 and D90, respectively. Finally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to provide a comprehensive proteomic landscape of dairy calves rumen development at tissue level.Conclusion: To conclude, our data indicated that keratinocyte differentiation, mitochondrion formation, the establishment of urea transport and innate immune system play central roles during rumen epithelium development. Tetrahydrobiopterin (BH4) presents an important role in rumen epithelial keratinization. The biological processes of BH4 biosynthesis and molecular function of nicotinamide adenine dinucleotide phosphate binding participate in mitochondrial cristae formation. The proposed datasets provide a useful basis for future studies to better comprehend dairy calves rumen epithelial development. more...

Background: The development of the rumen epithelium is a critical physiological challenge for sheep. However, the molecular mechanism underlying postnatal rumen development in sheep remains rarely understood.Results: Here, we used a shotgun approach and bioinformatics analyses to investigate and compare proteomic profiles of sheep rumen epithelium tissue on day 0, 15, 30, 45, and 60 of age. A total of 4,523 proteins were identified, in which we found 852, 342, 164, and 95 differentially expressed proteins (DEPs) between day 0 and day 15, between day 15 and day 30, between day 30 and day 45, between day 45 and day 60, respectively. Furthermore, subcellular localization analysis showed that the DEPs were majorly localized in mitochondrion between day 0 and day 15, after which nucleus proteins were the most DEPs. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that DEPs significantly enriched in mitochondrion, ubiquitination, histone modifications, glutathione synthase activity, and wnt and nortch signaling pathways.Conclusion: Our data indicate that the biogenesis of mitochondrion in rumen epithelial cell is essential for the initiation of rumen epithelial development. Glutathione, wnt signaling pathway and nortch signaling pathway participated in rumen epithelial growth. Ubiquitination, post-translational modifications of histone might be key molecular functions in regulating rumen epithelial development. more...

As eco–environmental effects have become important considerations in the construction and planning of production, living, and ecological spaces, we used a combination of quantitative and qualitative methods to analyze the eco–environmental effects’ spatiotemporal evolution and present appropriate ideas for ecological restoration based on the land use data. The results show that during the research period both an improvement and degradation of the regional eco–environment occurred simultaneously. In the earlier period, the ecological environment tended to be worse, while in the later period, the eco–environmental quality was dramatically enhanced. Pasture ecological land in the study area had the strongest positive impact on the eco–environmental quality, while the negative effect of agricultural production land was severe. The quality of the regional eco−environment was enhanced (degraded) due to the extension (contraction) of ecological land. The construction of an ecological environment is a complex engineering task. Although the eco−environment in most areas of the study area showed an improving trend, the overall eco–environment remains relatively fragile. In the course of supporting high–quality regional social and economic growth and pursuing high–level environmental preservation strategies, we should take corresponding measures to protect and repair the regional ecological environment. more...

In response to the strong coupling between line-of-sight angular rate and attitude error in the output of the strapdown rotating projectile, a method based on adaptive fading volume covariance Kalman filtering (AFCKF) is proposed. To achieve the decoupling of line-of-sight angular rate, the relative motion characteristics of missile and target are considered. A state model is constructed, which includes an estimation model of line-of-sight angular rate in the terminal guidance phase, and a measurement model including line-of-sight angle and attitude angular is established based on geometric relationships. To solve the problem of filter failure caused by the divergence of the line-of-sight angular rate estimation results in traditional CKF for rotating projectile, a fading factor based on residual sequence is introduced to adjust the predicted state covariance for quick convergence of the estimation results. To verify the effectiveness of AFCKF, typical interferences of attitude angle and measurement angle are considered. Simulation results show that the mean estimation errors of line-of-sight elevation rate and azimuth rate of the proposed method are 30.41% and 42.18% of the traditional EKF, respectively, effectively improving the accuracy of line-of-sight angular rate estimation for rotating projectil more...