Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).
We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of αvβ5 integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.
Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.
Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.
In this manuscript, we describe the development and application of a novel, rapid, and quantitative flow cytometry-based assay to assess photoreceptor outer segment phagocytosis by retinal pigment epithelium (RPE) cells in vitro and have used it to evaluate and compare human induced pluripotent stem (iPS) cell-derived RPE cells with ARPE-19 and human fetal RPE.
The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is an important forest pest as well as the principal vector of the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer), in mainland China. Despite the economic importance of this insect-disease complex, only a few studies are available on the population genetic structure of M. alternatus and the relationship between its historic dispersal pattern and various human activities. The aim of the present study was to further explore aspects of human activity on the population genetic structure of M. alternatus in mainland China. The molecular data based on the combined mitochondrial cox1 and cox2 gene fragments from 140 individuals representing 14 Chinese populations yielded 54 haplotypes. Overall, a historical (natural) expansion that originated from China’s eastern coast to the western interior was revealed by the haplotype network, as well as several recent, long-distant population exchanges. Correlation analysis suggested that regional economic status and proximity to marine ports significantly influenced the population genetic structure of M. alternatus as indicated by both the ratio of shared haplotypes and the haplotype diversity, however, the PWN distribution in China was significantly correlated with only the ratio of shared haplotypes. Our results suggested that the modern logistical network (i.e., the transportation system) in China is a key medium by which humans have brought about population exchange of M. alternatus in mainland China, likely through inadvertent movement of infested wood packaging material associated with trade, and that this genetic exchange was primarily from the economically well-developed east coast of China, westward, to the less-developed interior. In addition, this study demonstrated the existence of non-local M. alternatus in new PWN-infested localities in China, but not all sites with non-local M. alternatus were infested with PWN.
20(S)-protopanaxadiol (PPD), similar to several other anticancer agents, has low oral absorption and is extensively metabolized. These factors limit the use of PPD for treatment of human diseases.
In this study, we used cubic nanoparticles containing piperine to improve the oral bioavailability of PPD and to enhance its absorption and inhibit its metabolism. Cubic nanoparticles loaded with PPD and piperine were prepared by fragmentation of glyceryl monoolein (GMO)/poloxamer 407 bulk cubic gel and verified using transmission electron microscopy and differential scanning calorimetry. We evaluated the in vitro release of PPD from these nanoparticles and its absorption across the Caco-2 cell monolayer model, and subsequently, we examined the bioavailability and metabolism of PPD and its nanoparticles in vivo.
The in vitro release of PPD from these nanoparticles was less than 5% at 12 hours. PPD-cubosome and PPD-cubosome loaded with piperine (molar ratio PPD/piperine, 1:3) increased the apical to basolateral permeability values of PPD across the Caco-2 cell monolayer from 53% to 64%, respectively. In addition, the results of a pharmacokinetic study in rats showed that the relative bioavailabilities of PPD-cubosome [area under concentration–time curve (AUC)0–∞] and PPD-cubosome containing piperine (AUC0–∞) compared to that of raw PPD (AUC0–∞) were 166% and 248%, respectively.
The increased bioavailability of PPD-cubosome loaded with piperine is due to an increase in absorption and inhibition of metabolism of PPD by cubic nanoparticles containing piperine rather than because of improved release of PPD. The cubic nanoparticles containing piperine may be a promising oral carrier for anticancer drugs with poor oral absorption and that undergo extensive metabolism by cytochrome P450.
20(S)-protopanaxadiol; cubosome; piperine; Caco-2 cell monolayer; bioavailability; metabolites
Peroxisomes participate in various important metabolisms and are required in pathogenicity of fungal plant pathogens. Peroxisomal matrix proteins are imported from cytoplasm into peroxisomes through peroxisomal targeting signal 1 (PTS1) or peroxisomal targeting signal 2 (PTS2) import pathway. PEX5 and PEX7 genes participate in the two pathways respectively. The involvement of PEX7 mediated PTS2 import pathway in fungal pathogenicity has been documented, while that of PTS1 remains unclear. Through null mutant analysis of MoPEX5, the PEX5 homolog in Magnaporthe oryzae, we report the crucial roles of PTS1 pathway in the development and host infection in the rice blast fungus, and compared with those of PTS2. We found that MoPEX5 disruption specifically blocked the PTS1 pathway. Δmopex5 was unable to use lipids as sole carbon source and lost pathogenicity completely. Similar as Δmopex7, Δmopex5 exhibited significant reduction in lipid utilization and mobilization, appressorial turgor genesis and H2O2 resistance. Additionally, Δmopex5 presented some distinct defects which were undetected in Δmopex7 in vegetative growth, conidial morphogenesis, appressorial morphogenesis and melanization. The results indicated that the PTS1 peroxisomal import pathway, in addition to PTS2, is required for fungal development and pathogenicity of the rice blast fungus, and also, as a main peroxisomal import pathway, played a more predominant role than PTS2.
Mixed micelles are widely used to increase solubility and bioavailability of poorly soluble drugs. One promising antitumor drug candidate is 20(S)-protopanaxadiol (PPD), although its clinical application is limited by low water solubility and poor bioavailability after oral administration. In this study, we developed mixed micelles consisting of PPD–phospholipid complexes and Labrasol® and evaluated their potential for oral PPD absorption. Micelles were prepared using a solvent-evaporation method, and their physicochemical properties, including particle size, zeta potential, morphology, crystal type, drug loading, drug entrapment efficiency, and solubility, were characterized. Furthermore, in vitro release was investigated using the dialysis method, and transport and bioavailability of the mixed micelles were investigated through a Caco-2 cell monolayer and in vivo absorption studies performed in rats. Compared with the solubility of free PPD (3 μg/mL), the solubility of PPD in the prepared mixed micelles was 192.41 ± 1.13 μg/mL in water at room temperature. The in vitro release profiles showed a significant difference between the more rapid release of free PPD and the slower and more sustained release of the mixed micelles. At the end of a 4-hour transport study using Caco-2 cells, the apical-to-basolateral apparent permeability coefficients (Papp) increased from (1.12 ± 0.21) × 106 cm/s to (1.78 ± 0.16) × 106 cm/s, while the basolateral-to-apical Papp decreased from (2.42 ± 0.16) × 106 cm/s to (2.12 ± 0.32) × 106. In this pharmacokinetic study, compared with the bioavailability of free PPD (area under the curve [AUC]0–∞), the bioavailability of PPD from the micelles (AUC0–∞) increased by approximately 216.36%. These results suggest that novel mixed micelles can significantly increase solubility, enhance absorption, and improve bioavailability. Thus, these prepared micelles might be potential carriers for oral PPD delivery in antitumor therapies.
20(S)-protopanaxadiol; phospholipid complex; Labrasol; mixed micelles; Caco-2 cell monolayer; bioavailability
We explored the desaturase activities and the correlation of fatty acid desaturases (FADS) gene single nucleotide polymorphisms (SNPs) with plasma fatty acid in coronary artery disease (CAD) patients in a Chinese Han population.
Plasma fatty acids were measured by gas chromatography in CAD patients (n = 505) and a control group (n = 510). Five SNPs in the FADS gene were genotyped with high-resolution melting (HRM) methods.
After adjustment, D6D activity, assessed as arachidonic acid (AA, C20:4n-6)/linoleic acid (LA, C18:2n-6), was higher in CAD patients (p<0.001). D9D activity, which was estimated as the ratio of palmitoleic acid (C16:1)/palmitic acid (C16:0) or oleic acid (C18:1n-9) to stearic acid (C18:0), was also increased (p<0.001). The genotype distributions of rs174537 G>T and rs174460 C>T were different between the two groups. The rs174537 T allele was associated with a lower risk of CAD [OR 0.743, 95% CI (0.624, 0.884), p = 0.001]. Carriers of the rs174460 C allele were associated with a higher risk of CAD [OR 1.357, 95% CI (1.106, 1.665), p = 0.003].
We firstly report that the rs174460 C allele is associated with a higher risk of CAD, and confirm that the rs174537 T allele is associated with a lower risk of CAD. Our results indicate that FADS gene polymorphisms are likely to influence plasma fatty acid concentrations and desaturase activities.
Due to low tolerance to chemotherapy, the maximum number of cycles of postoperative adjuvant chemotherapy is 4 in adjuvant gastric clinical trials. The aim of this study is to retrospectively evaluate the safety and efficacy of adjuvant epirubicin-based triplet chemotherapy and radiotherapy in the treatment of resected locally advanced stomach or gastroesophageal junction adenocarcinoma.
From January 2004 to July 2008, ninety-seven consecutive gastric or gastroesophageal junction adenocarcinoma patients in stages T3–4/N+ were treated with postoperative radiotherapy and chemotherapy. The recommended treatment plan was radical resection followed by 1–2 cycles of adjuvant chemotherapy (ACT), postoperative chemoradiotherapy (CRT), and, finally, 4–5 cycles of ACT. The patients were classified into two groups depending on the number of cycles of ACT: group 1 received 4–6 cycles (n = 59), and group 2 received 0–3 cycles (n = 38). The detailed grouping is as follows: RT alone, 2; RT and CT, 18; concurrent RTCT and CT, 41; and CRT, 36. Of the 97 patients, 77 patients received concurrent therapy (CRT, (5-fluorouracil or capecitabine), and 20 received radiotherapy alone because of patient refusal (n = 15) or treatment toxicity (n = 5). After a median follow-up of 44 months, the 3-year disease free survival(DFS) and overall survival (OS) were 66.5% and 69.5% for group 1 and 45.5% and 50% for group 2, respectively (p = 0.005 and p = 0.024). Multivariate analysis revealed that 4–6 cycles of ACT, lymphovascular invasion, or peritoneal metastasis were independent prognostic factors for disease-free survival or overall survival (p<0.05).
This study demonstrates that concurrent chemoradiation with adjuvant epirubicin-based triplet chemotherapy is feasible and tolerable for gastric or gastroesophageal junction carcinoma patients. Patients can benefit from more cycles of ACT.
Anterior-posterior (A-P) patterning of the vertebrate limb is controlled by sonic hedgehog (SHH) signaling, and the precise restriction of Shh expression to the posterior limb bud is essential for its polarizing effect. Fibroblast growth factor (FGF) signaling, a key control of proximal-distal (P-D) limb outgrowth, is known to promote Shh expression in the posterior limb bud. Here we show that conditional knockout of FGF-activated transcription factor genes Etv4 and Etv5 in mouse led to ectopic Shh expression in the anterior limb bud, and a preaxial polydactyly (PPD) skeletal phenotype. These unexpected results suggest that ETV4 and ETV5 act downstream of FGF signaling to inhibit Shh expression in the anterior limb bud. This finding elucidates a novel aspect of the mechanism coordinating limb development along the A-P and P-D axes.
DICER is an enzyme that processes microRNAs (miRNAs) to their mature forms. As miRNAs were first discovered for their role in the control of developmental timing, we investigated their potential requirement in mouse somitogenesis, an event with precise temporal periodicity. To address the collective role of miRNAs in mesoderm development including somite formation, we used T (Brachyury)-Cre mouse line to inactivate Dicer in most cells of the mesoderm lineage. This Dicer mutant exhibits a reduced anterior-posterior axis. Somite number remains normal in mutant embryos up until the death of the embryos more than two days after Dicer inactivation. Consistent with this, the molecular machineries required for establishing segmentation, including clock and wave front, are not perturbed. However, somite size is reduced and later-formed somites are caudalized, coincident with increased cell death. Outside of the paraxial mesoderm and prior to apparent reduction of the axis in the mutant, the position of the hindlimb bud, a lateral plate mesoderm-derived structure, is posteriorly shifted and the timing of hindlimb bud initiation is delayed accordingly. We observed changes in the expression of genes critical for limb positioning, which include a shifted and delayed downregulation of Hand2 and Tbx3, and shifted and delayed upregulation of Gli3 in the prospective limb bud field. The 3′UTRs of both Hand2 and Tbx3 harbor target sites for a seed sequence-sharing cluster of miRNAs mir-25/32/92/363/367. As an example of the cluster, we show that mir-363, a miRNA with elevated expression in the prospective limb bud field, is capable of inhibiting Hand2/Tbx3 expression in vitro in a binding site-dependent manner. Together, our findings provide the first demonstration that in mouse embryonic mesoderm, while Dicer is dispensable for somite segmentation, it is essential for proper limb bud positioning.
limb; limb positioning; somite; Dicer; microRNA; mouse
Based on the hypothesis that first-line chemoradiation followed by chemotherapy was superior for primary tumor and non-inferior for distant lesions compared to chemotherapy alone in synchronous unresectable distant metastases rectal adenocarcinoma, this study was designed to assess the efficacy and safety of this strategy.
Materials and methods
Thirty two eligible patients received intensity modulated radiation therapy (45 Gy to the pelvis and a concomitant 10 Gy boost to the gross tumor), along with concurrent weekly capecitabine and oxaliplatin. Patients underwent radical surgery if all lesions were visually evaluated as resectable and received chemotherapy for a total of 6 months, whether pre- or post-operatively (definitive therapy group). The remaining patients received 6 months of consolidation chemotherapy followed by maintenance chemotherapy (non-definitive therapy group).
The toxicities were acceptable, with radiation-induced dermatitis around the anal verge being the most common (18.8%). Fourteen patients underwent surgical resection of the rectal tumor, with 5 (35.7%) experiencing a pathological complete response. Nine out of 14 received definitive treatment, defined as R0 resections of all visible tumors. At a median follow-up of 12 months (range, 4–23 months), 2 cases were evaluated as local failure, and the median overall survival (OS) and progression free survival (PFS) for all 32 patients were 17.5 and 12 months, respectively. OS differed significantly in the definitive and non-definitive therapy groups (p=0.045), and PFS tended to differ (p=0.274).
It was demonstrated that the strategy of first-line chemoradiation followed by chemotherapy was effective and tolerable, especially for local control. OS and PFS were superior in patients who did than did not undergo curative therapy.
Rectal cancer; Synchronous unresectable distant metastases; Chemoradiotherapy; Imtensity modulated radiation therapy
A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.
A sensitive HPLC method was developed for the quantitative determination of isoliquiritin apioside (ILA) and isoliquiritin (IL) in rat plasma. After protein precipitation with acetonitrile, chloroform was used to separate lipid-soluble impurities from the plasma samples and remove acetonitrile. A chromatography was carried out on Diamonsil C18 (150 × 4.6 mm; 5 μm) analytical column, using a mobile phase consisting of water (containing phosphoric acid 0.1%, v/v); acetonitrile (72 : 28, v/v) at a flow rate of 1.0 mL/min. The wavelength-switching technology was performed to determine ILA and IL at 360 nm and wogonoside (internal standard, IS) at 276 nm. The calibration curves of ILA and IL were fairly linear over the concentration ranges of 0.060–3.84 μg/mL (r = 0.9954) and 0.075–4.80 μg/mL (r = 0.9968), respectively. The average extract recoveries of ILA, IL, and IS were all over 80%. The precision and accuracy for all concentrations of quality controls and standards were within 15%. The lower limit of quantification (LLOQ) was 0.060 μg/mL for ILA and 0.075 μg/mL for IL. The method was used in pharmacokinetic study after an oral administration of Zhigancao extract to rats.
Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM.
Methods and Results
GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2.
Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.
Five populations segregated in isogenic backgrounds and three sets of near isogenic lines (NILs) overlapping in a 362.3-kb region covering heading date gene Hd1 were developed from the indica rice cross Zhenshan97 (ZS97)/Milyang 46 (MY46). They were used to analyze the effects of Hd1 on heading date, plant height and yield traits. In a background of the parental mixtures, the photoperiod-sensitive allele derived from ZS97 functioned in promoting and delaying flowering in the natural short-day and long-day conditions, respectively. In the background of ZS97, no response to the photoperiod was observed, whereas the photoperiod-insensitive allele derived from MY46 functioned in delaying flowering, increasing plant height, and enhancing grain productivity. The additive effects estimated in two NIL sets were 6.14 and 6.14 d for heading date, 4.46 and 5.55 cm for plant height, 10.82 and 11.54 for the number of spikelets per panicle, 6.82 and 8.00 for the number of grains per panicle, and 2.16 and 2.23 g for grain yield per plant, which explained 94.1% and 96.3%, 70.5% and 84.8%, 52.4% and 55.2%, 28.9% and 39.2%, and 36.5% and 26.9% of the phenotypic variances, respectively. Since the photoperiod-insensitive allele of Hd1 confers a long vegetative phase, it is a good candidate for breeding rice varieties with high yielding potential for low latitudes.
Plants have evolved a complicated resistance system and exhibit a variety of defense patterns in response to different attackers. Previous studies have shown that responses of plants to chewing insects and phloem-feeding insects are significantly different. Less is known, however, regarding molecular responses to leafminer insects. To investigate plant transcriptome response to leafminers, we selected the leafminer Liriomyza huidobrensis, which has a special feeding pattern more similar to pathogen damage than that of chewing insects, as a model insect, and Arabidopsis thaliana as a response plant.
We first investigated local and systemic responses of A. thaliana to leafminer feeding using an Affymetrix ATH1 genome array. Genes related to metabolic processes and stimulus responses were highly regulated. Most systemically-induced genes formed a subset of the local response genes. We then downloaded gene expression data from online databases and used hierarchical clustering to explore relationships among gene expression patterns in A. thaliana damaged by different attackers.
Our results demonstrate that plant response patterns are strongly coupled to damage patterns of attackers.
Plant defenses; Transcriptome; Microarray; Leaf miner; Pathogen; Systemic defense
Baicalein, a flavone present in Scutellaria baicalensis Georgi, has been demonstrated to possess antitumor activity in a variety of cancer cells in vitro. However, its effects on the growth inhibition and induction of apoptosis in human esophageal carcinoma cells remain unclear. The aims of this study were to determine whether cultured EC-109 esophageal squamous cell carcinoma (ESCC) cells undergo apoptosis when treated with baicalein and to investigate the underlying mechanisms in vitro. Cell growth was measured using MTT and plate colony formation assays. Induction of apoptosis was examined using Hoechst 33258 staining, flow cytometry analysis and a DNA fragmentation assay. The mechanisms underlying the observed growth suppression were examined using western blot analysis. The results demonstrated that treatment of EC-109 cells with baicalein for 48 h markedly decreased the rate of cell viability. Colony formation was almost fully suppressed at 40 μM baicalein. EC-109 cells underwent apoptosis in response to baicalein treatment, demonstrated by an increase in the percentage of cells stainable with Hoechst 33258 and Annexin V-FITC/PI, increased DNA fragmentation and activation of the intrinsic (mitochondrial) pathway for cell death. The latter was characterized by increased expression of the cleaved forms of caspase-9 and -3, and poly (ADP-ribose) polymerase (PARP). Additionally, baicalein was found to downregulate anti-apoptotic components and upregulate apoptotic components of the PI3K/Akt pathway. In conclusion, baicalein induces apoptosis in EC-109 cells through modulation of the PI3K/Akt pathway, thus providing further understanding of the molecular mechanisms of baicalein action in esophageal carcinoma. Therefore, the present study revealed that baicalein significantly inhibits growth and induces apoptosis in EC-109 human ESCC cells in vitro.
cell apoptosis; baicalein; esophageal squamous cell carcinoma; PI3K/Akt signaling pathway
Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently we have shown that Cd elevates intracellular free calcium ion ([Ca2+]i) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. Here we show that the effects of Cd elevated [Ca2+]i on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca2+ with BAPTA/AM or preventing Cd-induced [Ca2+]i elevation using 2-aminoethoxydiphenyl borate (2-APB) blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), JNK (SP600125) and Erk1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca2+]i elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca2+ level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.
cadmium; apoptosis; calcium ion; calcium/calmodulin-dependent protein kinase II; mitogen-activated protein kinase; mammalian target of rapamycin
There is a lack of large-scale studies on vitamin D status and its relationship to parathyroid hormone (PTH) and bone turnover markers in adults living in Shanghai. The objectives were to determine the prevalence of vitamin D insufficiency in Shanghai and to investigate the relationship of 25(OH)D with parathyroid function and bone turnover markers. This cross-sectional study involved 649 men and 1939 women aged 20–89 years who were randomly sampled in Shanghai. Serum concentrations of 25(OH)D, PTH, albumin, and bone turnover markers were measured. During the winter season, the prevalence of vitamin D insufficiency (<30 ng/mL) was 84% in males and 89% in females. The prevalence of vitamin D deficiency (<20 ng/mL) was 30% in males and 46% in females. With increasing serum 25(OH)D concentrations categorized as <10, 10–20, 20–30, and ≥30 ng/mL, the mean PTH and bone turnover markers levels gradually decreasd in both sexes (p<0.001). There was an inverse relationship between the serum 25(OH)D and PTH concentrations in both genders, but no threshold of 25(OH)D at which PTH levels plateaued was observed. There were modest but significantly inverse relationships between the levels of 25(OH)D and bone turnover markers, but no plateau was observed for serum 25(OH)D levels up to 40 ng/mL.
Endoscopic sphincterotomy (EST) is considered as a possible etiological factor for severe cholangitis. We herein report a case of severe cholangitis after endoscopic sphincterotomy induced by barium examination. An adult male patient presented with epigastric pain was diagnosed as having choledocholithiasis by ultrasonography. EST was performed and the stone was completely cleaned. Barium examination was done 3 d after EST and severe cholangitis appeared 4 h later. The patient was recovered after treated with tienam for 4 d. Barium examination may induce severe cholangitis in patients after EST, although rare, barium examination should be chosen cautiously. Cautions should be also used when EST is performed in patients younger than 50 years to avoid the damage to the sphincter of Oddi.
Endoscopic sphincterotomy; Severe cholangitis; Barium examination; Sphincter of Oddi
The phenomenon of pistil abortion widely occurs in Japanese apricot, and imperfect flowers with pistil abortion seriously decrease the yield in production. Although transcriptome analyses have been extensively studied in the past, a systematic study of differential gene expression has not been performed in Japanese apricot. To investigate genes related to the pistil development of Japanese apricot, high-throughput sequencing technology (Illumina) was employed to survey gene expression profiles from perfect and imperfect Japanese apricot flower buds. 3,476,249 and 3,580,677 tags were sequenced from two libraries constructed from perfect and imperfect flower buds of Japanese apricot, respectively. There were 689 significant differentially-expressed genes between the two libraries. GO annotation revealed that highly ranked genes were those implicated in small molecule metabolism, cellular component organisation or biogenesis at the cellular level and fatty acid metabolism. According to the results, we assumed that late embryogenesis abundant protein (LEA), Dicer-like 3 (DCL3) Xyloglucan endotransglucosylase/hydrolase 2 (XTH2), Pectin lyase-like superfamily protein (PPME1), Lipid transfer protein 3 (LTP3), Fatty acid biosynthesis 1 (FAB1) and Fatty acid desaturase 5 (FAD5) might have relationships with the pistil abortion in Japanese apricot. The expression patterns of 36 differentially expressed genes were confirmed by real-time (RT)-PCR. This is the first report of the Illumina RNA-seq technique being used for the analysis of differentially-expressed gene profiles related to pistil abortion that both computationally and experimentally provides valuable information for the further functional characterisation of genes associated with pistil development in woody plants.
On April 14, 2010, an earthquake registering 7.1 on the Richter scale shook Qinghai Province in southwest China. The earthquake caused numerous casualties and much damage. The epicenter, Yushu County, suffered the most severe damage. As a part of the psychological relief work, the present study evaluated the mental health statuses of the people affected and identified the mental disorder risk factors related to earthquakes.
Five hundred and five earthquake survivors living in Yushu County were investigated 3–4 months after the earthquake. Participant demographic data including gender, age, marital status, ethnicity, educational level, and religious beliefs were collected. The Earthquake-Specific Trauma Exposure Indicators assessed the intensity of exposure to trauma during the earthquake. The PTSD Checklist-Civilian version (PCL-C) and the Hopkins Symptoms Checklist-25 (HSCL-25) assessed the symptoms and prevalence rates of probable Posttraumatic Stress Disorder (PTSD) as well as anxiety and depression, respectively. The Perceived Social Support Scale (PSSS) evaluated subjective social support.
The prevalence rates of probable PTSD, anxiety, and depression were 33.7%, 43.8% and 38.6%, respectively. Approximately one fifth of participants suffered from all three conditions. Individuals who were female, felt initial fear during the earthquake, and had less social support were the most likely to have poor mental health.
The present study revealed that there are serious mental problems among the hard–hit survivors of the Yushu earthquake. Survivors at high risk for mental disorders should be specifically considered. The present study provides useful information for rebuilding and relief work.
Histidine kinase receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi and other protists. Typical histidine kinase receptors have an extracellular sensor portion to detect the signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While the kinase domains are highly conserved, sensor domains are diverse. Histidine kinase receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, Family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane histidine kinases. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated- and apo-state structures provide insights into signal transmission. Some HK1-family proteins prove to be sensors for chemotaxis proteins or for diguanylate cyclase receptors, which implies a combinatorial molecular evolution.
crystal structure; molecular evolution; PDC domain; signal transduction; two-component system
Baohuoside I is a potential anticancer drug for a variety of malignancies and has been approved for in vitro use. However, baohuoside I has very poor oral absorption.
In the present study, we prepared baohuoside I-phospholipid complexes of different diameters and determined their physicochemical properties using transmission electron microscopy, ultraviolet spectroscopy, and differential scanning calorimetry. The in vitro absorption of baohuoside I and baohuoside I-phospholipid complexes of different sizes were compared using the Caco-2 cell culture model, and subsequently, the bioavailability of baohuosidel and its complexes were estimated in vivo.
Compared with the large-sized phospholipid complexes, a nanoscale phospholipid complex improved the oral bioavailability of baohuoside I. In addition, our results suggest that the smaller the particle size, the faster the complexes crossed the Caco-2 monolayer and the faster they were resorbed after oral administration in rats. The relative oral bioavailability of a nanoscale size 81 ± 10 nm baohuoside I-phospholipid complex (area under the concentration-time curve [AUC]0–∞) was 342%, while that of baohuoside I and a 227.3 ± 65.2 μm baohuoside I-phospholipid complex was 165%.
We enhanced the oral bioavailability of baohuoside I by reducing the particle size of the phospholipid complex to the nanometer range, thereby improving its potential for clinical application.
nanoscale phospholipid complex; Caco-2 cell monolayer; bioavailability; oral absorption