We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.

In order to test the ability of peripheral blood gene expression profiles to predict future disease severity in patients with early rheumatoid arthritis (RA), a group of 17 patients (1 ± 0.2 years disease duration) was evaluated at baseline for gene expression profiles. Disease status was evaluated after a mean of 5 years using an index combining pain, global and recoded MHAQ scores. Unsupervised and supervised algorithms identified "predictor genes" whose combined expression levels correlated with follow-up disease severity scores. Unsupervised clustering algorithms separated patients into two branches. The only significant difference between these two groups was the disease severity score; demographic variables and medication usage were not different. Supervised T-Test analysis identified 19 "predictor genes" of future disease severity. Results were validated in an independent cohort of subjects of established RA with using Support Vector Machines and K-Nearest-Neighbor Classification. Our study demonstrates that peripheral blood gene expression profiles may be a useful tool to predict future disease severity in patients with early and established RA.

To determine whether docosahexaenoic acid can protect against hereditary retinal degenerations in transgenic mice expressing the V20G, P23H, and P27L (VPP) rhodopsin mutations.

The Tubby mouse is a phenotypic model for sensorineural deafness and retinal dystrophy including Usher syndrome type 1. Thioredoxin is a small 13kDa protein which, when ubiquitously expressed as a transgene in the mouse, provides protection against multiple disease states including light-induced and oxidative stress-induced neurodegeneration and is down-regulated in the Tubby retina. We tested if overexpression of human thioredoxin in the Tubby mouse inhibits retinal degeneration and loss of visual function. Electroretinography, immunocytochemistry, quantitative histology, RT-PCR and Western blots were used to obtain data which showed that thioredoxin overexpression prevented loss of photoreceptors and retinal function. Analysis of signal pathways showed that thioredoxin up-regulated neurotrophic factors BDNF and GDNF and activated survival signaling pathways Akt, Ras/Raf1/ and the ERKs while inhibiting the ASK1/JNK apoptosis pathway. Relationships between the Tubby gene, its pathological phenotype and regulation of the thioredoxin system remain to be established.

Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo.

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

For identification of genes responsible for varietal differences in flowering time and leaf morphological traits, we constructed a linkage map of Brassica rapa DNA markers including 170 EST-based markers, 12 SSR markers, and 59 BAC sequence-based markers, of which 151 are single nucleotide polymorphism (SNP) markers. By BLASTN, 223 markers were shown to have homologous regions in Arabidopsis thaliana, and these homologous loci covered nearly the whole genome of A. thaliana. Synteny analysis between B. rapa and A. thaliana revealed 33 large syntenic regions. Three quantitative trait loci (QTLs) for flowering time were detected. BrFLC1 and BrFLC2 were linked to the QTLs for bolting time, budding time, and flowering time. Three SNPs in the promoter, which may be the cause of low expression of BrFLC2 in the early-flowering parental line, were identified. For leaf lobe depth and leaf hairiness, one major QTL corresponding to a syntenic region containing GIBBERELLIN 20 OXIDASE 3 and one major QTL containing BrGL1, respectively, were detected. Analysis of nucleotide sequences and expression of these genes suggested possible involvement of these genes in leaf morphological traits.

Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3.

During the 2009 influenza (H1N1) pandemic, some countries used quarantine for containment or mitigation. Of 152 quarantined university students we studied, risk for illness was higher for students quarantined in a room with a person with a confirmed case; we found no difference between students quarantined in double or single rooms.

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.

Carbonic anhydrase IX(CA9)is a member of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide, and plays a key role in the regulation of pH. Although a large number of studies have shown that CA9 is strongly up-regulated by HIF1-α, little is known about the negative regulation mechanism of CA9 in cancer cells. Here we find that CREB is a key negative regulator of CA9 in gastric cancer. Over-expression of CREB can significantly repress the expression of CA9. Treating with anisomycin (ANS), an activator of p38, the phosphorylation and nuclear translocation of CREB are both promoted, while the transcription of CA9 is repressed. Besides, our results firstly identify that CREB can recruit SIRT1 (class III HDACS) by adaptor protein p300, then repress the expression of CA9. These findings may contribute to understand the negative regulation mechanisms of CA9 in gastric cancer.

Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs) targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX), monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT) were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.

Janus kinase 2 (JAK2) plays important roles in the regulation of varieties cellular processes including cell migration, proliferation and apoptosis. JAK2 I682F genetic mutation existed in the 4-8% of B-cell acute lymphoblastic leukemia (B-ALL). However, roles of this mutation in the development of B-ALL are still unknown. In order to investigation the mechanism of the JAK2 I682F mutation led to B-ALL, series of mutations were constructed. Mutations I682F, I682G, I682D and I682S significantly increased JAK2's activity and decreased its structural stability, while the I682L mutation almost had no effect on JAK2's activity and structural stability. Furthermore, the spectroscopic experiments implied that mutations I682F, I682G, I682D and I682S impaired the structure of JAK2 JH2 domain, and led JAK2 to the partially unfolded state. It may be this partially unfolded state that caused JAK2 I682F constitutive activation. This study provides clues in understanding the mechanism of the JAK2 I682F mutation caused B-ALL.

Cyclin-dependent kinases (CDKs) play important roles in the development of many types of cancers by binding with their paired cyclins. However, the function of CDK11 larger protein isomer, CDK11(p110), in the tumorigenesis of human breast cancer remains unclear. In the present study, we explored the effects and molecular mechanisms of CDK11(p110) in the proliferation and growth of breast cancer cells by determining the expression of CDK11(p110) in breast tumor tissues and examining the phenotypic changes of breast cancer cells after CDK11(p110) knockdown. We found that CDK11(p110) was highly expressed in breast tumor tissues and cell lines. Tissue microarray analysis showed that elevated CDK11(p110) expression in breast cancer tissues significantly correlated with poor differentiation, and was also associated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11(p110) by siRNA significantly inhibited cell growth and migration, and dramatically induced apoptosis in breast cancer cells. Flow cytometry demonstrated that cells were markedly arrested in G1 phase of the cell cycle after CDK11(p110) downregulation. These findings suggest that CDK11(p110) is critical for the proliferation and growth of breast cancer cells, which highlights CDK11(p110) may be a promising therapeutic target for the treatment of breast cancer.

Mesenchymal stem cell (MSC) therapy shows considerable promise for the treatment of myocardial infarction (MI). However, the inefficient migration and homing of MSCs after systemic infusion have limited their therapeutic applications. Ultrasound-targeted microbubble destruction (UTMD) has proven to be promising to improve the homing of MSCs to the ischemic myocardium, but the concrete mechanism remains unclear. We hypothesize that UTMD promotes MSC homing by upregulating SDF-1/CXCR4, and this study was aimed at exploring this potential mechanism. We analyzed SDF-1/CXCR4 expression after UTMD treatment in vitro and in vivo and counted the number of homing MSCs in MI areas. The in vitro results demonstrated that UTMD not only led to elevated secretion of SDF-1 but also resulted in an increased proportion of MSCs that expressed surface CXCR4. The in vivo findings show an increase in the number of homing MSCs and higher expression of SDF-1/CXCR4 in the UTMD combined with MSCs infusion group compared to other groups. In conclusion, UTMD can increase SDF-1 expression in the ischemic myocardium and upregulate the expression of surface CXCR4 on MSCs, which provides a molecular mechanism for the homing of MSCs assisted by UTMD via SDF-1/CXCR4 axis.

Microrchidia (MORC) family CW-type zinc-finger 2 (MORC2) regulates chromatin remodeling during the DNA-damage response, represses gene transcription, promotes lipogenesis. Here, we found that MORC2 down-regulated p21 by recruiting HDAC1 to the p21 promoter, in a p53-independent manner. MORC2-mediated down-regulation of p21 in turn promoted cell cycle progression in gastric cancer cells. Furthermore, MORC2 expression correlated negatively with p21 expression in gastric tumors in patients. We suggest that MORC2 may be a potential therapeutic target in cancer.

Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated whether garcinol, a polyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin. We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines, enhanced the apoptotic effect of cisplatin, suppressed constitutive as well as cisplatin-induced NF-κB activation, and downregulated the expression of various oncogenic gene products (cyclin D1, Bcl-2, survivin and VEGF). In vivo study showed that administration of garcinol alone (0.5 mg/kg body weight, i.p. five times/week) significantly suppressed the growth of the tumor, and this effect was further increased by cisplatin. Both the markers of proliferation index (Ki-67) and microvessel density (CD31) were downregulated in tumor tissues by the combination of cisplatin and garcinol. The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after i.p. administration of garcinol at 0.5 mg/kg and 2 mg/kg with mean peak concentration (Cmax) of 1825.4 and 6635.7 nM in the mouse serum, respectively. Overall, our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.

Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease.

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.

Mesenchymal stem cell (MSC) therapy has been considered a promising strategy to cure diabetic nephropathy (DN). However, insufficient MSCs can settle in injured kidneys, which constitute one of the major barriers to the effective implementation of MSC therapy. Stromal cell-derived factor-1 (SDF-1) plays a vital role in MSC migration and involves activation, mobilization, homing, and retention, which are presumably related to the poor homing in DN therapy. Ultrasound-targeted microbubble destruction has become one of the most promising strategies for the targeted delivery of drugs and genes. To improve MSC homing to DN kidneys, we present a strategy to increase SDF-1 via ultrasound-targeted microbubble destruction. In this study, we developed SDF-1-loaded microbubbles (MB(SDF-1)) via covalent conjugation. The characterization and bioactivity of MB(SDF-1) were assessed in vitro. Target release in the targeted kidneys was triggered with diagnostic ultrasound in combination with MB(SDF-1). The related bioeffects were also elucidated. Early DN was induced in rats with streptozotocin. Green fluorescent protein-labeled MSCs were transplanted intravenously following the target release of SDF-1 in the kidneys of normal and DN rats. The homing efficacy was assessed by detecting the implanted exogenous MSCs at 24 hours. The in vitro results showed an impressive SDF-1 loading efficacy of 79% and a loading content of 15.8 μg/mL. MB(SDF-1) remained bioactive as a chemoattractant. In the in vivo study, SDF-1 was successfully released in the targeted kidneys. The homing efficacy of MSCs to DN kidneys after the target release of SDF-1 was remarkably ameliorated at 24 hours compared with control treatments in normal rats and DN rats. In conclusion, ultrasound-targeted MB(SDF-1) destruction could promote the homing of MSCs to early DN kidneys and provide a novel potential therapeutic approach for DN kidney repair.