It is well established that activation of NMDARs plays an essential role in spinal cord synaptic plasticity (i.e., central sensitization) and pain hypersensitivity after tissue injury. Despite prominent expression of NMDARs in DRG primary sensory neurons, the unique role of peripheral NMDARs in regulating intrinsic neuronal excitability and pain sensitivity is not well understood, in part due to the lack of selective molecular tools. To address this problem, we used Advillin-Cre driver to delete the NR1 subunit of NMDARs selectively in DRG neurons. In NR1 conditional knock-out (NR1-cKO) mice, NR1 expression is absent in DRG neurons but remains normal in spinal cord neurons; NMDA-induced currents are also eliminated in DRG neurons of these mice. Surprisingly, NR1-cKO mice displayed mechanical and thermal hypersensitivity compared with wild-type littermates. NR1-deficient DRG neurons show increased excitability, as indicated by increased frequency of action potentials, and enhanced excitatory synaptic transmission in spinal cord slices, as indicated by increased frequency of miniature EPSCs. This hyperexcitability can be reproduced by the NMDAR antagonist APV and by Ca2+-activated slow conductance K+ (SK) channel blocker apamin. Furthermore, NR1-positive DRG neurons coexpress SK1/SK2 and apamin-sensitive afterhyperpolarization currents are elevated by NMDA and suppressed by APV in these neurons. Our findings reveal the hitherto unsuspected role of NMDARs in controlling the intrinsic excitability of primary sensory neurons possibly via Ca2+-activated SK channels. Our results also call attention to potential opposing effects of NMDAR antagonists as a treatment for pain and other neurological disorders.
This study was to examine the breast cancer-overexpressed gene 1 (BCOX1) expression in invasive ductal carcinomas (IDC) of the breast and its value in the prognosis of the disease. The levels of BCOX1 expression in 491 paired IDC and surrounding non-tumor breast tissues as well as 40 paired fresh specimens were evaluated by tissue microarray, immunohistochemistry and quantitative RT-PCR. The potential associations of high BCOX1 expression with clinicopathological variables and the overall survival of these patients were analyzed. The relative levels of BCOX1 mRNA transcripts in the IDC breast tissues were significantly higher than that in the corresponding non-tumor tissues (P = 0.005). The anti-BCOX1 was predominantly stained in the cytoplasm of breast tissue cells and the levels of BCOX1 expression in the majority of breast cancer tissues were obviously higher than that in the corresponding non-tumor breast tissues. High levels of BCOX1 expression were found in 59.5% (292/491) of breast cancer tissues. The high BCOX1 expression was significantly associated with high histological grade (P = 0.037), positive expression of human epidermal growth factor receptor 2 (HER2, P = 0.031) and triple negative breast cancer (P = 0.027). The high BCOX1 expression in breast cancers was significantly associated with a shorter overall survival of these patients (P = 0.023), particularly in patients with triple negative breast cancer (P = 0.005). Therefore, the high BCOX1 expression may serve as a novel marker of poor prognosis and a potential therapeutic target for patients with IDC of the breast.
Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers in vitro and in vivo. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.
Mesenchymal stem cells (MSCs) represent one of the most promising stem cells for a number of degenerative conditions due to their multipotency, immunoprivileged properties, and easy expansion in vitro. However, the limited life span of primary MSCs during in vitro expansion greatly hampers their use in clinical applications and basic research. Immortalization of MSCs will overcome this problem and may provide a very useful tool with which to study MSC biology. Here we showed that silencing p53 expression with lentivirus-mediated small interfering RNA delayed the senescence by extended passage number, but was not sufficient to immortalize primary MSCs. However, combination of p53 knockdown and human telomerase reverse transcriptase (hTERT) overexpression was sufficient to immortalize MSCs. The effects of p53 knockdown and hTERT overexpression on MSCs, including proliferation, colony formation, and differentiation, were determined. The resultant immortal MSCs displayed similar surface antigen profile to primary MSCs and retained MSC differentiation potential. Gene expression profile showed high similarity between immortalized MSCs and primary MSCs. In addition, immortalization-associated genes were also identified. Our data suggested immortalization of MSCs related to upregulation of cell cycle regulator and DNA repair genes enabling them to bypass cell crisis and complete mitosis. This study provides a new cellular model for basic studies of MSCs and understanding of the molecular basis of MSC immortalization.
Peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) data are often used to qualitatively support models for protein structure. We have developed and validated a method (DXCOREX) by which exchange data can be used to quantitatively assess the accuracy of 3-D models of protein structure. The method utilizes the COREX algorithm to predict a protein’s amide hydrogen exchange rates by reference to a hypothesized structure, and these values are used to generate a virtual data set (deuteron incorporation per peptide) that can be quantitatively compared to the deuteration level of the peptide probes measured by hydrogen exchange experimentation. The accuracy of DXCOREX was established in studies performed with thirteen proteins for which both high-resolution structures and experimental data were available. The DXCOREX-calculated and experimental data for each protein was highly correlated. We then employed correlation analysis of DXCOREX-calculated vs. DXMS experimental data to assess the accuracy of a recently proposed structural model for the catalytic domain of a Ca2+- independent phospholipase A2. The model’s calculated exchange behavior was highly correlated with the experimental exchange results available for the protein, supporting the accuracy of the proposed model. This method of analysis will substantially increase the precision with which experimental hydrogen exchange data can help decipher challenging questions regarding protein structure and dynamics.
The sweetpotato whitefly, Bemisia tabaci biotype B is extremely polyphagous with >600 species of host plants. We hypothesized that previous experience of the whitefly on a given host plant affects their host selection and performance on the plants without previous experience. We investigated the host selection for feeding and oviposition of adults and development and survival of immatures of three host-plant-experienced populations of B. tabaci, namely Bemisia-eggplant, Bemisia-tomato and Bemisia-cucumber, on their experienced host plant and each of the three other plant species (eggplant, tomato, cucumber and pepper) without previous experience. We found that the influence of previous experience of the whiteflies varied among the populations. All populations refused pepper for feeding and oviposition, whereas the Bemisia-cucumber and the Bemisia-eggplant strongly preferred cucumber. Bemisia-tomato did not show strong preference to any of the three host palnts. Development time from egg to adult eclosion varied among the populations, being shortest on eggplant, longest on pepper, and intermediate on tomato and cucumber except for the Bemisia-cucumber developed similarly on tomato and pepper. The survivorship from egg to adult eclosion of all populations was highest on eggplant (80-98%), lowest on pepper (0-20%), and intermediate on tomato and cucumber. In conclusion, the effects of previous experience of whiteflies on host selection for feeding and oviposition, development, and survivorship varied depending on host plants, and host plants play a stronger role than previous experience. Preference of feeding and oviposition by adults may not accurately reflect host suitability of immatures. These results provided important information for understanding whitefly population dynamics and dispersal among different crop systems.
Breast cancers are heterogeneous and complex diseases, and subtypes of breast cancers may involve unique molecular mechanisms. The p16INK4a and p53 pathways are two of the major pathways involved in control of the cell cycle. They also play key roles in tumorigenesis. However, whether the roles of these pathways differ in the subtypes of breast cancer is unclear. Therefore, p16 and p53 expression were investigated in different breast cancer subtypes to ascertain their contributions to these cancers. A total of 400 cases of non-invasive ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), including the major molecular subtypes luminal-A, luminal-B, Her-2, and triple-negative subtypes, and 50 cases of normal controls were compared. Luminal-A cancers expressed the lowest level of p16 among the subtypes in DCIS, and the level of p16 expression was up-regulated in the luminal-A of IDC (P<0.008). Triple-negative breast cancers were characterized by a correlation of p53 overexpression with a high level of p16 expression. Luminal lesion types with high p16 expression in DCIS were found to be more likely to develop into aggressive breast cancers, possibly promoted by p53 dysfunction. Taken together, the present study suggest that p16 expression in luminal-A breast cancers is associated with their progression from DCIS to IDC, and both p53 and p16 expressions are important for the development of triple-negative breast cancers in DCIS and IDC.
Objective. In our previous study, we found that some miRNAs were deregulated in hepatocellular carcinoma (HCC), including miR-183. However, the expression of miR-183 in the progression of benign liver diseases to HCC and its correlation with clinicopathologic factors remain undefined. Methods. MiR-183 expression was measured in normal controls (NC) (n = 21), chronic viral hepatitis B or C (CH) tissues (n = 10), liver cirrhosis (LC) tissues (n = 18), HCC tissues (n = 92), and adjacent nontumor tissues (NT) (n = 92) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Results. The expression levels of miR-183 were significantly higher in HCC than in NT, LC, CH, and NL (P = 0.001, P < 0.001, P = 0.011, P < 0.001, resp.). The upregulated miR-183 in HCC was correlated with TNM stage (P = 0.042) and cirrhosis (P = 0.025). The Kaplan-Meier survival analysis showed that miR-183 expression was not associated with the survival of HCC patients. However, miR-183 yielded an area under the curve (AUC) of 0.808 with 59.8% sensitivity and 91.8% specificity in discriminating HCC from benign liver diseases (CH and LC) or NC. Conclusions. The upregulated miR-183 may associate with onset and progression of HCC, but not with the patient survival. A further research is needed to determine the potential of miR-183 as biomarker for HCC.
In this study, the two-step PV method of immunohistochemistry was used to determine livin protein expression in HCC tissues, pericarcinoma tissues, hepatitis/hepatic cirrhosis tissues, and normal hepatic tissues, and livin protein expression was detected in the blood plasma of patients with HCC before and after surgery, subjects with hepatic cirrhosis and hepatitis, and healthy blood donors using ELISA. Livin protein expression was significantly higher in HCC tissues than that in normal hepatic tissues and hepatitis/hepatic cirrhosis tissues, with no significant difference between HCC tissues and pericarcinoma tissues. The HCC patients with positive livin protein expression had a significantly higher survival rate than those with negative livin protein expression. Livin protein expression was significantly higher in the blood plasma of patients with HCC before and after surgery and in patients with hepatic cirrhosis and hepatitis than that in healthy blood donors, whereas livin protein expression in the blood plasma of patients with HCC was not significantly different from that of patients with hepatic cirrhosis and hepatitis. Livin protein expression in HCC tissues did not correlate with that in the blood plasma of the same HCC patients. Livin protein expression may be a potential, effective indicator for assessing prognosis in patients with HCC.
Familial melanoma is associated with point mutations in the cyclin-dependent kinase (CDK) inhibitor p16INK4A (p16). We recently reported that p16 regulates intracellular oxidative stress in a cell cycle-independent manner. Here, we constructed 12 different familial melanoma-associated point mutants spanning the p16 coding region and analyzed their capacity to regulate cell-cycle phase and suppress reactive oxygen species (ROS). Compared to wild-type p16 which fully restored both functions in p16-deficient fibroblasts, various p16 mutants differed in their capacity to normalize ROS and cell cycle profiles. While some mutations did not impair either function, others impaired both. Interestingly, several impaired cell-cycle (R24Q, R99P, V126D) or oxidative function (A36P, A57V, P114S) selectively, indicating that these two functions of p16 can be uncoupled. Similar activities were confirmed with selected mutants in human melanoma cells. Many mutations impairing both cell-cycle and oxidative functions, or only cell cycle function, localize to the third ankyrin repeat of the p16 molecule. Alternatively, most mutations impairing oxidative but not cell-cycle function, or those not impairing either function, lie outside this region. These results demonstrate that particular familial melanoma-associated mutations in p16 can selectively compromise these two independent tumor-suppressor functions, which may be mediated by distinct regions of the protein.
p16; melanoma; oxidative stress; cell cycle
The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with the polyester, poly-(R)-3hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3–S4 linker. Removal of PHB by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function.
Transient Receptor Potential Melastatin 8 channel (TRPM8); Cold; Menthol; Icilin-sensation; Poly-(R)-3-hydroxybutyrate (PHB); PHB-depolymerase PhaZ7; Post-translational modification (PTM); Human Embryonic Kidney 293 cells (HEK-293)
Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways.1,2 The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation.3 A more recent discovery is that VP24 also binds STAT1 directly,4 suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses.
Ebola virus; IFNα; IFNβ; IFNγ; STAT1; VP24; VP35; X-ray crystallography; crystal structures; ebolavirus; interferon antagonist; karyopherin α proteins
The aim of this study was to report a rare case of non-functional ectopic thyroid tissue in the neck with a coexisting normal thyroid. A 36-year-old female presented with a mass in the anterior neck. The thyroid function of the patient was normal. Ultrasonography and computed tomography (CT) revealed a normal thyroid gland and a solid mass in the middle lower neck. Scintigraphy showed a normal thyroid and the mass did show any uptake of 99mTcO4−. The patient underwent en bloc resection. During surgery, the mass was observed to be well encapsulated and completely separated from the thyroid gland. Histology revealed it as ectopic thyroid tissue. The patient had an uneventful recovery.
ectopic; thyroid; cervical mass; non-functional; histology
Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-β and β-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFβ alone, TGFβ with transient β-catenin coactivation, or TGFβ with continuous β-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFβ activation, transient coactivation of TGFβ-induction with β-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFβ signaling pathway with β-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.
Regression analysis is applied to quantitatively analyze the impact of different ambient temperature characteristics on the transformer life at different locations of Chinese mainland. 200 typical locations in Chinese mainland are selected for the study. They are specially divided into six regions so that the subsequent analysis can be done in a regional context. For each region, the local historical ambient temperature and load data are provided as inputs variables of the life consumption model in IEEE Std. C57.91-1995 to estimate the transformer life at every location. Five ambient temperature indicators related to the transformer life are involved into the partial least squares regression to describe their impact on the transformer life. According to a contribution measurement criterion of partial least squares regression, three indicators are conclusively found to be the most important factors influencing the transformer life, and an explicit expression is provided to describe the relationship between the indicators and the transformer life for every region. The analysis result is applicable to the area where the temperature characteristics are similar to Chinese mainland, and the expressions obtained can be applied to the other locations that are not included in this paper if these three indicators are known.
Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.
Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.
To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc).
Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor β1 (TGF-β1), and costimulated with PDGF-AA and TGF-β1, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression.
Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-β1 stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA1495 had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05).
PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-β1. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts.
Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.
Cryptococcus neoformans is an AIDS-associated human fungal pathogen that annually causes over 1 million cases of meningitis world-wide, and more than 600,000 attributable deaths. Cryptococcus often causes lung and brain infection and is the leading cause of fungal meningitis in immunosuppressed patients. Why Cryptococcus frequently infects the central nervous system to cause fatal meningitis is an unanswered critical question. Our previous studies revealed a sophisticated inositol acquisition system in Cryptococcus that plays a central role in utilizing environmental inositol to complete its sexual cycle. Here we further demonstrate that inositol acquisition is also important for fungal infection in the brain, where abundant inositol is available. We found that inositol promotes the traversal of Cryptococcus across the blood-brain barrier (BBB), and such stimulation is fungal inositol transporter dependent. We also identified the effects of host inositol on fungal cellular functions that contribute to the stimulation of fungal penetration of the BBB. We propose that inositol utilization is a novel virulence factor for CNS cryptococcosis. Our work lays an important foundation for understanding how fungi respond to available host inositol and indicates the impact of host inositol acquisition on the development of cryptococcal meningitis.
Trimeric sodium channels of the DEG/ENaC family have important roles in neurons, but the specific functions of different subunits present in heteromeric channels are poorly understood. We previously reported that the Drosophila DEG/ENaC subunit Ppk25 is essential in a small subset of gustatory neurons for activation of male courtship behavior, likely through detection of female pheromones. Here we show that, like mutations in ppk25, mutations in another Drosophila DEG/ENaC subunit, nope, specifically impair male courtship of females. nope regulatory sequences drive reporter gene expression in gustatory neurons of the labellum, wings and legs, including all gustatory neurons in which ppk25 function is required for male courtship of females. In addition, gustatory-specific knockdown of nope impairs male courtship. Further, the impaired courtship response of nope mutant males to females is rescued by targeted expression of nope in the subset of gustatory neurons in which ppk25 functions. However, nope and ppk25 have non-redundant functions, as targeted expression of ppk25 does not compensate for the lack of nope and vice versa. Moreover, Nope and Ppk25 form specific complexes when co-expressed in cultured cells. Together, these data indicate that the Nope and Ppk25 subunits have specific, non-redundant functions in a subset of gustatory neurons required for activation of male courtship in response to females, and suggest the hypothesis that Nope and Ppk25 function as subunits of a heteromeric DEG/ENaC channel required for gustatory detection of female pheromones.
Transition metal-transporting P1B-type CPx ATPases play crucial roles in mediating metal homeostasis and resistance in all cells. The degree to which N-terminal metal binding domains (MBDs) confer metal specificity to the transporter is unclear. We show that the two MBDs of the Zn/Cd/Pb effluxing pump Anabaena AztA are functionally nonequivalent, but only with respect to zinc resistance. Inactivation of the a-MBD largely abrogates resistance to high intracellular Zn(II) levels, whereas inactivation of the b-MBD is not as deleterious. In contrast, inactivation of either the a- or b-MBD has little measurable impact on Cd(II) and Pb(II) resistance. The membrane proximal b-MBD binds Zn(II) with a higher affinity than the distal N-terminal a-MBD. Facile Zn(II)-specific intermolecular transfer from the a-MBD to the higher-affinity b-MBD is readily observed by 1H–15N HSQC spectroscopy. Unlike Zn(II), Cd(II) and Pb(II) form saturated 1:1 S4 or S3(O/N) complexes with AztAaHbH, where a single metal ion bridges the two MBDs. We propose that the tandem MBDs enhance Zn(II)-specific transport, while stabilizing a non-native inter-MBD Cd/Pb cross-linked structure that is a poor substrate and/or regulator for the transporter.
AIM: To explore the optimal steroid therapeutic strategy for autoimmune pancreatitis (AIP).
METHODS: This study was conducted retrospectively in two large institutions in China. Patients with clinically, radiologically and biochemically diagnosed AIP were enrolled. The performed radiological investigations and biochemical tests, the regimen of the given steroid treatment, remission and relapse whether with and without steroid therapy were analyzed.
RESULTS: Twenty-eight patients with AIP received steroid treatment, while 40 patients were treated surgically by pancreatoduodenectomy, distal pancreatectomy and choledochojejunostomy, radiofrequency ablation for the enlarged pancreatic head, percutaneous transhepatic biliary drainage and endoscopic biliary drainage. The starting oral prednisolone dose was 30 mg/d in 18 (64.3%) patients and 40 mg/d in 10 (35.7%) patients administered for 3 wk. The remission rate of AIP patients with steroid treatment (96.4%) was significantly higher than in those without steroid treatment (75%). Maintenance therapy (oral prednisolone dose 5 mg/d) was performed after remission for at least 6-12 mo to complete the treatment course. Similarly, the relapse rate was significantly lower in AIP patients with steroid treatment (28.6%) than in those without steroid treatment (42.5%). Steroid re-treatment was effective in all relapsed patients with or without steroid therapy.
CONCLUSION: Steroid therapy should be considered in all patients with active inflammatory phase of AIP. However, the optimal regimen still should be trailed in larger numbers of patients with AIP.
Autoimmune pancreatitis; Chinese population; Steroid therapy; Remission; Relapse
ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.