Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth.

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations.

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

Bcl6 is a subset-defining transcription factor of lymphoid tissue inducer-like ILC3.

Innate lymphoid cells (ILCs) are tissue-resident effector cells with roles in tissue homeostasis, protective immunity, and inflammatory disease. Group 3 ILCs (ILC3s) are classically defined by the master transcription factor RORγt. However, ILC3 can be further subdivided into subsets that share type 3 effector modules that exhibit significant ontological, transcriptional, phenotypic, and functional heterogeneity. Notably lymphoid tissue inducer (LTi)-like ILC3s mediate effector functions not typically associated with other RORγt-expressing lymphocytes, suggesting that additional transcription factors contribute to dictate ILC3 subset phenotypes. Here, we identify Bcl6 as a subset-defining transcription factor of LTi-like ILC3s in mice and humans. Deletion of Bcl6 results in dysregulation of the LTi-like ILC3 transcriptional program and markedly enhances expression of interleukin-17A (IL-17A) and IL-17F in LTi-like ILC3s in a manner in part dependent upon the commensal microbiota-and associated with worsened inflammation in a model of colitis. Together, these findings redefine our understanding of ILC3 subset biology.

The comparable tumour microenvironment in sporadic and NF2-related schwannomatosis vestibular schwannoma.

Bilateral vestibular schwannoma is the hallmark of NF2-related schwannomatosis, a rare tumour predisposition syndrome associated with a lifetime of surgical interventions, radiotherapy and off-label use of the anti-angiogenic drug bevacizumab. Unilateral vestibular schwannoma develops sporadically in non-NF2-related schwannomatosis patients for which there are no drug treatment options available. Tumour-infiltrating immune cells such as macrophages and T-cells correlate with increased vestibular schwannoma growth, which is suggested to be similar in sporadic and NF2-related schwannomatosis tumours. However, differences between NF2-related schwannomatosis and the more common sporadic disease include NF2-related schwannomatosis patients presenting an increased number of tumours, multiple tumour types and younger age at diagnosis. A comparison of the tumour microenvironment in sporadic and NF2-related schwannomatosis tumours is therefore required to underpin the development of immunotherapeutic targets, identify the possibility of extrapolating ex vivo data from sporadic vestibular schwannoma to NF2-related schwannomatosis and help inform clinical trial design with the feasibility of co-recruiting sporadic and NF2-related schwannomatosis patients. This study drew together bulk transcriptomic data from three published Affymetrix microarray datasets to compare the gene expression profiles of sporadic and NF2-related schwannomatosis vestibular schwannoma and subsequently deconvolved to predict the abundances of distinct tumour immune microenvironment populations. Data were validated using quantitative PCR and Hyperion imaging mass cytometry. Comparative bioinformatic analyses revealed close similarities in NF2-related schwannomatosis and sporadic vestibular schwannoma tumours across the three datasets. Significant inflammatory markers and signalling pathways were closely matched in NF2-related schwannomatosis and sporadic vestibular schwannoma, relating to the proliferation of macrophages, angiogenesis and inflammation. Bulk transcriptomic and imaging mass cytometry data identified macrophages as the most abundant immune population in vestibular schwannoma, comprising one-third of the cell mass in both NF2-related schwannomatosis and sporadic tumours. Importantly, there were no robust significant differences in signalling pathways, gene expression, cell type abundance or imaging mass cytometry staining between NF2-related schwannomatosis and sporadic vestibular schwannoma. These data indicate strong similarities in the tumour immune microenvironment of NF2-related schwannomatosis and sporadic vestibular schwannoma.

Early preclinical plasma protein biomarkers of brain trauma are influenced by early seizures and levetiracetam.

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Frizzled 7 modulates goblet and Paneth cell fate, and maintains homeostasis in mouse intestine.

Intestinal homeostasis depends on interactions between the intestinal epithelium, the immune system and the microbiota. Because of these complicated connections, there are many problems that need to be solved. Current research has indicated that genes targeted by Wnt signaling are responsible for controlling intestinal stem cell fate and for modulating intestinal homeostasis. Our data show that loss of frizzled 7 (Fzd7), an important element in Wnt signaling, interrupts the differentiation of mouse intestinal stem cells into absorptive progenitors instead of secretory progenitors (precursors of goblet and Paneth cells). The alteration in canonical Wnt and Notch signaling pathways interrupts epithelial homeostasis, resulting in a decrease in physical protection in the intestine. Several phenotypes in our Fzd7-deleted model were similar to the features of enterocolitis, such as shortened intestines, decreased numbers of goblet cells and Paneth cells, and severe inflammation. Additionally, loss of Fzd7 exacerbated the defects in a chemical-induced colitis model and could initiate tumorigenesis. These findings may provide important information for the discovery of efficient therapeutic methods to treat enterocolitis and related cancers in the intestines.

Endurance exercise ameliorates Western diet-induced atherosclerosis through modulation of microbiota and its metabolites.

The World Health Organization determined cardiovascular disease to be the leading cause of death globally; atherosclerosis is the primary cause of the high morbidity and mortality rates. Regular physical activity is an effective strategy for maintaining endothelial health and function to prevent the development of atherosclerosis. Obesity is also a crucial risk factor for atherosclerotic progression in combination with various complications and systemic inflammation. Physiological homeostasis is modulated by the intestinal microbiota, but the mechanisms through which exercise attenuates atherosclerosis through the microbiota have not been elucidated. Therefore, we investigated the effects of endurance exercise on atherosclerosis induced by a Western diet (WD) and apolipoprotein E (ApoE) knockout in terms of microbiota parameters and metabolites. Genetically modified ApoE knockout mice (C57BL/6-Apoeem1Narl/Narl, ApoEKO) and wild-type mice (C57BL6/J) were divided into the following four groups (n = 6), namely, wild-type mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX), for a 12-week intervention. The WD significantly induced obesity and atherosclerotic syndrome in the ApoE WD group. Severe atherosclerotic lesions and arterial thickness were significantly elevated and accompanied by increases in VCAM-1, MCP-1, TNF-α, and IL-1ß for immune cell chemotaxis and inflammation during atherosclerotic pathogenesis in the ApoE WD group. In addition, dysbiosis in the ApoE WD group resulted in the lowest short-chain fatty acid (SCFA) production. Endurance exercise intervention (ApoE WD EX) significantly alleviated atherosclerotic syndrome by reducing obesity, significantly inhibiting VCAM-1, MCP-1, TNF-α, and IL-1ß expression, and increasing the production of SCFAs. Modulation of the microbiota associated with inflammation, such as Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge, and increased SCFA production, particularly through an abundance of Rikenellaceae and Dubosiella, were also observed after exercise intervention. Endurance exercise can alleviate WD-induced atherosclerosis through the amelioration of obesity, inflammation, and chemotaxis signaling, which are modulated by the microbiota and derived SCFAs.

The ADAM9/UBN2/AKR1C3 axis promotes resistance to androgen-deprivation in prostate cancer.

Metastatic and castration-resistant disease is a fatal manifestation of prostate cancer (PCa). The mechanism through which resistance to androgen deprivation in PCa is developed remains largely unknown. Our understanding of the tumor microenvironment (TME) and key signaling pathways between tumors and their TME is currently changing in light of the generation of new knowledge with regard to cancer progression. A disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) is a membranous bridge forming cell-cell and cell-matrix connections that regulate tumor aggressiveness and metastasis. However, it is not known whether ADAM9 expressed in the TME contributes to the CRPC phenotype. In this study, we aimed to investigate the expression patterns of ADAM9 in prostate cancer-associated fibroblasts (CAFs). We also intended to elucidate the effects of both stromal cell- and cancer cell-derived ADAM9 on the progression of CRPC and the implicated molecular pathways. By using both clinical specimens and cell lines, we herein showed that unlike the membrane anchored ADAM9 overexpressed by both PCa cells and prostate CAFs, the secreted isoform of ADAM9 (sADAM9) was strongly detected in CAFs, but rarely in tumor cells, and that could be a serum marker for PCa patients. We demonstrated that functionally sADAM9 are characterized as chemoattractant for the directed movement of androgen-independent PCa cells through integrin downstream FAK/AKT pathway, supporting that elevated sADAM9 by prostate CAFs could be responsible for the promotion of CRPC metastasis. Moreover, by stimulating PCa cells with sADAM9, we found that ubinuclein-2 (UBN2) expression was increased. A positive correlation of ADAM9 and UBN2 expression was observed in androgen receptor-expressing PCa cell lines and further confirmed in clinical PCa specimens. Using a genetic modification approach, we identified UBN2 as a downstream target gene of ADAM9 that is critical for the survival of androgen-dependent PCa cells in response to androgen deprivation, through the induction and effect of the aldo-keto reductase family 1 member C3 (AKR1C3). Collectively, our results reveal a novel action of ADAM9 on the transition of androgen-dependent PCa cells into an androgen-independent manner through the UBN2/AKR1C3 axis; the aforementioned action could contribute to the clinically-observed acquired androgen-deprivation therapy resistance.

Distinct transcriptional programs stratify ovarian cancer cell lines into the five major histological subtypes.

BACKGROUND: Epithelial ovarian cancer (OC) is a heterogenous disease consisting of five major histologically distinct subtypes: high-grade serous (HGSOC), low-grade serous (LGSOC), endometrioid (ENOC), clear cell (CCOC) and mucinous (MOC). Although HGSOC is the most prevalent subtype, representing 70-80% of cases, a 2013 landmark study by Domcke et al. found that the most frequently used OC cell lines are not molecularly representative of this subtype. This raises the question, if not HGSOC, from which subtype do these cell lines derive? Indeed, non-HGSOC subtypes often respond poorly to chemotherapy; therefore, representative models are imperative for developing new targeted therapeutics. METHODS: Non-negative matrix factorisation (NMF) was applied to transcriptomic data from 44 OC cell lines in the Cancer Cell Line Encyclopedia, assessing the quality of clustering into 2-10 groups. Epithelial OC subtypes were assigned to cell lines optimally clustered into five transcriptionally distinct classes, confirmed by integration with subtype-specific mutations. A transcriptional subtype classifier was then developed by trialling three machine learning algorithms using subtype-specific metagenes defined by NMF. The ability of classifiers to predict subtype was tested using RNA sequencing of a living biobank of patient-derived OC models. RESULTS: Application of NMF optimally clustered the 44 cell lines into five transcriptionally distinct groups. Close inspection of orthogonal datasets revealed this five-cluster delineation corresponds to the five major OC subtypes. This NMF-based classification validates the Domcke et al. analysis, in identifying lines most representative of HGSOC, and additionally identifies models representing the four other subtypes. However, NMF of the cell lines into two clusters did not align with the dualistic model of OC and suggests this classification is an oversimplification. Subtype designation of patient-derived models by a random forest transcriptional classifier aligned with prior diagnosis in 76% of unambiguous cases. In cases where there was disagreement, this often indicated potential alternative diagnosis, supported by a review of histological, molecular and clinical features. CONCLUSIONS: This robust classification informs the selection of the most appropriate models for all five histotypes. Following further refinement on larger training cohorts, the transcriptional classification may represent a useful tool to support the classification of new model systems of OC subtypes.

Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth.

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

Gastroprotective effect of Lycium barbarum polysaccharides and C-phycocyanin in rats with ethanol-induced gastric ulcer.

This study investigated the gastroprotective effect of Lycium barbarum polysaccharides (LBP) and C-phycocyanin (C-PC) in rats with ethanol-induced gastric ulcer. Rats were divided into 5 groups: normal, ulcer, ulcer treated with 100 mg/kg bw LBP, ulcer treated with 50 mg/kg bw C-PC, and ulcer treated with 50 mg/kg bw LBP and 25 mg/kg bw C-PC. Pretreatment with LBP and/or C-PC was given a week before ulcer induction. Ulcer induction was produced by 50% ethanol administration orally every other day for 4 weeks. After 5-week treatment, the histopathological observation showed that LBP or C-PC attenuated the severity of gastric mucosal damage. LBP decreased serum malondialdehyde (MDA) levels and gastric interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) levels, and myeloperoxidase (MPO) activity. C-PC decreased serum MDA levels and gastric tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, ICAM-1 levels, and MPO activity. Combined LBP and C-PC decreased serum MDA levels and gastric TNF-α, IL-1ß, IL-6, and ICAM-1 levels. LBP and/or C-PC increased gastric heat shock protein 70 and non-protein sulfhydryl compounds. Rats with ulcer and treatment had enriched with the family Bacillaceae. Therefore, pretreatment with LBP and/or C-PC attenuated ethanol-induced gastric ulcer in rats via suppressing oxidation and inflammation and increasing gastroprotection.

Long-term Proton Pump Inhibitor Administration Caused Physiological and Microbiota Changes in Rats.

Proton pump inhibitors (PPIs) are used for the long-term treatment of gastroesophageal disorders and the non-prescription medicines for acid reflux. However, there is growing concerns about PPI misuse, overuse and abuse. This study aimed to develop an animal model to examine the effects of long-term use of PPI in vivo. Twenty one Wistar rats were given omeprazole orally or intravenously for 30 days, and caerulein as a positive control. After euthanization, the serum and stool were collected to perform MS-based quantitative analysis of metabolites. We carried out 16S-based profiling of fecal microbiota, assessed the expression of bile acid metabolism regulators and examined the immunopathological characteristics of bile ducts. After long-term PPI exposure, the fecal microbial profile was altered and showed similarity to those observed in high-fat diet studies. The concentrations of several metabolites were also changed in various specimens. Surprisingly, morphological changes were observed in the bile duct, including ductal epithelial proliferation, micropapillary growth of biliary epithelium, focal bile duct stricture formation and bile duct obstruction. These are characteristics of precancerous lesions of bile duct. FXR and RXRα expressions were significantly reduced, which were similar to that observed in cholangiocarcinoma in TCGA and Oncomine databases. We established a novel animal model to examine the effects of long-term use of omeprazole. The gut microbes and metabolic change are consequences of long-term PPI exposure. And the results showed the environment in vivo tends to a high-fat diet. More importantly, we observed biliary epithelial hyperplasia, which is an indicator of a high-fat diet.

Fish Oil, but Not Olive Oil, Ameliorates Depressive-Like Behavior and Gut Microbiota Dysbiosis in Rats under Chronic Mild Stress.

BACKGROUND: This study investigated the effects of fish oil and olive oil in improving dysbiosis and depressive-like symptoms. METHODS AND RESULTS: Male rats were fed normal, fish oil-rich or olive oil-rich diets for 14 weeks. Chronic mild stress (CMS) was administered from week 2. The sucrose preference test (SPT) and forced swimming test (FST) were used to determine depressive-like behavior. The SPT results revealed that the CMS, CMS with imipramine (CMS+P) treatment, and CMS with olive oil diet (CMS+O) groups exhibited significantly reduced sucrose intake from week 8, whereas the fish oil diet (CMS+F) group exhibited significantly reduced sucrose intake from week 10. The FST results showed that the immobile time of the CMS+F group was significantly less than that of the CMS-only group. Next generation sequencing (NGS) results showed CMS significantly reduced the abundance of Lactobacillus and increased that of Marvinbryantia and Ruminiclostridium_6. However, the CMS+F group showed an increase in the abundance of Eisenbergiella, Ruminococcaceae_UCG_009, and Holdemania, whereas the CMS+O group showed an increase in the abundance of Akkermansia. CONCLUSIONS: CMS stimuli altered the gut microbiome in depressed rats. Fish oil and olive oil exerted part of a prebiotic-like effect to ameliorate dysbiosis induced by CMS. However, only fish oil ameliorated depressive-like symptoms.

A high linoleic acid diet exacerbates metabolic responses and gut microbiota dysbiosis in obese rats with diabetes mellitus.

Dietary polyunsaturated fatty acid (PUFA) levels may affect inflammatory responses and lipid metabolism. Gut microbiota diversity is strongly associated with chronic inflammatory disease, diabetes mellitus (DM), and obesity through abnormal energy homeostasis. In this study, the association between metabolic responses and gut microbiota diversity at different dietary n-6/n-3 PUFA ratios was evaluated in DM rats. Obesity and DM were induced in rats by using a high-fat diet and streptozotocin (STZ), respectively. The obese DM rats were assigned to three groups and administered regular (R), high (H), and low (L) n-6/n-3 ratio diets (n-6/n-3 = 6.39, 3.02, and 9.29, respectively) for 6 weeks. Some metabolic parameters and gut microbiota of the rats were analysed. The results revealed that a high linoleic acid diet increased the plasma and kidney interleukin 6 levels, whereas a low n-6/n-3 ratio diet ameliorated blood glucose homeostasis, reduced plasma tumour necrosis factor α levels, and inhibited systematic inflammation. DM rats exhibited low gut microbiota diversity; however, compared with the R group, the L and H groups did not exhibit alterations in the α-diversity (Observed, Chao 1, Shannon and Simpson). The percentage of Firmicutes was lower in the DM groups than in the non-DM group; however, the L group showed a nonsignificantly higher Firmicutes/Bacteroidetes ratio than did the other groups. Thus, a low n-6/n-3 ratio diet can improve blood glucose homeostasis, reduce systematic inflammation, ameliorate glomerular basal membrane thickening, reduce the expression of receptors of advanced glycation end products in renal vessel walls, and prevent diabetic nephropathies.

DNA primase polypeptide 1 (PRIM1) involves in estrogen-induced breast cancer formation through activation of the G2/M cell cycle checkpoint.

The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real-time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 103 /µg) expression was 4.7-fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40-fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER- (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1-siRNA in the ER+ BT-474 cells-xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti-tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT-474-xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

Regulation of IL-20 Expression by Estradiol through KMT2B-Mediated Epigenetic Modification.

Cytokines are low molecular weight regulatory proteins, or glycoproteins, with both tumor-promoting and inhibitory effects on breast cancer growth. Different cytokines play important roles in breast cancer initiation and progression. Here, we show that of the 39 interleukin (IL) genes, IL-20 is the only gene over-expressed in MCF-7 cells treated with estradiol (E2) and that induction of IL-20 expression by estrogen was epigenetically regulated. Methylation of histone H3K4 in the IL-20 promoter was shown to occur via the specific recruitment of KMT2B by estrogen receptor alpha (ERα), but not by other members of the mixed-lineage leukemia (MLL) family of histone methyltransferases. Depletion of KMT2B, or IL-20, disrupts estrogen signaling, attenuates cell proliferation, reduces colony formation, and results in cell cycle arrest. Furthermore, we demonstrated that KMT2B-mediated epigenetic modification also affected the expression of several ERα target genes. IL-20 and KMT2B expression were also associated with ERα-positive breast cancer tissues. We have revealed an important role for KMT2B in the epigenetic transcriptional regulation of cytokine IL-20, and other ERα-responsive genes, in breast cancer cells. Inhibition of IL-20 and KMT2B may have therapeutic benefits in ERα-positive breast cancer.

An H2A Histone Isotype, H2ac, Associates with Telomere and Maintains Telomere Integrity.

Telomeres are capped at the ends of eukaryotic chromosomes and are composed of TTAGGG repeats bound to the shelterin complex. Here we report that a replication-dependent histone H2A isotype, H2ac, was associated with telomeres in human cells and co-immunoprecipitates with telomere repeat factor 2 (TRF2) and protection of telomeres protein 1 (POT1), whereas other histone H2A isotypes and mutations of H2ac did not bind to telomeres or these two proteins. The amino terminal basic domain of TRF2 was necessary for the association with H2ac and for the recruitment of H2ac to telomeres. Depletion of H2ac led to loss of telomeric repeat sequences, the appearance of dysfunctional telomeres, and chromosomal instability, including chromosomal breaks and anaphase bridges, as well as accumulation of telomere-associated DNA damage factors in H2ac depleted cells. Additionally, knockdown of H2ac elicits an ATM-dependent DNA damage response at telomeres and depletion of XPF protects telomeres against H2ac-deficiency-induced G-strand overhangs loss and DNA damage response, and prevents chromosomal instability. These findings suggest that the H2A isotype, H2ac, plays an essential role in maintaining telomere functional integrity.

Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing.

The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-RasV12-transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-RasV12-induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.