A deregulated m6A writer complex axis driven by BRD4 confers an epitranscriptomic vulnerability in combined DNA repair-targeted therapy.

Aberrant transcripts expression of the m6A methyltransferase complex (MTC) is widely found across human cancers, suggesting a dysregulated signaling cascade which integrates m6A epitranscriptome to drive tumorigenesis. However, the responsible transcriptional machinery directing the expression of distinct MTC subunits remains unclear. Here, we identified an unappreciated interplay between the histone acetyl-lysine reader BRD4 and the m6A writer complex across human cancers. BRD4 directly stimulates transcripts expression of seven MTC subunits, allowing the maintenance of the nuclear writer complex integrity. Upon BET inhibition, this BRD4-MTC signaling cascade accounts for global m6A reduction and the subsequent dynamic alteration of BRD4-dependent transcriptome, resulting in impaired DNA damage response that involves activation of homologous recombination (HR) repair and repression of apoptosis. We further demonstrated that the combined synergy upon BET/PARP inhibition largely relies on disrupted m6A modification of HR and apoptotic genes, counteracting PARP inhibitor (PARPi) resistance in patient-derived xenograft models. Our study revealed a widespread active cross-talk between BRD4-dependent epigenetic and MTC-mediated epitranscriptomic networks, which provides a unique therapeutic vulnerability that can be leveraged in combined DNA repair-targeted therapy.

Deficiency of leucine-rich repeat kinase 2 aggravates thioacetamide-induced acute liver failure and hepatic encephalopathy in mice.

BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.

Species selection as a key factor in the afforestation of coastal salt-affected lands: Insights from pot and field experiments.

Soil salinization is a significant global issue that leads to land degradation and loss of ecological function. In coastal areas, salinization hampers vegetation growth, and forestation efforts can accelerate the recovery of ecological functions and enhance resilience to extreme climates. However, the salinity tolerance of tree species varies due to complex biological factors, and results between lab/greenhouse and field studies are often inconsistent. Moreover, in salinized areas affected by extreme climatic and human impacts, afforestation with indigenous species may face adaptability challenges. Therefore, it is crucial to select appropriate cross-species salinity tolerance indicators that have been validated in the field to enhance the success of afforestation and reforestation efforts. This study focuses on five native coastal tree species in Taiwan, conducting afforestation experiments on salt-affected soils mixed with construction and demolition waste. It integrates short-term controlled experiments with potted seedlings and long-term field observations to establish growth performance and physiological and biochemical parameters indicative of salinity tolerance. Results showed that Heritiera littoralis Dryand. exhibited the highest salinity tolerance, accumulating significant leaf proline under increased salinity. Conversely, Melia azedarach Linn. had the lowest tolerance, evidenced by complete defoliation and reduced biomass under salt stress. Generally, the field growth performance of these species aligns with the results of short-term pot experiments. Leaf malondialdehyde content from pot experiments proved to be a reliable cross-species salinity tolerance indicator, correlating negatively with field relative height growth and survival rates. Additionally, parameters related to the photosynthetic system or water status, measured using portable devices, also moderately indicated field survival, aiding in identifying potential salt-tolerant tree species. This study underscores the pivotal role of species selection in afforestation success, demonstrating that small-scale, short-term salinity control experiments coupled with appropriate assessment tools can effectively identify species suitable for highly saline and degraded environments. This approach not only increases the success of afforestation but also conserves resources needed for field replanting and maintenance, supporting sustainable development goals.

[Efficacy of bronchoalveolar lavage combined with prone positioning in children with Mycoplasma pneumoniae pneumonia and atelectasis: a prospective randomized controlled study]. / æ”¯æ°”ç®¡è‚ºæ³¡çŒæ´—è”åˆä¿¯å§ä½æ²»ç–—å„¿ç«¥è‚ºç‚Žæ”¯åŽŸä½“è‚ºç‚Žä¼´è‚ºä¸å¼ ç–—æ•ˆçš„å‰çž»æ€§éšæœºå¯¹ç §ç ”ç©¶.

OBJECTIVES: To study the efficacy of bronchoalveolar lavage (BAL) combined with prone positioning in children with Mycoplasma pneumoniae pneumonia (MPP) and atelectasis and its effect on pulmonary function. METHODS: A prospective study was conducted on 94 children with MPP and atelectasis who were hospitalized in Ordos Central Hospital of Inner Mongolia from November 2020 to May 2023. The children were randomly divided into a treatment group and a control group, with 47 children in each group. The children in the treatment group were given conventional treatment, BAL, and prone positioning, and those in the control group were given conventional treatment and BAL. The two groups were compared in terms of fever, pulmonary signs, length of hospital stay, lung recruitment, and improvement in pulmonary function. RESULTS: Compared with the control group, the treatment group had significantly shorter time to improvement in pulmonary signs and length of hospital stay and a significantly higher rate of lung recruitment on day 7 of hospitalization, on the day of discharge, and at 1 week after discharge (P<0.05). Compared with the control group, the treatment group had significantly higher levels of forced vital capacity (FVC) as a percentage of the predicted value, forced expiratory volume (FEV) in 1 second as a percentage of the predicted value, ratio of FEV in 1 second to FVC, forced expiratory flow at 50% of FVC as a percentage of the predicted value, forced expiratory flow at 75% of FVC as a percentage of the predicted value, and maximal mid-expiratory flow as a percentage of the predicted value on the day of discharge and at 1 week after discharge (P<0.05). There was no significant difference in the time for body temperature to return to normal between the two groups (P>0.05). CONCLUSIONS: In the treatment of children with MPP and atelectasis, BAL combined with prone positioning can help to shorten the time to improvement in pulmonary signs and the length of hospital stay and promote lung recruitment and improvement in pulmonary function.

Effect of modification of polystyrene nanoparticles with different bile acids on their oral transport.

Bile acid-modified nanomedicine is a promising strategy to improve oral bioavailability. However, the efficiencies of different bile acids have not been clarified. To clarify this issue, deoxycholic acid (DCA) and cholic acid (CA) and glycocholic acid (GCA) were conjugated to carboxylated polystyrene nanoparticle (CPN). The endocytosis, intracellular and transcellular transport among the NPs were compared in Caco-2 cells, and their oral pharmacokinetics profiles were studied in C57BL/6 J mice. It was found that DCPN demonstrated higher uptake and transcytosis rate. With modification by different bile acids, the transport pathways of the NPs were altered. In mice, GCPN showed the highest absorption speed and oral bioavailability. It was found that the synergic effect of hydrophobicity and ASBT affinity might lead to the difference between in vitro and in vivo transport. This study will build a basis for the rational design of bile acid-modified nanomedicines.

Twinfilin-1 is an essential regulator of myogenic differentiation through the modulation of YAP in C2C12 myoblasts.

Actin cytoskeletal dynamics play a critical role in the regulation of myogenesis through mechanotransduction, Hippo signaling modulation, cell proliferation, and morphological changes. Although Twinfilin-1 (TWF1), a highly conserved actin-depolymerizing factor, is known to regulate actin filament assembly by sequestering actin monomer and capping barbed ends, the biological significance of TWF1 during the differentiation of myogenic progenitor cells has not been investigated. In this study, we unveiled the roles played by TWF1 in the proliferation and differentiation of C2C12 myoblasts. TWF1 was the predominant isoform in myoblasts, and its expression was induced during the early stage of differentiation. Knockdown of TWF1 by siRNA (siTWF1) induced the accumulation of actin filaments (F-actin) and promoted the nuclear translocation of Yes-associated protein (YAP) in the Hippo signaling pathway. TWF1 depletion activated transcription of YAP target genes and induced cell cycle and proliferation in myoblasts. Furthermore, TWF1 knockdown markedly reduced the expressions of myogenic regulatory factors, such as MyoD and MyoG, and drastically hindered myoblast differentiation, fusion, and myotube formation. Collectively, this study highlights the essential role of TWF1 in the myogenic differentiation of progenitor cells via modulation of F-actin and YAP, and suggests TWF1 as a potential therapeutic target for muscle wasting and myopathies.

Emergence of aac(6')-Ie-aph(2'')-Ia-positive enterococci with non-high-level gentamicin resistance mediated by IS1216V: adaptation to decreased aminoglycoside usage in Taiwan.

OBJECTIVES: To explore the mechanisms mediating the different levels of gentamicin resistance in enterococci. METHODS: Susceptibility testing with gentamicin and PCR of resistance determinants were performed in 149 enterococcal isolates. Genetic relatedness was characterized by MLST and PFGE analysis. Sequences of the aac(6')-Ie-aph(2'')-Ia gene and its surrounding environment were determined by Illumina sequencing. Stability assays of gentamicin resistance were carried out to evaluate the probability of loss of the high-level gentamicin resistance (HLGR) phenotype. RESULTS: A total of 17 (11.4%) aac(6')-Ie-aph(2'')-Ia-positive enterococcal isolates (2 Enterococcus faecalis and 15 Enterococcus faecium) with non-HLGR phenotype were found. MLST analysis revealed that the 2 E. faecalis belonged to ST116 and ST618, while all the 15 E. faecium belonged to clonal complex 17. Sequence analysis demonstrated that IS1216V was inserted into the 5'-end of aac(6')-Ie-aph(2'')-Ia, leading to loss of HLGR phenotype. Three IS1216V insertion types were found, and type II and III were frequently found in E. faecium. Interestingly, a total of 38 aac(6')-Ie-aph(2'')-Ia-positive E. faecium with HLGR phenotype also had type II or type III IS1216V insertion. Sequencing of the aac(6')-Ie-aph(2'')-Ia-positive HLGR E. faecium E37 revealed that an intact aac(6')-Ie-aph(2'')-Ia was located adjacent to IS1216V-disrupted aac(6')-Ie-aph(2'')-Ia. In a non-antibiotic environment, E37 tended to lose HLGR phenotype with a probability of 1.57 × 10-4, which was largely attributed to homologous recombination between the intact and disrupted aac(6')-Ie-aph(2'')-Ia. CONCLUSIONS: This is first study to elucidate that the E. faecium is capable of changing its HLGR phenotype, which may contribute to adaptation to hospital environments with decreased usage of gentamicin.

Selected Factors Affecting Oral Bioavailability of Nanoparticles Surface-Conjugated with Glycocholic Acid via Intestinal Lymphatic Pathway.

Here, we describe the absorption pathways of nanoparticles whose surface is modified with bile acid and present environmental factors that influence oral bioavailability (BA) from the gastrointestinal tract (GIT). The approach utilized 100 nm sized fluorescence-labeled, carboxylated polystyrene nanoparticles (CPN) conjugated with glycocholic acid (G/CPN) to exclude potential artifacts, if existing, and instability issues in evaluating the transit of G/CPN in the GIT and measuring BA. The in vitro study using SK-BR-3 that expresses the apical sodium bile acid transporter showed that once G/CPN is internalized, it stayed 2.9 times longer in the cells than CPN, indirectly suggesting that G/CPN takes intracellular trafficking pathways different from CPN in SK-BR-3 cells. In a Caco-2 cell monolayer, G/CPN passed through the monolayer without damaging the tight junction. G/CPN, when administered orally in rodents, showed sustained transit time in the GIT for at least 4 h and was absorbed into the intestinal lymphatic system and circulated into the blood. Ingestion of food before and after oral administration delays G/CPN absorption and decreases BA. A decrease in gastrointestinal motility by anesthetic condition increased the relative BA of G/CPN by up to 74%. Thus, the oral BA of G/CPN can be optimized by taking food ingestion and gastrointestinal motility into account.

Is There a Difference in Condyle Position Changing Pattern Between Deviated and Non-Deviated Sides After Intraoral Vertical Ramus Osteotomy in Facial Asymmetry?

PURPOSE: The position changing pattern of the condyles after intraoral vertical ramus osteotomy (IVRO) on the deviated and non-deviated sides is not clearly known. This study was conducted to evaluate the changes in condylar position after IVRO in patients with facial asymmetry and to compare the deviated and non-deviated sides using computed tomography imaging. MATERIALS AND METHODS: This retrospective cohort study investigated patients with a diagnosis of mandibular prognathism with facial asymmetry who had undergone bilateral IVRO with Le Fort I osteotomy. Condylar positions were recorded on the non-deviated and deviated sides in the midaxial, midsagittal, and coronal planes at 3 time points using 3-dimensional analysis software: preoperatively (T1), at 6 months postoperatively (T2), and at 12 months postoperatively (T3). Linear and angular changes in condyle position were measured and analyzed between T1, T2, and T3. Reliability and comparative analyses were conducted. RESULTS: Thirty-two patients were involved in this study. At T2, the most superior point of the condyle moved to 1.15 ± 0.24 mm (inferiorly) and 0.88 ± 0.23 mm (anteriorly) on the deviated side (P = .0002 and P = .0005, respectively) and to 0.99 ± 0.25 mm (inferiorly) and 1.08 ± 0.34 mm (anteriorly) on the non-deviated side, showing significant differences (P < .0001 and P = .0007, respectively) compared with T1. The condyle position showed a tendency to recover to its original position by T3. However, there were no statistically significant differences between T2 and T3 (P > .05). Furthermore, there were no statistically significant differences between the deviated and non-deviated sides over the entire follow-up period (P > .05). CONCLUSIONS: The condyles did not completely recover to their preoperative positions until 12 months postoperatively. There was no significant difference between the deviated and non-deviated sides in mandibular prognathism with facial asymmetry.

ΔNp63α is a common inhibitory target in oncogenic PI3K/Ras/Her2-induced cell motility and tumor metastasis.

Activation of phosphatidylinositol 3 kinase (PI3K), Ras, and Her2 signaling plays a critical role in cancer development. Hotspot constitutive activating mutations in oncogenes, such as PIK3CA encoding the p110α catalytic subunit or RAS, as well as overexpression of Her2, are frequently found in human tumors and cancers. It has been well established that activation of these oncogenes profoundly promotes tumor metastasis, whereas decreased expression of ΔNp63α, the major protein isoform of the p53-related p63 expressed in epithelial cells, has been associated with cancer metastasis. In this study, we demonstrate that hotspot oncogenic mutations on PIK3CA and RAS, including p110αH1047R, K-RasG12V, and H-RasG12V, as well as activation of Her2, all led to suppression of ΔNp63α expression via Akt-fork-head transcription factor 3a (Akt-FOXO3a) signaling, resulting in increased cell motility and tumor metastasis. Expression of ΔNp63α effectively reversed p110αH1047R-, K-RasG12V-, H-RasG12V-, or Her2-induced cell motility in vitro and tumor metastasis in mouse models. We show that ΔNp63α was a direct FOXO3a transcriptional target and that expression of FOXO3a and ΔNp63α was correlated in human cancer biopsy samples. Together, these results demonstrate that ΔNp63α is a common inhibitory target of oncogenic PI3K, Ras, and Her2, and that ΔNp63α may function as a critical integrator of oncogenic signaling in cancer metastasis.

Glycogen Synthase Kinase-3 Modulates Cbl-b and Constrains T Cell Activation.

The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K-protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K-PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K-PKB-GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.

Immense Insulin Intestinal Uptake and Lymphatic Transport Using Bile Acid Conjugated Partially Uncapped Liposome.

We provide immense insulin absorption from the gastrointestinal tract, combining apical sodium-dependent bile acid transporter-mediated intestinal uptake and the lymphatic transport pathway. This strategy has proven to employ chondroitin sulfate- g-taurocholic acid coated, insulin-loaded partially uncapped liposome (IPUL-CST) for type 1 diabetes mellitus (T1DM) treatment. The loading efficiency of insulin in IPUL-CST increased significantly from 33% to 75% via the partially uncapped liposome preparation method. Moreover, the IPUL-CST revealed an improved insulin protection efficacy in GIT simulated pH and digestive enzyme conditions. The high dose of IPUL-CST in the small intestine was detected 4 h post-oral administration using ex vivo optical imaging and fluorescence intensity. The IPUL-CST exhibited significantly enhanced intestinal absorption (oral bioavailability, 34%; Tmax, 9 h) and reduced blood glucose levels for 16 h in T1DM. The results demonstrated that the new investigated IPUL-CST is a promising carrier for oral insulin delivery.

FoxO3 inactivation promotes human cholangiocarcinoma tumorigenesis and chemoresistance through Keap1-Nrf2 signaling.

UNLABELLED: FoxO transcription factors have been reported to play pivotal roles in tumorigenesis and drug resistance. The mechanisms underlying the tumor suppression function of FoxOs in human cancers remain largely unknown. Aberrant expression and activation of Nrf2 often correlate with chemoresistance and poor prognosis. Here, we report that FoxO3 directs the basal transcription of Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein that bridges Nrf2 to Cul3 for degradation. FoxO3 depletion resulted in Keap1 down-regulation, thereby activating Nrf2 signaling. We further demonstrated that inhibition of the FoxO3-Keap1 axis accounts for Nrf2 induction and activation induced by constitutively active AKT signaling or tumor necrosis factor α treatment. Unlike previous findings, FoxO3 silencing led to decreased reactive oxygen species production, therefore protecting cells from oxidative stress-induced killing in an Nrf2-dependent manner. Importantly, FoxO3 deficiency strongly potentiated tumor formation in nude mice and rendered cholangiocarcinoma xenografts resistant to cisplatin-induced cell death by activating Nrf2. Additionally, we found that clinical cholangiocarcinoma samples displayed FoxO3-Keap1 down-regulation and Nrf2 hyperactivation, underscoring the essential roles of these proteins in cholangiocarcinoma development. CONCLUSION: Our results unravel a unique mechanism underlying the tumor suppressor function of FoxO3 through constraining Nrf2 signaling. (Hepatology 2016;63:1914-1927).

Tempo-spatial Activation of Sequential Quadruple Stimuli for High Gene Expression of Polymeric Gene Nanocomplexes.

The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications.

Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71.

Enterovirus 71 (EV71) is the causative agent of hand, foot and mouth disease and can spread its infections to the central nervous and other systems with severe consequences. The replication of EV71 depends on its 3C proteinase (3Cpro ), a significant drug target. By X-ray crystallography and functional assays, the interactions between inhibitors and EV71 3Cpro were evaluated. It was shown that improved interactions at S4 for the substrate binding could significantly enhance the potency. A new series of potent inhibitors with high ligand efficiency was generated for developing antivirals to treat and control the EV71-associated diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Arabidopsis RAV1 transcription factor, phosphorylated by SnRK2 kinases, regulates the expression of ABI3, ABI4, and ABI5 during seed germination and early seedling development.

The phytohormone abscisic acid (ABA) modulates a number of processes during plant growth and development. In this study, the molecular mechanism of Arabidopsis RAV (Related to ABI3/VP1) transcription factor RAV1 involving ABA signaling was investigated. RAV1-underexpressing lines were more sensitive to ABA than wild-type plants during seed germination and early seedling development, whereas RAV1-overexpressing lines showed strong ABA-insensitive phenotypes. Overexpression of RAV1 repressed ABI3, ABI4, and ABI5 expression, and RAV1 bound to the ABI3, ABI4, and ABI5 promoters in vitro and in vivo, indicating that RAV1 directly down-regulates the expression of ABI3, ABI4, and ABI5. The interruption of ABI5 function in RAV1-U abi5 plants abolished the ABA-hypersensitive phenotype of RAV1-U plants, demonstrating that ABI5 is epistatic to RAV1. RAV1 interacted with SNF1-RELATED PROTEIN KINASE SnRK2.2, SnRK2.3 and SnRK2.6 in the nucleus. In vitro kinase assays showed that SnRK2.2, SnRK2.3 and SnRK2.6 phosphorylated RAV1. Transient expression assays revealed that SnRK2.2, SnRK2.3 and SnRK2.6 reduced the RAV1-dependent repression of ABI5, and the ABA-insensitive phenotype of the RAV1-overexpressing line was impaired by overexpression of SnRK2.3 in the RAV1 OE3 plants. Together, these results demonstrated that the Arabidopsis RAV1 transcription factor plays an important role in ABA signaling by modulating the expression of ABI3, ABI4, and ABI5, and that its activity is negatively affected by SnRK2s.

Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation.

The WRKY transcription factor family has more than 70 members in the Arabidopsis (Arabidopsis thaliana) genome, and some of them are involved in plant responses to biotic and abiotic stresses. This study evaluated the role of WRKY45 in regulating phosphate (Pi) uptake in Arabidopsis. WRKY45 was localized in the nucleus and mainly expressed in roots. During Pi starvation, WRKY45 expression was markedly induced, typically in roots. WRKY45 overexpression in Arabidopsis increased Pi content and uptake, while RNA interference suppression of WRKY45 decreased Pi content and uptake. Furthermore, the WRKY45-overexpressing lines were more sensitive to arsenate, the analog of Pi, compared with wild-type seedlings. These results indicate that WRKY45 positively regulates Arabidopsis Pi uptake. Quantitative real-time polymerase chain reaction and ß-glucuronidase staining assays showed that PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression was enhanced in the WRKY45-overexpressing lines and slightly repressed in the WRKY45 RNA interference line. Chromatin immunoprecipitation and electrophoretic mobility shift assay results indicated that WRKY45 can bind to two W-boxes within the PHT1;1 promoter, confirming the role of WRKY45 in directly up-regulating PHT1;1 expression. The pht1;1 mutant showed decreased Pi content and uptake, and overexpression of PHT1;1 resulted in enhanced Pi content and uptake. Furthermore, the PHT1;1-overexpressing line was much more sensitive to arsenate than WRKY45-overexpressing and wild-type seedlings, indicating that PHT1;1 overexpression can enhance Arabidopsis Pi uptake. Moreover, the enhanced Pi uptake and the increased arsenate sensitivity of the WRKY45-overexpressing line was impaired by pht1;1 (35S:WRKY45-18::pht1;1), demonstrating an epistatic genetic regulation between WRKY45 and PHT1;1. Together, our results demonstrate that WRKY45 is involved in Arabidopsis response to Pi starvation by direct up-regulation of PHT1;1 expression.

DNA Polyplexes as Combinatory Drug Carriers of Doxorubicin and Cisplatin: An in Vitro Study.

Double helix nucleic acids were used as a combination drug carrier for doxorubicin (DOX), which physically intercalates with DNA double helices, and cisplatin (CDDP), which binds to DNA without an alkylation reaction. DNA interacting with DOX, CDDP, or both was complexed with positively charged, endosomolytic polymers. Compared with the free drug, the polyplexes (100-170 nm in size) delivered more drug into the cytosol and the nucleus and demonstrated similar or superior (up to a 7-fold increase) in vitro cell-killing activity. Additionally, the gene expression activities of most of the chemical drug-loaded plasmid DNA (pDNA) polyplexes were not impaired by the physical interactions between the nucleic acid and DOX/CDDP. When a model reporter pDNA (luciferase) was employed, it expressed luciferase protein at 0.7- to 1.4-fold the amount expressed by the polyplex with no bound drugs (a control), which indicated the fast translocation of the intercalated or bound drugs from the "carrier DNA" to the "nuclear DNA" of target cells. The proposed concept may offer the possibility of versatile combination therapies of genetic materials and small molecule drugs that bind to nucleic acids to treat various diseases.

Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis.

The tumour-suppressor phosphatase with tensin homology (PTEN) is the most important negative regulator of the cell-survival signalling pathway initiated by phosphatidylinositol 3-kinase (PI3K). Although PTEN is mutated or deleted in many tumours, deregulation of the PI3K-PTEN network also occurs through other mechanisms. Crosstalk between the PI3K pathways and other tumorigenic signalling pathways, such as those that involve Ras, p53, TOR (target of rapamycin) or DJ1, can contribute to this deregulation. How does the PI3K pathway integrate signals from numerous sources, and how can this information be used in the rational design of cancer therapies?

Regulation of cell cycle progression by forkhead transcription factor FOXO3 through its binding partner DNA replication factor Cdt1.

To ensure genome stability, DNA must be replicated once and only once during each cell cycle. Cdt1 is tightly regulated to make sure that cells do not rereplicate their DNA. Multiple regulatory mechanisms operate to ensure degradation of Cdt1 in S phase. However, little is known about the positive regulators of Cdt1 under physiological conditions. Here we identify FOXO3 as a binding partner of Cdt1. FOXO3 forms a protein complex with Cdt1, which in turn blocks its interaction with DDB1 and PCNA. Conversely, FOXO3 depletion facilitated the proteolysis of Cdt1 in unperturbed cells. Intriguingly, FOXO3 deficiency resulted in impaired S-phase entry and reduced cell proliferation. We provide data that FOXO3 knockdown mimics Cdt1 down-regulation and affects G1/S transitions. Our results demonstrate a unique role of FOXO3 in binding to Cdt1 and maintaining its level required for cell cycle progression.