TTN Mutation in Endometrial Endometrioid Carcinoma Is Associated with Poor Clinical Outcomes and High Tumor Mutation Burden.

Endometrioid endometrial carcinoma (EEC) stands as a prevalent gynecologic malignancy in developed regions. However, predicting relapse cases remains challenging, necessitating the identification of a novel biomarker for EEC relapse. The assessment of tumor mutational burden (TMB) is pivotal for immunotherapy in EEC patients. However, both whole-exome sequencing (WES) and targeted sequencing encountered application-related difficulties. In light of this, standardized and simplified techniques for TMB measurement are imperative. In this study, we employed WES on 25 EEC patients (12 relapsed cases and 13 non-relapsed cases) who accepted hysterectomy surgery (CHCAMS cohort). We additionally obtained a total of 391 tumor samples with clinicopathological features from TCGA website to broaden the study cohort. In the CHCAMS cohort, the TTN mutant group showed shorter progression-free survival (p < 0.001) and overall survival (p < 0.001) than TTN wild-type group. Additionally, we discovered that the number of TTN mutations per sample was significantly linked with TMB-WES in CHCAMS cohort and TCGA cohort (p < 0.05). And the number of TTN mutations per sample in POLE mutant group was greater than in the POLE wild-type group (p < 0.0001). In conclusion, TTN mutation may serve as a biomarker for EEC prognosis. TTN mutation is also associated with WES-TMB, and could be a simplified TMB measurement technique.

Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction.

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 µM). PE5 (50, 100, 200 µM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.

Alternative splicing of the human rhomboid family-1 gene RHBDF1 inhibits epidermal growth factor receptor activation.

The human rhomboid-5 homolog-1 (RHBDF1) is a multi-transmembrane protein present mainly on the endoplasmic reticulum. RHBDF1 has been implicated in the activation of epidermal growth factor receptor (EGFR)-derived cell growth signals and other activities critical to cellular responses to stressful conditions, but details of this activation mechanism are unclear. Here, we report a RHBDF1 mRNA transcript alternative splicing variant X6 (RHBDF1 X6 or RHX6) that antagonizes RHBDF1 activities. We found that while the RHBDF1 gene is marginally expressed in breast tumor-adjacent normal tissues, it is markedly elevated in the tumor tissues. In sharp contrast, the RHX6 mRNA represents the primary RHBDF1 variant in normal breast epithelial cells and tumor-adjacent normal tissues but is diminished in breast cancer cells and tumors. We demonstrate that, functionally, RHX6 acts as an inhibitor of RHBDF1 activities. We show that artificially overexpressing RHX6 in breast cancer cells leads to retarded proliferation, migration, and decreased production of epithelial-mesenchymal transition-related adhesion molecules. Mechanically, RHX6 is able to inhibit the maturation of TACE, a protease that processes pro-TGFα, a pro-ligand of EGFR, and to prevent intracellular transportation of pro-TGFα to the cell surface. Additionally, we show that the production of RHX6 is under the control of the alternative splicing regulator RNA binding motif protein-4 (RBM4). Our findings suggest that differential splicing of the RHBDF1 gene transcript may have a regulatory role in the development of epithelial cell cancers.

RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

Melanoma is a dangerous form of skin cancer, making it important to investigate new mechanisms and approaches to enhance the effectiveness of treatment. Here, we establish a positive correlation between the human rhomboid family-1 (RHBDF1) protein and melanoma malignancy. We demonstrate that the melanoma RHBDF1 decrease dramatically inhibits tumor growth and the development of lung metastases, which may be related to the impaired glycolysis. We show that RHBDF1 function is essential to the maintenance of high levels of glycolytic enzymes, especially glucose-6-phosphate isomerase (GPI). Additionally, we discover that the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) mediates the K27/K63-linked ubiquitination of GPI and the ensuing lysosomal degradation process. We prove that the multi-transmembrane domain of RHBDF1 is in competition with GPI, preventing the latter from interacting with NCL1-HT2A-LIN41 (NHL) domain of TRIM32. We also note that the mouse RHBDF1's R747 and Y799 are crucial for competitive binding and GPI protection. Artificially silencing the Rhbdf1 gene in a mouse melanoma model results in declined lactic acid levels, elevated cytotoxic lymphocyte infiltration, and improved tumor responsiveness to immunotherapy. These results provide credence to the hypothesis that RHBDF1 plays a significant role in melanoma regulation and suggest that blocking RHBDF1 may be an efficient technique for reestablishing the tumor immune microenvironment (TIME) in melanoma and halting its progression.

The effects of ethical leadership on creativity: A conservation of resources perspective.

The pursuit of novelty can be a challenging experience that often comes with stress. Thinking outside the box can even lead to ethical dilemmas, particularly when innovators are under the pressure to meet deadlines. In this study, we examine creativity as a stress-inducing process, especially when employees encounter setbacks during their pursuit of novelty. Our aim was to explore the relationship between ethical leadership and creativity from a Conservation of Resources (COR) perspective. Using two distinct research samples, we discovered that help seeking behavior during the pursuit of novelty is crucial for acquiring resources in the workplace and serves as a mediator in the relationship between ethical leadership and creativity. We also discuss the theoretical and practical implications of these findings.

TL1A inhibits atherosclerosis in apoE-deficient mice by regulating the phenotype of vascular smooth muscle cells.

TNF ligand-related molecule 1A (TL1A) is a vascular endothelial growth inhibitor to reduce neovascularization. Lack of apoE a expression results in hypercholesterolemia and atherosclerosis. In this study, we determined the precise effects of TL1A on the development of atherosclerosis and the underlying mechanisms in apoE-deficient mice. After 12 weeks of pro-atherogenic high-fat diet feeding and TL1A treatment, mouse aorta, serum, and liver samples were collected and used to assess atherosclerotic lesions, fatty liver, and expression of related molecules. We found that TL1A treatment significantly reduced lesions and enhanced plaque stability. Mechanistically, TL1A inhibited formation of foam cells derived from vascular smooth muscle cells (VSMCs) but not macrophages by activating expression of ABC transporter A1 (ABCA1), ABCG1, and cholesterol efflux in a liver X receptor-dependent manner. TL1A reduced the transformation of VSMCs from contractile phenotype into synthetic phenotypes by activating expression of contractile marker α smooth muscle actin and inhibiting expression of synthetic marker osteopontin, or osteoblast-like phenotype by reducing calcification. In addition, TL1A ameliorated high-fat diet-induced lipid metabolic disorders in the liver. Taken together, our work shows that TL1A can inhibit the development of atherosclerosis by regulating VSMC/foam cell formation and switch of VSMC phenotypes and suggests further investigation of its potential for atherosclerosis treatment.

Impact of Intracerebral Hematoma Evacuation During Decompressive Hemicraniectomy on Functional Outcomes.

BACKGROUND AND PURPOSE: Decompressive hemicraniectomy has been used to treat spontaneous intracerebral hemorrhage, but the benefit of evacuating the hematoma during the procedure is unclear. We aim to evaluate the utility of performing clot evacuation during hemicraniectomy for spontaneous intracerebral hemorrhage. METHODS: Retrospective cohort of consecutive patients (2010-2019) treated with decompressive hemicraniectomy for a spontaneous supratentorial intracerebral hemorrhage at the University of Iowa. We compared hemicraniectomy alone to hemicraniectomy plus hematoma evacuation. We analyzed clinical features and hematoma characteristics. The outcomes at 6 months were dichotomized into unfavorable (Glasgow Outcome Scale score 1-3) and favorable (Glasgow Outcome Scale score 4-5). RESULTS: Eighty-three patients underwent decompressive hemicraniectomy for spontaneous intracerebral hemorrhage, 52 with hematoma evacuation, and 31 without hematoma evacuation. There were no statistically significant differences in clinical and radiographic characteristics between the 2 groups. Evacuating the hematoma in addition to hemicraniectomy did not change the odds of favorable outcome at 6 months (P=0.806). CONCLUSIONS: In this retrospective study, the performance of hematoma evacuation during decompressive hemicraniectomy for spontaneous intracerebral hemorrhage may not change functional outcomes over performing the hemicraniectomy alone.

Concentration of Lp(a) (Lipoprotein[a]) in Aneurysm Sac Is Associated With Wall Enhancement of Unruptured Intracranial Aneurysm.

BACKGROUND AND PURPOSE: Atherosclerotic remodeling of the aneurysm wall, which could be detected as aneurysm wall enhancement (AWE) by magnetic resonance-vessel wall imaging, is a part of degenerative change of unruptured intracranial aneurysms (UIAs). The purpose of this study was to determine whether the luminal concentrations of atherosclerotic proteins in the aneurysm sac were associated with increased wall enhancement of UIAs in vessel wall imaging. METHODS: We performed a prospective study of subjects undergoing endovascular treatments for UIAs. All subjects underwent evaluation using 3T-magnetic resonance imaging, including pre/postcontrast vessel wall imaging of the UIAs. Blood samples were collected from the aneurysm sac and the parent artery during endovascular procedures. Presence/absence of AWE was correlated with the delta difference in concentration for each atherosclerotic protein between the lumen of UIA and in the parent artery. RESULTS: A total of consecutive 17 patients with 19 UIAs were enrolled. The delta difference of lipoprotein(a) was significantly higher in UIAs with AWE compared with those without AWE (-6.9±16.0 versus -45.4±44.9 µg/mL, P=0.03). CONCLUSIONS: Higher luminal concentrations of lipoprotein(a) in the aneurysm sac were significantly associated with increased wall enhancement of UIAs. A larger study is needed to confirm these findings.

Genetic screening and multipotency in rhesus monkey haploid neural progenitor cells.

Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, a mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium show naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period in vitro High-throughput trapping mutations can be efficiently introduced into haNPCs via piggyBac transposons. This system proves useful when identifying gene targets of neural toxicants via a proof-of-concept experiment. Using CRISPR/Cas9 editing, we confirmed that B4GALT6, from the candidate gene list, is a resistance gene of A803467 (a tetrodotoxin-like toxicant). This model is the first non-human primate haploid somatic cell line with proliferative ability, multipotency and an intact genome, thus providing a cellular resource for recessive genetic and potential drug screening.

Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus.

Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J.IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

RHBDF2 gene functions are correlated to facilitated renal clear cell carcinoma progression.

BACKGROUND: The rhomboids are a family of multi-transmembrane proteins, many of which have been implicated in facilitating tumor progression. Little is yet known, however, about rhomboid-associated biomarkers in cancers. An analysis of such biomarkers could yield important insights into the role of the rhomboids in cancer pathology. METHODS: In this study, we carried out the univariate Cox regression analysis and compared gene expression patterns of several rhomboid genes in 30 types of cancers by using The Cancer Genome Atlas (TCGA) database and the methods delineated in Gene Expression Profiling Interactive Analysis (GEPIA). We then used datasets GSE47032, GSE126964, GSE68417 and 75 paired pathological specimens to verify the influences of the rhomboid genes in cancer progression. Moreover, we carried out Weighted Gene Correlation Network Analysis (WGCNA) to investigate gene-related functions and we exploited potential correlations between rhomboid genes expression and immune cell infiltration in cancer tissues. Furthermore, we constructed gene-knockdown cancer cell lines to investigate rhomboid gene functions. RESULTS: We find that kidney renal clear cell carcinoma (KIRC) disease progression is affected by fluctuations in the expression of a number of the rhomboid family of genes and, more specifically, high levels of RHBDF2 gene expression are a good indicator of poor prognosis of the disease, as patients with high RHBDF2 expression levels exhibit less favorable survival rates compared to those with low RHBDF2 levels. Silencing of the RHBDF2 gene in KIRC cell lines leads to significantly diminished cell proliferation and migration; this is in good agreement with the identification of an enhanced presence of a number of cell growth and migration promoting signaling molecules in KIRC tumors. We found that, although high level of RHBDF2 correlated with increased infiltration of lymphocytes in cancer tissues, artificially overexpressed RHBDF2 led to an inhibition of the activity of the infiltrated immune cells through sustaining PD-L1 protein level. Furthermore, we show that RHBDF2 related cell migration and PD-L1 regulation were potentially mediated by EGFR signaling pathway. CONCLUSIONS: RHBDF2 gene functions are correlated to facilitated renal clear cell carcinoma progression and may serve as a critical prognostic biomarker for the disease.

A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Effect of Coformer Selection on In Vitro and In Vivo Performance of Adefovir Dipivoxil Cocrystals.

PURPOSE: This study aimed to improve the in vitro dissolution, permeability and oral bioavailability of adefovir dipivoxil (ADD) by cocrystal technology and clarify the important role of coformer selection on the cocrystal's properties. METHODS: ADD was cocrystallized with three small molecules (i.e., paracetamol (PA), saccharin (SAC) and nicotinamide (NIC)), respectively. The obtained ADD-PA cocrystal was characterized by DSC, TGA, PXRD and FTIR. Comparative study on dissolution rates among the three ADD cocrystals were conducted in water and pH 6.8 phosphate buffer. Besides, effects of coformers on intestinal permeability of ADD were evaluated via in vitro Caco-2 cell model and in situ single-pass intestinal perfusion model in rats. Furthermore, in vivo pharmacokinetic study of ADD cocrystals was also compared. RESULTS: Dissolution rates of ADD cocrystals were improved with the order of ADD-SAC cocrystal > ADD-PA cocrystal > ADD-NIC cocrystal. The permeability studies on Caco-2 cell model and single-pass intestinal perfusion model indicated that PA could enhance intestinal absorption of ADD by P-gp inhibition, while SAC and NIC did not. Further in vivo pharmacokinetic study showed that ADD-SAC cocrystal exhibited higher Cmax (1.4-fold) and AUC0-t (1.3-fold) of ADD than administration of ADD alone, and Cmax and AUC0-t of ADD-PA cocrystal were significantly enhanced by 2.1-fold and 2.2-fold, respectively, which was attributed to its higher dissolution and improved intestinal permeability. CONCLUSION: Coformer selection had an important role on cocrystal's properties, and cocrystallization of ADD with a suitable coformer was an effective approach to enhance both dissolution and bioavailability of ADD.

Synthesis and biological evaluation of all possible inosine-mixed cyclic dinucleotides that activate different hSTING variants.

Cyclic dinucleotides (CDNs) could activate stimulator of interferon genes (STING) protein to produce type I interferon and other pro-inflammation cytokines in mammalian cells. To explore new types of potentially efficient STING activators targeting all five major hSTING variants (WT, R232H, HAQ, AQ and R293Q), we here reported the synthesis of a total of 19 inosine-containing CDNs based on the combinations of hypoxanthine with four natural bases (A, G, C and U) and three phosphodiester linkage backbones (3'-3', 2'-3', 2'-2'). The IFN-ß induction results showed that all of the 2'-3' and 2'-2' CDNs linked by inosine and purine nucleosides favored the stacking interaction with Y167 and R238 residues of hSTING protein, and several CDNs constructed by hypoxanthine and pyrimidine like c[I(2',5')U(2',5')] could also activate all five hSTING variants. The molecular dynamic simulation and the isothermal titration calorimetric (ITC) assay further demonstrated the potential of cAIMP isomers with 2'-5' phosphate to form the hydrogen binding with R232 and R238 residues of hSTING in an entropically driven manner compared to cGAMP isomers. It would be promising to exploit novel inosine-mixed CDNs as activators of hSTING variants in immune therapy.

TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway.

The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB-derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)-derived HSC. TNFSF15-treated UCB-HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi-SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle-treated group. Additionally, signal proteins of the Notch pathway are highly up-regulated in TNFSF15-treated UCB-HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB-HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB-HSC application or study.

Inhibition of intracranial hemangioma growth and hemorrhage by TNFSF15.

Hemangioblastoma (HB) is an abnormal intracranial buildup of blood vessels that exhibit a great potential for hemorrhage. Surgical options are limited, and few medications are available for treatment. We show here by immunohistochemical analysis that HB lesions display highly increased levels of VEGF expression and macrophage/microglia infiltration compared with those in normal brain tissues. In the meantime, TNF superfamily 15 (TNFSF15) (also known as vascular endothelial growth inhibitor), an antiangiogenic cytokine, is highly expressed in normal brain blood vessels but diminished in HB lesions. We set up a brain hemangioma model by using mouse bEnd.3 cells of a T antigen-transformed endothelial cell line that produce a large amount of VEGF. When implanted in mouse brains, these cells form lesions that closely resemble the pathologic characteristics of HB. Retroviral infection of bEnd.3 cells with TNFSF15 leads to inhibition of VEGF production and retardation of hemangioma formation. Similar results are obtained when wild-type bEnd.3 cells are implanted in the brains of transgenic mice overexpressing TNFSF15. Additionally, TNFSF15 treatment results in enhanced pericyte coverage of the blood vessels in the lesions together with reduced inflammatory cell infiltration and decreased hemorrhage. These findings indicate that the ability of TNFSF15 to counterbalance the abnormally highly angiogenic and inflammatory potential of the microenvironment of HB is of therapeutic value for the treatment of this disease.-Yang, G.-L., Han, Z., Xiong, J., Wang, S., Wei, H., Qin, T.-T., Xiao, H., Liu, Y., Xu, L.-X., Qi, J.-W., Zhang, Z.-S., Jiang, R., Zhang, J., Li, L.-Y. Inhibition of intracranial hemangioma growth and hemorrhage by TNFSF15.

Perturbation of epithelial apicobasal polarity by rhomboid family-1 gene overexpression.

The human rhomboid family (RHBDF)1 gene is highly expressed in breast cancer under clinical conditions but not in normal mammary gland tissues. Silencing the RHBDF1 gene in breast cancer xenograft tumors leads to inhibition of tumor growth. We show in this study that artificially raising RHBDF1 protein levels in the mammary epithelial cells MCF-10A results in severe perturbations of the ability of the cells to form lumen-containing acini, either in 3-dimensional cell cultures or implanted in mouse mammary fat pads. Knocking down RHBDF1 with short hairpin (sh)RNA leads to restoration of acinus formation. Consistently, RHBDF1 overexpression gives rise to disordered distribution of polarity markers GM130 and laminin-5, which otherwise are located in apical and basal positions, respectively, in the acini. Further investigations reveal that RHBDF1 directly binds to Par6a, a component of a protein complex consisting of partitioning-defective scaffold protein (Par)6, Par3, renin-angiotensin system-related C3 botulinum toxin substrate (Rac)1, and cell-division cycle (Cdc)42, which is structurally critical to the formation of apicobasal polarity. RHBDF1 binding to Par6a results in collapse of the protein complex and thus disruption of polarity formation. Since early stages of breast cancer are characterized by the loss of mammary gland epithelial cell polarity, our findings indicate that perturbations of apicobasal polarity by high levels of RHBDF1 is a significant attribute in the development of breast neoplasia.-Peng, X.-M., Gao, S., Deng, H.-T., Cai, H.-X., Zhou, Z., Xiang, R., Zhang, Q.-Z., Li, L.-Y. Perturbation of epithelial apicobasal polarity by rhomboid family-1 gene overexpression.

CRISPR RNA Array-Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1.

To achieve multisite-targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA-array-guided FnCpf1 nuclease showed target-activated, nonspecific DNase activity on both target DNA and nontarget DNA at random sites. Through optimization of the ratio of nuclease and CRIPSR RNAs, CRISPRay should further enhance gene interference in cells. This work presents a simple approach through which to improve multisite-directed gene disruption by fusing four guide RNAs (sgRNAs or crRNAs) into a CRISPR RNA string.

Activation of hepatic Nogo-B receptor expression-A new anti-liver steatosis mechanism of statins.

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or ß-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression.

A target-triggered biosensing platform for detection of HBV DNA based on DNA walker and CHA.

Hepatitis B virus (HBV), one of the causative agents of viral hepatitis, may lead to chronic hepatitis, cirrhosis, and liver cancer. In this work, we designed a sensitive and modular biosensing platform for detecting HBV DNA based on a DNA walker that hangs on to surfaces and a catalyst-triggered catalyzed hairpin assembly (CHA). In the presence of HBV DNA, strand displacement reaction between target and double-stranded complex caused the release of walker strand to trigger the DNA walker. Then, a catalyst was free to open the trapped hairpins to form a new double-strand complex, driving the CHA reaction. Thus, a powerful cascade amplification reaction realized in DNA walker and CHA based on toehold-mediated strand displacement reaction in this system. To achieve quantitative detection of HBV DNA, a fluorescent-quencher signaling pair was employed, the turn-on fluorescence provided an analytical signal. A wide detection range from 0.5 nM to 50 nM with a detection limit as low as 0.20 nM was reached on the condition of acceptable specificity and reproducibility. We could also further apply it to multiple different bioanalysis by changing adjustable elements. This reported biosensor opened a new avenue for sensitivity and modularity of DNA detection.