Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.
The co-infection of rice caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) was widely found at many regions, such as Yunnan Province, China, and North and Central Vietnam. These rice viral diseases lead to seriously yield loss of rice. In this study, the proteomics technology of shotgun and label free combined with multiple reaction monitoring (MRM) was developed to detect rice sample of a single or/and co-infection. The shotgun assay indicated that some proteins coded by SRBSDV and RRSV were detected via the mode of in-gel digestion, except for P5-2, P7-2 and P9-2 of SRBSDV and P4b, P5, P6, P8a and P8b of RRSV. The technology of label free combined with MRM indicated that P2, P5-1, P4, P8, P7-1, P6, P9-1 and P10 of SRBSDV and P1, P3 and P9 of RRSV were higher abundance in rice plant, and P5-2, P7-2 and P9-2 of SRBSDV and P4b and P5 of RRSV were lower abundance in viruliferous-rice plant. So SRBSDV P9-1 and RRSV P3 was selected as marker molecule to be used in detection technology, and the label free combined with MRM technology was established to detect two kinds of rice virus.
Electronic supplementary material
The online version of this article (doi:10.1007/s13337-014-0195-y) contains supplementary material, which is available to authorized users.
SRBSDV; RRSV; Shotgun protein proteomic; Multi reaction monitoring; Label free; Bio-informatics
Telocytes (Tcs) are cells with telopodes (Tps), which are very long cellular extensions with alternating thin segments (podomers) and dilated bead-like thick regions known as podoms. Tcs are a distinct category of interstitial cells and have been identified in many mammalian organs including heart, lung and kidney. The present study investigates the existence, ultrastructure, distribution and contacts of Tcs with surrounding cells in the uterus (shell gland) of the oviduct of the Chinese soft-shelled turtle, Pelodiscus sinensis. Samples from the uterine segment of the oviduct were examined by transmission electron microscopy. Tcs were mainly located in the lamina propria beneath the simple columnar epithelium of the uterus and were situated close to nerve endings, capillaries, collagen fibres and secretory glands. The complete morphology of Tcs and Tps was clearly observed and our data confirmed the existence of Tcs in the uterus of the Chinese soft-shelled turtle Pelodiscus sinensis. Our results suggest these cells contribute to the function of the secretory glands and contraction of the uterus.
telocytes; uterus; Chinese soft-shelled turtle (Pelodiscus sinensis); ultrastructure
Bone marrow stromal cells (BMSCs) play an important role in ischemic limb angiogenesis. BMSCs cultured in vitro can be exposed to oxygen tension much higher than that experienced in vivo. This study assessed oxygen tension in bone marrow and ischemic muscle in vivo, and then identified an appropriate oxygen concentration for culturing BMSCs.
Unilateral hind limb ischemia was surgically induced in 30 mice, and tissue oxygen tension in bilateral gastrocnemius muscles and femoral bone marrow was monitored in vivo using a micro-electrode at 24 hours, 1 week, 2 weeks, and 3 weeks after modeling. Media used for culturing normal marrow, muscle, and artery tissue were incubated with various oxygen concentrations, and O2 tension was continuously monitored. Oxygen tension in aortic arterial blood was monitored using a micro-electrode and blood gas analyzer, and the results were compared.
Oxygen tension in ischemic gastrocnemius muscle reached a nadir at 1 week after ischemic modeling, when histological changes were most noticeable. Culture media incubated with 3%, 6%, and 14% oxygen (the normal oxygen levels of bone marrow, muscle, and arterial blood, respectively) required 9, 6, and 2 hours, respectively, to reach an equilibrated oxygen tension, and oxygen tension was elevated by 1.6-, 1.2-, and 0.4-fold, respectively, upon re-exposure of the media to air.
Physiological oxygen tension differs in different tissues. A 3% O2 concentration mimics the physiological O2 exposure experienced by BMSCs and represents the hypoxic concentration. Culture medium incubated under hypoxic conditions requires a prolonged period of time to regain equilibrated oxygen tension.
Arterial Pressure; Cell Hypoxia; Mesenchymal Stromal Cells
To simplify the architecture of a neuromorphic system, it is extremely desirable to develop synaptic cells with the capacity of low operation power, high density integration, and well controlled synaptic behaviors. In this study, we develop a resistive switching device (ReRAM)-based synaptic cell, fabricated by the CMOS compatible nano-fabrication technology. The developed synaptic cell consists of one vertical gate-all-around Si nano-pillar transistor (1T) and one transition metal-oxide based resistive switching device (1R) stacked on top of the vertical transistor directly. Thanks to the vertical architecture and excellent controllability on the ON/OFF performance of the nano-pillar transistor, the 1T1R synaptic cell shows excellent characteristics such as extremely high-density integration ability with 4F2 footprint, ultra-low operation current (<2 nA), fast switching speed (<10 ns), multilevel data storage and controllable synaptic switching, which are extremely desirable for simplifying the architecture of neuromorphic system.
Folate is a nutrient crucial for rapidly growing tissues, including developing embryos and cancer cells. Folate participates in the biosynthesis of nucleic acids, proteins, amino acids, S-adenosylmethionine, many neurotransmitters, and some vitamins. The intracellular folate pool consists of different folate adducts, which carry one-carbon units at three different oxidative states and participate in distinct biochemical reactions. Therefore, the content and dynamics of folate adducts will affect the homeostasis of the metabolites generated in these folate-mediated reactions. Currently, the knowledge on the level of each individual folate adduct in developing embryos is limited. With an improved high-performance liquid chromatography protocol, we found that tetrahydrofolate (THF), the backbone of one-carbon carrier, gradually increased and became dominant in developing zebrafish embryos. 5-methyl-tetrahydrofolate (5-CH3-THF) was abundant in unfertilized eggs but decreased rapidly when embryos started to proliferate and differentiate. 10-formyltetrahydrofolate at first increased after fertilization, and then dropped dramatically before reaching a sustained level at later stages. Dihydrofolate (DHF) slightly decreased initially and remained low throughout embryogenesis. Exposure to methotrexate significantly decreased 5-CH3-THF levels and increased DHF pools, besides causing brain ventricle anomaly. Rescuing with leucovorin partly reversed the abnormal phenotype. Unexpectedly, the level of 5-CH3-THF remained low even when leucovorin was added for rescue. Our results show that different folate adducts fluctuated significantly and differentially in concert with the physiological requirement specific for the corresponding developmental stages. Furthermore, methotrexate lowered the level of 5-CH3-THF in developing embryos, which could not be reversed with folate supplementation and might be more substantial to cellular methylation potential and epigenetic control than to nucleotide synthesis.
The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here, we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested substantial stability. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses.
IMPORTANCE The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described.
In liver transplantation, the efficacy of mycophenolate mofetil (MMF) has been confirmed in clinical trials and studies. However, therapeutic drug monitoring for mycophenolic acid (MPA) has not been fully accepted in liver transplantation as no long-term prospective study of concentration controlled vs fixed-dose prescribing of MMF has been done. This review addressed MPA measurement, pharmacokinetic variability and reasons of this variation, exposure related to acute rejection and MMF-associated side effects in liver transplant recipients. Limited sampling strategies to predict MPA area under the concentration-time curve have also been described, and the value of clinical use needs to be investigated in future. The published data suggested that a fixed-dosage MMF regimen might not be suitable and monitoring of MPA exposure seems helpful in various clinical settings of liver transplantation.
Mycophenolate mofetil; Mycophenolic acid; Pharmacokinetics; Therapeutic drug monitoring; Liver transplantation
To evaluate the accuracy of 20 MHz immersion B-scan ultrasonography in observing lens and to investigate the value of this noninvasive preoperative diagnosis method in alkali burn eyes.
It was a comparative study. Fifty-six cases (56 eyes) of alkali burn eyes were examined by ultrasound biomicroscopy (UBM) and immersion 20 MHz B-scan ultrasonography from June 2011 to April 2013, the images were analyzed, and the ultrasonographic diagnosis compared with the operation results.
In 56 alkali burn eyes examined by UBM, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; the anterior lens capsule surface or/and the front lens could be detected in 18 eyes, and lens opacification in 3 eyes of them; suspected abnormal lens were detected in the other 20 eyes. In all the same eyes examined by immersion 20 MHz B-scan ultrasonography, the lens were not detected in 16 eyes; the IOL could be detected in 2 eyes; 24 abnormal lens (opacity, lens expansion, shrinkage) and 14 normal lens were found. Compared with the intraoperative findings, the diagnostic accordance rate of the immersion 20 MHz B-scan appearance of lens was 100% (56/56), which was significantly higher than examined by UBM 57.14% (32/56) (χ2=30.55, P=0.0000).
Immersion 20 MHz B-scan ultrasonography can observe the lens accurately in alkali burn eyes. It has important clinical value to combine with UBM in eyes of alkali burn.
immersion; B-scan; ultrasonography; alkali burn; ultrasound biomicroscopy
This study aimed to investigate changes in the expression levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and hypoxia-inducible factor-1α (HIF-1α) in the trophoblasts of patients who had experienced missed abortions. The missed abortion group included 28 patients with missed abortions. The control group was comprised of 35 women who had elected to undergo surgically induced abortion in their first trimester, and whose embryos were confirmed to be alive before surgery. No woman in either group had any known causative factor for missed or spontaneous abortion. As soon as the diagnosis of “missed abortion” was definitively made, the chorionic trophoblast was obtained by induced abortion operation. The same method was used for individuals in the control group, who were at 7-10 weeks of pregnancy. Levels of ROS, SOD, and HIF-1α in the chorionic trophoblasts from women in both groups were examined within 1 hour by fluorescent staining, chemiluminometry, and enzyme immunoassay methods. The SOD and HIF-1α levels were lower and the ROS level was higher in the trophoblasts from women in the missed abortion group compared to levels in the control group (P < 0.05). ROS, SOD and HIF-1α levels in the chorionic trophoblasts from patients with missed abortion are altered compared to levels in control patients. Changes in these factors should be evaluated further for their potential role in missed abortion.
Hypoxia-inducible factor-1α (HIF-1α); reactive oxygen species (ROS); super oxide dismutase (SOD); missed abortion
CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.
Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.
Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the −124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBPα was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBPα helps relieve embryonic oxidative stress induced by ethanol exposure.
Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3β-hydroxysteroid dehydrogenase–positive cells) were seen in testes of postnatal day (PND) 14–90 Igf1−/− mice compared with age-matched Igf1+/+ controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1−/− and Igf1+/+ mice. In contrast, the labeling index of PLCs was reduced in the Igf1−/− mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1−/− mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1+/+ mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1−/− mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1−/− mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1−/− testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult testes of Igf1−/− mice result at least in part from altered proliferation and differentiation of ALC precursor cells, but not of the stem cells that give rise to these cells.
Developmental lineage; proliferation; testosterone
Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.
Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.
Objective: To compare outcomes of vaginal and abdominal hysterectomy procedures in women with benign gynaecological diseases.
Methods: This was a prospective study of outcomes of consecutive patients who underwent total vaginal hysterectomy (VH) or abdominal hysterectomy (AH) for benign gynaecological diseases. Patient characteristics before, during, and after the operations were reviewed. Patients were followed up for three months to evaluate postoperative complications.
Results: This study included a total of 313 patients. 143 patients underwent AH and 170 patients underwent VH. Baseline characteristics were similar between the two groups. There were no intraoperative complications in either group. Operation time, intraoperative blood loss, first postoperative flatus time, time to out-of-bed activity, mean maximum postoperative body temperature, and duration of fever were all significantly shorter and less severe in the VH group compared with the AH group. In addition, vaginal length in the VH group was significantly shorter than in the AH group.
Conclusions: Vaginal hysterectomy has advantages over AH in the treatment of benign gynaecological diseases, providing greater efficacy and safety with minimal invasiveness.
Abdominal hysterectomy; Gynaecological benign diseases; Laparotomy; Vaginal hysterectomy
To evaluate the accuracy of axial length (AL) measurements obtained from immersion B-scan ultrasonography (immersion B-scan) for intraocular lens (IOL) power calculation in patients with high myopia and cataracts.
Immersion B-scan, contact A-scan ultrasonography (contact A-scan), and the IOLMaster were used to preoperatively measure the AL in 102 eyes from 102 patients who underwent phacoemulsification and IOL implantation. Patients were divided into two groups according to the AL: one containing patients with 22 mm≤AL<26 mm(group A) and the other containing patients with AL≥26 mm (group B). The mean error (ME) was calculated from the difference between the AL measurement methods predicted refractive error and the actual postoperative refractive error.
In group A, ALs measured by immersion B-scan (23.48±1.15) didn't differ significantly from those measured by the IOLMaster (23.52±1.17) or from those by contact A-scan (23.38±1.20). In the same group, the standard deviation (SD) of the mean error (ME) of immersion B-scan (-0.090±0.397 D) didn't differ significantly from those of IOLMaster (-0.095±0.411 D) and contact A-scan (-0.099±0.425 D). In group B, ALs measured by immersion B-scan (27.97±2.21 mm) didn't differ significantly from those of the IOLMaster (27.86±2.18 mm), but longer than those measured by Contact A-scan (27.75±2.23 mm, P=0.009). In the same group, the standard deviation (SD) of the mean error (ME) of immersion B-scan (-0.635±0.157 D) didn't differ significantly from those of the IOLMaster (-0.679±0.359 D), but differed significantly from those of contact A-scan (-0.953±1.713 D, P=0.028).
Immersion B-scan exhibits measurement accuracy comparable to that of the IOLMaster, and is thus a good alternative in measuring AL in eyes with high myopia when the IOLMaster can't be used, and it is more accurate than the contact A-scan.
axial length; measurement; immersion; ultrasonography; high myopia
Reprogramming energy metabolism has been an emerging hallmark of cancer cells. MicroRNAs play important roles in glucose metabolism.
The targets of microRNA-26a (miR-26a) were predicted by bioinformatics tools. The efficacy of miR-26a binding the 3′-untranslated region (UTR) of pyruvate dehydrogenase protein X component (PDHX) mRNA was evaluated using a dual-luciferase reporter assay. The PDHX expression at the mRNA and protein level in several colon cancer cell lines was quantified with real-time PCR and Western blot analysis respectively. The effects of miR-26a on glucose metabolism were determined by detecting the content of glucose consumption, production of lactate, pyruvate, and acetyl-coenzyme A.
The expression of miR-26a is inversely associated with the level of its targeting protein PDHX in several colon cancer cell lines with different malignancy potentials. MiR-26a inhibits PDHX expression by direct targeting the 3′-UTR of PDHX mRNA. The glucose consumption and lactate concentration were both greatly increased in colon cancer cells than the normal colon mucosal epithelia under physiological conditions. The overexpression of miR-26a in HCT116 cells efficiently improved the accumulation of pyruvate and decreased the production of acetyl coenzyme A. Meanwhile the inhibition of miR-26a expression induced inverse biological effects.
MiR-26a regulates glucose metabolism of colorectal cancer cells by direct targeting the PDHX, which inhibits the conversion of pyruvate to acetyl coenzyme A in the citric acid cycle.
MicroRNA-26a; PDHX; Colorectal cancer; Glucose metabolism
To investigate how auditory working memory relates to speech perception performance by Mandarin-speaking cochlear implant (CI) users.
Auditory working memory and speech perception was measured in Mandarin-speaking CI and normal-hearing (NH) participants. Working memory capacity was measured using forward digit span and backward digit span; working memory efficiency was measured using articulation rate. Speech perception was assessed with: (a) word-in-sentence recognition in quiet, (b) word-in-sentence recognition in speech-shaped steady noise at +5 dB signal-to-noise ratio, (c) Chinese disyllable recognition in quiet, (d) Chinese lexical tone recognition in quiet. Self-reported school rank was also collected regarding performance in schoolwork.
There was large inter-subject variability in auditory working memory and speech performance for CI participants. Working memory and speech performance were significantly poorer for CI than for NH participants. All three working memory measures were strongly correlated with each other for both CI and NH participants. Partial correlation analyses were performed on the CI data while controlling for demographic variables. Working memory efficiency was significantly correlated only with sentence recognition in quiet when working memory capacity was partialled out. Working memory capacity was correlated with disyllable recognition and school rank when efficiency was partialled out. There was no correlation between working memory and lexical tone recognition in the present CI participants.
Mandarin-speaking CI users experience significant deficits in auditory working memory and speech performance compared with NH listeners. The present data suggest that auditory working memory may contribute to CI users' difficulties in speech understanding. The present pattern of results with Mandarin-speaking CI users is consistent with previous auditory working memory studies with English-speaking CI users, suggesting that the lexical importance of voice pitch cues (albeit poorly coded by the CI) did not influence the relationship between working memory and speech perception.
Insulin resistance (IR) is closely correlated with cardiovascular disease (CVD). Retinol-binding protein 4 (RBP4) is a novel adipokine that modulates the action of insulin in various diseases. This study addressed the relationship between RBP4 and IR in newly diagnosed essential hypertension.
Serum RBP4, anthropometric and metabolic parameters were determined in 267 newly diagnosed essential hypertensive patients not taking antihypertensive medications. The patients along with 64 control (NC) normotensive and lean subjects paired by age and sex were divided into two groups depending on body mass index (BMI), hypertension with obesity (HPO) and hypertension without obesity (HP).
A striking difference was observed in RBP4 levels between the HP and NC groups. Significantly higher levels were noted in the HP group compared with the NC group; slightly, but not significantly, lower levels were observed in the HPO group compared with the HP group. After adjusting for BMI, WC and WHR, a modestly linear relationship was observed between RBP4 levels and SBP (r = 0.377; p = 0.00), DBP (r = 0.288; p = 0.00) and HOMA-β(r = 0.121; p = 0.028). Multiple stepwise regression analysis showed that SBP, WHR and drinking were independently related with serum RBP4 levels.
The results of this study indicated that RBP4 levels were increased in naive hypertensive patients; however, no differences were observed in obese or non-obese hypertensive subjects. Our data suggest for the first time that RBP4 levels are significantly increased but do not contribute to the development of IR in newly diagnosed hypertensive Chinese patients.
Retinol-binding protein 4; Insulin resistance; Obesity; Essential hypertension
Trials on sling exercise (SE), commonly performed to manage chronic low back pain (LBP), yield conflicting results. This study aimed to review the effects of SE on chronic LBP.
The randomized controlled trials comparing SE with other treatments or no treatment, published up to August 2013, were identified by electronic searches. Primary outcomes were pain, function, and return to work. The weighted mean difference (WMD) and 95% confidence interval (CI) were calculated, using a random-effects model.
Risk of bias was rated as high in 9 included trials, where some important quality components such as blinding were absent and sample sizes were generally small. We found no clinically relevant differences in pain or function between SE and other forms of exercise, traditional Chinese medical therapy, or in addition to acupuncture. Based on two trials, SE was more effective than thermomagnetic therapy at reducing pain (short-term: WMD –13.90, 95% CI –22.19 to –5.62; long-term: WMD –26.20, 95% CI –31.32 to –21.08) and improving function (short-term: WMD –10.54, 95% CI –14.32 to –6.75; long-term: WMD –25.75, 95% CI –30.79 to –20.71). In one trial we found statistically significant differences between SE and physical agents combined with drug therapy (meloxicam combined with eperisone hydrochloride) but of borderline clinical relevance for pain (short-term: WMD –15.00, 95% CI –19.64 to −10.36) and function (short-term: WMD −10.00; 95% CI −13.70 to −6.30). There was substantial heterogeneity among the two trials comparing SE and thermomagnetic therapy; both these trials and the trial comparing SE with physical agents combined with drug therapy had serious methodological limitations.
Based on limited evidence from 2 trials, SE was more effective for LBP than thermomagnetic therapy. Clinically relevant differences in effects between SE and other forms of exercise, physical agents combined with drug therapy, traditional Chinese medical therapy, or in addition to acupuncture could not be found. More high-quality randomized trials on the topic are warranted.
Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis.
This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.
AIM: To investigate the rate of Helicobacter pylori (H. pylori) resistance to clarithromycin among ethnic minority patients in Guangxi, explore the underlying mechanisms, and analyze factors influencing genotype distribution of H. pylori isolates.
METHODS: H. pylori strains were isolated, cultured and subjected to drug sensitivity testing. The 23S rRNA gene of H. pylori isolates was amplified by PCR and analyzed by PCR-RFLP and direct sequencing to detect point mutations. REP-PCR was used for genotyping of H. pylori isolates, and NTsys_2 software was used for clustering analysis based on REP-PCR DNA fingerprints. Factors potentially influencing genotype distribution of H. pylori isolates were analyzed.
RESULTS: The rate of clarithromycin resistance was 31.3%. A2143G and A2144G mutations were detected in the 23S rRNA gene of all clarithromycin-resistant H. pylori isolates. At a genetic distance of 78%, clarithromycin-resistant H. pylori isolates could be divided into six groups. Significant clustering was noted among H. pylori isolates from patients with peptic ulcer or gastritis.
CONCLUSION: The rate of clarithromycin resistance is relatively high in ethnic minority patients in Guangxi. Main mechanisms of clarithromycin resistance are A2143G and A2144G mutations in the 23S rRNA gene. Clarithromycin-resistant H. pylori isolates can be divided into six groups based on REP-PCR DNA fingerprints. Several factors such as disease type may influence the genotype distribution of H. pylori isolates.
Helicobacter pylori; Antibiotic resistance; Mechanism; Clarithromycin; Genotype
Mitochondria are crucial to the hypoxia response of aerobic organisms. However, mitochondrial mechanisms for hypoxia adaptation remain largely unknown. We conducted a comparative study on the mitochondrial hypoxia response and adaptation of the Tibetan Plateau and North China lowland populations of migratory locusts, Locusta migratoria. Compared with lowland locusts, Tibetan locusts presented significantly higher hypoxia tolerance and a better-maintained mitochondrial structure in flight muscles under oxygen partial pressure of 1.6 kPa. The hypoxic treatment inhibited the NADH-linked oxidative phosphorylation (OXPHOS) significantly in both locust populations, but to a less extent in Tibetan locusts. Among the critical components of OXPHOS, only cytochrome c oxidase (COX) exhibited significantly higher activity in Tibetan locusts under normoxia and hypoxia. Pharmacological interventions using NaN3 confirmed that COX activity inhibition reduced hypoxia tolerance by downregulating OXPHOS in both locust populations. The enhanced COX activity was caused not by protein content, but by elevated catalytic efficiency resulting from the increased ferrocytochrome c affinity of COX and the increased electron transport rate via catalytic redox centres. These findings reveal a novel mechanism that confers mitochondrial robustness against hypoxia by modulating the COX activity, which represents an adaptation to permanent hypoxia in the Tibetan Plateau.
Tibetan plateau; mitochondria; hypoxia response; hypoxia adaptation; catalytic efficiency