TGFß1-RCN3-TGFBR1 loop facilitates pulmonary fibrosis by orchestrating fibroblast activation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) bears high mortality due to unclear pathogenesis and limited therapeutic options. Therefore, identifying novel regulators is required to develop alternative therapeutic strategies. METHODS: The lung fibroblasts from IPF patients and Reticulocalbin 3 (RCN3) fibroblast-selective knockdown mouse model were used to determine the importance of Rcn3 in IPF; the epigenetic analysis and protein interaction assays, including BioID, were used for mechanistic studies. RESULTS: Reticulocalbin 3 (RCN3) upregulation is associated with the fibrotic activation of lung fibroblasts from IPF patients and Rcn3 overexpression blunts the antifibrotic effects of pirfenidone and nintedanib. Moreover, repressing Rcn3 expression in mouse fibroblasts ameliorates bleomycin-induced lung fibrosis and pulmonary dysfunction in vivo. Mechanistically, RCN3 promotes fibroblast activation by maintaining persistent activation of TGFß1 signalling via the TGFß1-RCN3-TGFBR1 positive feedback loop, in which RCN3 upregulated by TGFß1 exposure detains EZH2 (an epigenetic methyltransferase) in the cytoplasm through RCN3-EZH2 interaction, leading to the release of the EZH2-H3K27me3 epigenetic repression of TGFBR1 and the persistent expression of TGFBR1. CONCLUSIONS: These findings introduce a novel regulating mechanism of TGFß1 signalling in fibroblasts and uncover a critical role of the RCN3-mediated loop in lung fibrosis. RCN3 upregulation may cause resistance to IPF treatment and targeting RCN3 could be a novel approach to ameliorate pulmonary fibrosis.

Reticulocalbin 3 deficiency in alveolar epithelium attenuated LPS-induced ALI via NF-κB signaling.

Acute respiratory distress syndrome (ARDS) is characterized by acute lung injury (ALI) secondary to an excessive alveolar inflammatory response. Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein in the secretory pathway. We previously reported the indispensable role of Rcn3 in type II alveolar epithelial cells (AECIIs) during lung development and the lung injury repair process. In the present study, we further observed a marked induction of Rcn3 in the alveolar epithelium during LPS-induced ALI. In vitro alveolar epithelial (MLE-12) cells consistently exhibited a significant induction of Rcn3 accompanied with NF-κB activation in response to LPS exposure. We examined the role of Rcn3 in the alveolar inflammatory response by using mice with a selective deletion of Rcn3 in alveolar epithelial cells upon doxycycline administration. The Rcn3 deficiency significantly blunted the ALI and alveolar inflammation induced by intratracheal LPS instillation but not that induced by an intraperitoneal LPS injection (secondary insult); the alleviated ALI was accompanied by decreases in NF-κB activation and NLRP3 levels but not in GRP78 and cleaved caspase-3 levels. The studies conducted in MLE-12 cells consistently showed that Rcn3 knockdown blunted the activations of NF-κB signaling and NLRP3-dependent inflammasome upon LPS exposure. Collectively, these findings suggest a novel role for Rcn3 in regulating the alveolar inflammatory response to pulmonary infection via the NF-κB/NLRP3/inflammasome axis and shed additional light on the mechanism of ARDS/ALI.

Complete genome of a unicellular parasite (Antonospora locustae) and transcriptional interactions with its host locust.

Microsporidia are a large group of unicellular parasites that infect insects and mammals. The simpler life cycle of microsporidia in insects provides a model system for understanding their evolution and molecular interactions with their hosts. However, no complete genome is available for insect-parasitic microsporidian species. The complete genome of Antonospora locustae, a microsporidian parasite that obligately infects insects, is reported here. The genome size of A. locustae is 3 170 203 nucleotides, composed of 17 chromosomes onto which a total of 1857 annotated genes have been mapped and detailed. A unique feature of the A. locustae genome is the presence of an ultra-low GC region of approximately 25 kb on 16 of the 17 chromosomes, in which the average GC content is only 20 %. Transcription profiling indicated that the ultra-low GC region of the parasite could be associated with differential regulation of host defences in the fat body to promote the parasite's survival and propagation. Phylogenetic gene analysis showed that A. locustae, and the microsporidian family in general, is likely at an evolutionarily transitional position between prokaryotes and eukaryotes, and that it evolved independently. Transcriptomic analysis showed that A. locustae can systematically inhibit the locust phenoloxidase PPO, TCA and glyoxylate cycles, and PPAR pathways to escape melanization, and can activate host energy transfer pathways to support its reproduction in the fat body, which is an insect energy-producing organ. Our study provides a platform and model for studies of the molecular mechanisms of microsporidium-host interactions in an energy-producing organ and for understanding the evolution of microsporidia.

Epitope-directed antibody selection by site-specific photocrosslinking.

Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1ß and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.

Expression, location and biological effects of four and a half LIM domain protein 2 (FHL2) on granulosa cells in ovine.

Previous studies have shown that four and a half LIM domain protein 2 (FHL2) plays an essential role in the regulation of follicular development in mammals. Although the FHL2 genes of human and mouse have been well characterized, the expression and location of FHL2 in ovary and the biological functions of FHL2 on granulosa cells (GCs) of ovine are still not clear. In this study, full-length complementary DNA (cDNA) of FHL2 from ovine follicular GCs was amplified by real-time PCR (RT-PCR). The expression and location of FHL2 in ovary and GCs of ovine were studied by immunohistochemistry and immunofluorescence, and the biological effects of FHL2 on the cell proliferation, cell apoptosis, cell cycles and expression level of related genes of ovine GCs were also explored by overexpression or knockdown of FHL2. The results indicated that FHL2 was expressed in ovine follicular GCs and the sequence of the FHL2 cDNA was consistent with that predicted in GenBank, which did not cause an amino acid change. According to the results, FHL2 was expressed in ovine ovary and mainly located in the cytoplasm and nucleus of GCs. In addition, overexpression of FHL2 significantly reduced the cell viability, promoted the cell apoptosis and decreased the percentage of G0/G1 and S phase cells. RT-PCR showed that overexpression of FHL2 significantly increased the mRNA expression level of Bax and decreased the expression of Bcl-2 and the Bcl-2/Bax mRNA ratio compared with the control group. Besides, the knockdown of FHL2 gene in ovine GCs significantly improved the cell viability, suppressed the cell apoptosis, decreased the mRNA expression level of Caspase-3 gene, increased the Bcl-2/Bax mRNA ratio and increased the percentage of S and G2/M phase cells. Our results suggest that FHL2 may play an important role in the biological functions of GCs in ovine.

Organization of the Addax Major Histocompatibility Complex Provides Insights Into Ruminant Evolution.

Ruminants are critical as prey in transferring solar energy fixed by plants into carnivorous species, yet the genetic signature of the driving forces leading to the evolutionary success of the huge number of ruminant species remains largely unknown. Here we report a complete DNA map of the major histocompatibility complex (MHC) of the addax (Addax nasomaculatus) genome by sequencing a total of 47 overlapping BAC clones previously mapped to cover the MHC region. The addax MHC is composed of 3,224,151 nucleotides, harboring a total of 150 coding genes, 50 tRNA genes, and 14 non-coding RNA genes. The organization of addax MHC was found to be highly conserved to those of sheep and cattle, highlighted by a large piece of chromosome inversion that divided the MHC class II into IIa and IIb subregions. It is now highly possible that all of the ruminant species in the family of Bovidae carry the same chromosome inversion in the MHC region, inherited from a common ancestor of ruminants. Phylogenetic analysis indicated that DY, a ruminant-specific gene located at the boundary of the inversion and highly expressed in dendritic cells, was possibly evolved from DQ, with an estimated divergence time ~140 million years ago. Homology modeling showed that the overall predicted structure of addax DY was similar to that of HLA-DQ2. However, the pocket properties of P1, P4, P6, and P9, which were critical for antigen binding in the addax DY, showed certain distinctive features. Structural analysis suggested that the populations of peptide antigens presented by addax DY and HLA-DQ2 were quite diverse, which in theory could serve to promote microbial regulation in the rumen by ruminant species, contributing to enhanced grass utilization ability. In summary, the results of our study helped to enhance our understanding of the MHC evolution and provided additional supportive evidence to our previous hypothesis that an ancient chromosome inversion in the MHC region of the last common ancestor of ruminants may have contributed to the evolutionary success of current ruminants on our planet.

Network-Based Functional Prediction Augments Genetic Association To Predict Candidate Genes for Histamine Hypersensitivity in Mice.

Genetic mapping is a primary tool of genetics in model organisms; however, many quantitative trait loci (QTL) contain tens or hundreds of positional candidate genes. Prioritizing these genes for validation is often ad hoc and biased by previous findings. Here we present a technique for prioritizing positional candidates based on computationally inferred gene function. Our method uses machine learning with functional genomic networks, whose links encode functional associations among genes, to identify network-based signatures of functional association to a trait of interest. We demonstrate the method by functionally ranking positional candidates in a large locus on mouse Chr 6 (45.9 Mb to 127.8 Mb) associated with histamine hypersensitivity (Histh). Histh is characterized by systemic vascular leakage and edema in response to histamine challenge, which can lead to multiple organ failure and death. Although Histh risk is strongly influenced by genetics, little is known about its underlying molecular or genetic causes, due to genetic and physiological complexity of the trait. To dissect this complexity, we ranked genes in the Histh locus by predicting functional association with multiple Histh-related processes. We integrated these predictions with new single nucleotide polymorphism (SNP) association data derived from a survey of 23 inbred mouse strains and congenic mapping data. The top-ranked genes included Cxcl12, Ret, Cacna1c, and Cntn3, all of which had strong functional associations and were proximal to SNPs segregating with Histh. These results demonstrate the power of network-based computational methods to nominate highly plausible quantitative trait genes even in challenging cases involving large QTL and extreme trait complexity.

A high-density BAC physical map covering the entire MHC region of addax antelope genome.

BACKGROUND: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world. RESULTS: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing. CONCLUSIONS: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.

Human Papillomavirus 16 oncoprotein E7 retards mitotic progression by blocking Mps1-MAP4 signaling cascade.

Human epithelial cells can be infected by more than 200 types of human papilloma viruses (HPVs), and persistent HPV infections lead to cervical cancer or other deadly cancers. It has been established that mitotic progression is critical for HPV16 infection, but the underlying mechanism remains unknown. Here, we report that oncoprotein E7 of HPV16 but not HPV18 retards mitotic progression in host cell by direct binding to the C terminus of Microtubule-Associated Protein 4 (MAP4). MAP4 is a novel bona fide target of HPV16E7 protein which binds and recruits the latter to spindle microtubule in mitosis. HPV16E7 protein promotes MAP4 stability by inhibiting MAP4 phosphorylation- mediated degradation to increase the stability of microtubule polymerization and cause an extend mitotic progression. We further uncovered that Mps1 is a kinase of MAP4, and E7-MAP4 binding blocks Mps1 phosphorylation of MAP4, thereby interrupting phosphorylation-dependent MAP4 degradation. Mutations of MAP4 at T927ES928E partially abolished E7-binding capacity and rescued mitotic progression in host cells. In conclusion, our study reveals a molecular mechanism by which HPV16E7 perturbs host mitotic progression by interfering Mps1-MAP4 signaling cascade, which results in an extended infection window and may facilitate the persistent HPV16 infection.

Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

SHON expression predicts response and relapse risk of breast cancer patients after anthracycline-based combination chemotherapy or tamoxifen treatment.

BACKGROUND: SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). METHODS: SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα- early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. RESULTS: As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc- [HR (95% CI) = 0.52 (0.34-0.78), p = 0.002]. Meanwhile, in ERα- patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto- [HR (95% CI) = 0.50 (0.34-0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc- or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28-27.03), p = 0.012], or SHON-Cyto- [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18-25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc- [HR (95% CI) = 0.41 (0.19-0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto- patients [HR (95% CI) = 4.63 (1.05-20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13-44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. CONCLUSION: SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.

Reticulocalbin 3 Deficiency in Alveolar Epithelium Exacerbated Bleomycin-induced Pulmonary Fibrosis.

Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein localized to the secretory pathway. We have reported that Rcn3 plays a critical role in alveolar epithelial type II cell maturation during perinatal lung development, but its biological role in the adult lung is largely unknown. In this study, we found marked induction of Rcn3 expression in alveolar epithelium during bleomycin-induced pulmonary fibrosis, which is most obvious in alveolar epithelial type II cells (AECIIs). To further examine Rcn3 in pulmonary injury remodeling, we generated transgenic mice to selectively delete Rcn3 in AECIIs in adulthood. Although Rcn3 deletion did not cause obvious abnormalities in the lung architecture and mechanics, the exposure of Rcn3-deleted mice to bleomycin led to exacerbated pulmonary fibrosis and reduced lung mechanics. These Rcn3-deleted mice also displayed enhanced alveolar epithelial cell (AEC) apoptosis and ER stress after bleomycin treatment, which was confirmed by in vitro studies both in primary AECIIs and mouse lung epithelial cells. Consistently, Rcn3 deficiency also enhanced ER stress and apoptosis induced by ER stress inducers, tunicamycin and thapsigargin. In addition, Rcn3 deficiency caused blunted wound closure capability of AECs, but not altered proliferation and bleomycin-induced epithelial-mesenchymal transition process. Collectively, these findings indicate that bleomycin-induced upregulation of Rcn3 in AECIIs appears to contribute to AECII survival and wound healing. These observations, for the first time, suggest a novel role of Rcn3 in regulating pulmonary injury remodeling, and shed additional light on the mechanism of idiopathic pulmonary fibrosis.

Extensive diversity and evolution of hepadnaviruses in bats in China.

To better understand the evolution of hepadnaviruses, we sampled bats from Guizhou, Henan and Zhejiang provinces, China, and rodents from Zhejiang province. Genetically diverse hepadnaviruses were identified in a broad range of bat species, with an overall prevalence of 13.3%. In contrast, no rodent hepadnaviruses were identified. The newly discovered bat hepadnaviruses fell into two distinct phylogenetic groups. The viruses within the first group exhibited high diversity, with some closely related to viruses previously identified in Yunnan province. Strikingly, the newly discovered viruses sampled from Jiyuan city in the second phylogenetic group were most closely related to those found in bats from West Africa, suggestive of a long-term association between bats and hepadnaviruses. A co-phylogenetic analysis revealed frequent cross-species transmission among bats from different species, genera, and families. Overall, these data suggest that there are likely few barriers to the cross-species transmission of bat hepadnaviruses.

Epigenetic silencing of TET2 and TET3 induces an EMT-like process in melanoma.

Epithelial-Mesenchymal Transition (EMT) is a critical step in the progression of cancer. Malignant melanoma, a cancer developed from pigmented melanocytes, metastasizes through an EMT-like process. Ten-eleven translocation (TET) enzymes, catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5-hmC), are down regulated in melanoma. However, their roles in the progression and the EMT-like process of melanoma are not fully understood. Here we report that DNA methylation induced silencing of TET2 and TET3 are responsible for the EMT-like process and the metastasis of melanoma. TET2 and TET3 are down regulated in the TGF-ß1-induced EMT-like process, and the knocking down of TET2 or TET3 induced this EMT-like process. A DNA demethylating agent antagonized the TGF-ß-induced suppression of TET2 and TET3. Furthermore, a ChIP analysis indicated that enhanced recruitment of DNMT3A (DNA Methyltransferase 3A) is the mechanism by which TGF-ß induces the silencing of TET2 and TET3. Finally, the overexpression of the TET2 C-terminal sequence partially rescues the TGF-ß1-induced EMT-like process in vitro and inhibits tumor growth and metastasis in vivo. Hence, our data suggest an epigenetic circuitry that mediates the EMT activated by TGF-ß. As an effector, DNMT3A senses the TGF-ß signal and silences TET2 and TET3 promoters to induce the EMT-like process and metastasis in melanoma.

A Preliminary Study on RCN3 Protein Expression in Non-small Cell Lung Cancer.

BACKGROUND: Reticulocalbin 3 (RCN3), a member of CREC (Cab45/reticulocalbin/ ERC-45/calumenin) family protein, is located in the secretory pathway of endoplasmic reticulum (ER) of living cells. Disruption of RCN3 leads to failure of lung function in the mouse model. Although ER stress has been associated with the development of a variety of tumors, the role of RCN3 in development of non-small cell lung cancer (NSCLC) in human is unknown at present. METHODS: In this study a total of 41 paired NSCLC specimens (cancer group) and the adjacent normal tissues (control group) were obtained from patients undergoing lung lobectomy or pneumonectomy surgeries in Beijing Shijitan Hospital, Capital Medical University. The RCN3 mRNA and protein level in each clinical sample was determined using quantitative real time-PCR and immunoblotting, respectively. Immunohistochemistry analysis was utilized to compare the protein expressional patterns of RCN3 between the two clinical sample groups. RESULTS: Immunoblotting showed that levels of RCN3 protein in the NSCLC tissues were significantly lower than those in the control group (p < 0.001), suggesting ER stress is closely associated with the cancer cells. Accordingly, the ER stress protein GRP78 (glucose-regulated protein 78, also known as BIP) was remarkably upregulated in the cancer group (p < 0.05). Within the cancer group, a significant difference in RCN3 protein expression was observed in squamous cell carcinoma versus adenocarcinoma (p < 0.05). In the lung cancer group, however, RCN3 protein levels were not correlated with the age and the gender. In addition, RCN3 mRNA levels showed no significant difference between the cancer and the control groups, suggesting that the differential regulation of RCN3 is likely at post-transcription stage in NSCLC. CONCLUSIONS: Our study showed that RCN3 protein level was significantly down regulated in NSCLC, suggesting a potential correlation between RCN3 protein depletion and development of NSCLC. Although the exact cause-effect relationship between RCN3 and NSCLC needs to be further investigated, the study helps to shed additional lights on the molecular regulation of the lung cancer.

Neonatal Respiratory Failure with Retarded Perinatal Lung Maturation in Mice Caused by Reticulocalbin 3 Disruption.

Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum lumen protein localized to the secretory pathway. As a Ca2t-binding protein of 45 kDa (Cab45)/Rcn/ER Ca2t-binding protein of 55 kDa (ERC45)/calumenin (CREC) family member, Rcn3 is reported to function as a chaperone protein involved in protein synthesis and secretion; however, the biological role of Rcn3 is largely unknown. The results presented here, for the first time, depict an indispensable physiological role of Rcn3 in perinatal lung maturation by using an Rcn3 gene knockout mouse model. These mutant mice die immediately at birth owing to atelectasis-induced neonatal respiratory distress, although these embryos are produced with grossly normal development. This respiratory distress results from a failure of functional maturation of alveolar epithelial type II cells during alveogenesis. This immaturity of type II cells is associated with a dramatic reduction in surfactant protein A and D, a disruption in surfactant phospholipid homeostasis, and a disorder in lamellar body. In vitro studies further show that Rcn3 deficiency blunts the secretion of surfactant proteins and phospholipids from lung epithelial cells, suggesting a decrease in availability of surfactants for their surface activity. Collectively, these observations indicate an essential role of Rcn3 in perinatal lung maturation and neonatal respiratory adaptation as well as shed additional light on the mechanism of neonatal respiratory distress syndrome development.

Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice.

Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3(-/-)) and wild-type (AC3(+/+)) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3(-/-) mice was significantly altered, compared to AC3(+/+) mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3(-/-) and AC3(+/+) mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

Spina bifida in fetus is associated with an altered pattern of DNA methylation in placenta.

Failure in closure of neural tube leads to neural tube defects (NTDs), which are among the most common symptoms of human birth defects. Although epigenetic status in placenta is linked to fetal development, the mechanism behind this remains unknown. Because of the importance of DNA methylation in gene function, we set to explore whether or not DNA methylation in human placenta is also linked to fetal NTDs. Here we show for the first time that alteration of DNA methylation in placenta is closely associated with the phenotypes of fetal spina bifida (Sb). We found that patterns of DNA methylation for genes in neurological system process were differentially altered in the Sb placenta. In particular, the transcription regulatory regions of TRIM26 and GANS were kept at the hypomethylation status in Sb placenta alone. Accordingly, the protein levels of TRIM26 and GNAS were significantly elevated only in the Sb placenta but not in the Sb-affected fetuses. In cellular model of CHO cells deficient in Dihydrofolate reductase and treated with 5-aza-2'-deoxycytidine, the protein levels of GNAS and TRIM26 were significantly higher than those in normal control cells. These findings suggested that epigenetic status of genes in placenta have profound impacts on the development of NTDs.

A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

BACKGROUND: Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear. RESULTS: In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. CONCLUSION: Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

Overexpression of Mps1 in colon cancer cells attenuates the spindle assembly checkpoint and increases aneuploidy.

The spindle assembly checkpoint kinase Mps1 is highly expressed in several types of cancers, but its cellular involvement in tumorigenesis is less defined. Herein, we confirm that Mps1 is overexpressed in colon cancer tissues. Further, we find that forced expression of Mps1 in the colon cancer cell line SW480 enables cells to become resistant to both Mps1 inhibition-induced checkpoint depletion and cell death. Overexpression of Mps1 also increases genome instability in tumor cells owing to a weakened spindle assembly checkpoint. Collectively, our findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint.