Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-Escherichia-Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn(2)Cys(6) transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn(2)Cys(6)TF genes. A large variation in biological functions of Zn(2)Cys(6)TF genes was observed under the conditions tested. Sixty-one of 104 Zn(2)Cys(6) TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.
Lycopene ?-cyclase (?-LCY) is a key enzyme that catalyzes the synthesis of ?-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ?-LCY (designated Nt?-LCY1 and Nt?-LCY2) were cloned from Nicotiana tabacum. Nt?-LCY1 and Nt?-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Nt?-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Nt?-LCY1-GFP fusion protein demonstrated that mature Nt?-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Nt?-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ?-LCY in Nicotiana benthamiana resulted in an increase of ?-branch carotenoids and a reduction in the levels of ?-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of ?-OHase in the TRV-?-lcy line. Suppression of ?-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ?-LCY in plant carotenoid biosynthesis and suggest a role for ?-LCY in positively modulating low temperature stress responses.
Failure of remyelination in diseases, such as multiple sclerosis (MS), leads to permanent axonal damage and irreversible functional loss. The mechanisms controlling remyelination are currently poorly understood. Recent studies implicate the cyclin-dependent kinase 5 (Cdk5) in regulating oligodendrocyte (OL) development and myelination in CNS. In this study, we show that Cdk5 is also an important regulator of remyelination. Pharmacological inhibition of Cdk5 inhibits repair of lysolecithin lesions. This inhibition is a consequence of Cdk5 disruption in neural cells because remyelination in slice cultures is blocked by Cdk5 inhibitors, whereas specific deletion of Cdk5 in OLs inhibits myelin repair. In CNP-Cre;Cdk5(fl/fl) conditional knock-out mouse (Cdk5 cKO), myelin repair was delayed significantly in response to focal demyelinating lesions compared with wild-type animals. The lack of myelin repair was reflected in decreased expression of MBP and proteolipid protein and a reduction in the total number of myelinated axons in the lesion. The number of CC1(+) cells in the lesion sites was significantly reduced in Cdk5 cKO compared with wild-type animals although the total number of oligodendrocyte lineage cells (Olig2(+) cells) was increased, suggesting that Cdk5 loss perturbs the transition of early OL lineage cell into mature OL and subsequent remyelination. The failure of remyelination in Cdk5 cKO animals was associated with a reduction in signaling through the Akt pathway and an enhancement of Gsk-3? signaling pathways. Together, these data suggest that Cdk5 is critical in regulating the transition of adult oligodendrocyte precursor cells to mature OLs that is essential for myelin repair in adult CNS.
In this paper, a novel method for respiratory monitoring is presented. The method is based on Trichel pulses (TPs) using a simple field ionization sensor which consists of a needle electrode and a plate electrode. Experiments have been conducted to demonstrate that different respiratory patterns, including normal, ultra-fast, deep breaths, and apnea could be easily monitored in real time by detecting the changes in the TP frequency. The vital capacity could also be assessed by calculating the variation of TP frequency. It is found that the operation principle of the proposed sensor is based on the effects of breath airflow and the atomized water in exhaled air on the TP frequency by changing the ionization process and the dynamics of charged particles in the short gap. The influences of applied voltage and ambient parameters have also been investigated.
Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H. parasuis and has neutralizing activity. The constructed mouse-pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H. parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P < 0.05). Importantly, the chimeric antibody partially protected piglets against H. parasuis infection according to the clinical lesion scores and PCR results of H. parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse-pig chimeric antibody could be a therapeutic candidate to prevent the H. parasuis infection and control the prevalence of disease.
MicroRNA (miRNAs) is demonstrated to be present in the blood of humans and has been increasingly suggested as a novel biomarker for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In this study, we aim to evaluate the diagnostic and prognostic value of circulating miR-328 and miR-134 in patients with acute myocardial infarction (AMI). Circulating levels of miR-328 and miR-134 were detected by quantitative real-time PCR in plasma samples from 359 AMI patients and 30 healthy volunteers. Concentrations of high-sensitivity cardiac troponin T (hs-cTnT) were measured using electrochemiluminescence-based methods. MiRNAs were assessed for discrimination of a clinical diagnosis of AMI and for association with primary clinical endpoint defined as a composite of cardiogenic death and development of heart failure within 6 months after infarction. Results showed that levels of plasma miR-328 and miR-134 were significantly higher in AMI patients than in healthy controls. Receiver operating characteristic curve analyses showed significant diagnostic value of miR-328 and miR-134 for AMI. However, neither of them was superior to hs-cTnT for the diagnosis. Additionally, increased miRNA levels were strongly associated with increased risk of mortality or heart failure within 6 months for miR-328 (OR 7.35, 95 % confidence interval 1.07-17.83, P < 0.001) and miR-134 (OR 2.28, 95 % confidence interval 1.03-11.32 P < 0.001). In conclusion, circulating miR-328 and miR-134 could be potential indicators for AMI, and the miRNA levels are associated with increased risk of mortality or development of heart failure.
Activin A is a critical regulator in human embryonic stem cells (hESCs) maintenance and differentiation. Different concentrations of Activin A affect hESC maintenance and differentiation in different ways. A high concentration favors anterior primitive streak and gives rise to DE if the stimulation persists. hESCs were cultured with and without 10?ng/mL Activin A supplement respectively. The two groups of cells were differentiated into endoderm cells with 100?ng/mL Activin A and other reagents. Microarray-based DNA methylation was analyzed with the Infinium Human Methylation450 BeadChip on these two groups. There was a significant difference in endoderm differentiation efficiency (average efficiency: 71 vs. 58.5%, P?0.05). hESCs cultured with Activin A supplement had an increased propensity to form definitive endoderm cells in response to Activin A and Wnt signal. Differentially Methylated Regions (DMRs) between these two groups were found. DMRs were related to the stem cell maintenance and gene regulation by peroxisome proliferators via PPAR?, indicating that hESCs maintained with Activin A supplement had stronger "stemness."
The absolute stability of large-scale Lurie direct control systems with time-varying coefficients is investigated. Based on the decomposition method for large-scale systems and technique of the nonsingular M-matrix, a suitable scalar Lyapunov function as a weighted sum is constructed. By estimating its total time derivative, some absolute stability criteria and practical corollaries are derived. Furthermore, the results are extended to multiple nonlinearities. The salient feature of this paper is that the criteria which we propose allow for the situation that the norms of time-varying coefficients are unbounded. The main idea of the methodology is that even if the coefficients are norm-unbounded, by restricting their relative magnitude, the problem of negative definiteness for the derivative can also be changed into the problem of stability for a constant matrix. Finally, some numerical examples are included to illustrate the effectiveness of the proposed criteria.
Left ventricular remodeling after acute myocardial infarction (AMI) is associated with adverse prognosis. It is becoming increasingly clear that circulating miRNAs could be promising biomarkers for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In the present study, a total of 359 consecutive patients were recruited. Plasma samples were collected on admission. Echocardiographic studies were performed during the admission and at six months follow-up after AMI. Remodeling was defined as an at least 10% increase from baseline in the left ventricular end-diastolic volume. Plasma miRNA levels were assessed for association with six months mortality or development of heart failure. Results showed that levels of plasma miR-208b and miR-34a were significantly higher in patients with remodeling than those without. Increased miRNA levels were strongly associated with increased risk of mortality or heart failure within six months for miR-208b (OR 17.91, 95% confidence interval=2.07-98.81, p=0.003), miR-34a (OR 4.18, 95% confidence interval=1.36-12.83, p=0.012) and combination of the two miRNAs (OR 18.73, 95% confidence interval=1.96-101.23, p=0.000). The two miRNA panels reclassified a significant proportion of patients with a net reclassification improvement of 11.7% (p=0.025) and an integrated discrimination improvement of 7.7% (p=0.002). These results demonstrated that circulating miR-208b and miR-34a could be useful biomarkers for predicting left ventricular remodeling after AMI, and the miRNA levels are associated with increased risk of mortality or heart failure.
Myasthenia gravis (MG) is an autoimmune disease caused by circulating antibodies that block acetylcholine receptor (AchR) at the neuromuscular junction. There is the cognitive and memory impairment in patients with MG. However, the molecular mechanisms underlying the alteration of central nervous system in MG remain unknown. In the present study, we found that the level of malondialdehyde (MDA) was increased in the brain of experimental autoimmune myasthenia gravis (EAMG). Furthermore, the expression of thioredoxin-1 (Trx-1) and the activity of cAMP response element-binding protein (CREB) were significantly decreased in frontal lobe and hippocampus of mice with EAMG. We also found that the expression of pro-apoptotic C/EBP homologous protein (CHOP) was increased in the frontal lobe and hippocampus of mice. However, the expressions of glucose regulated protein 78 (GRP78/Bip) was not changed in same areas. Inversely, the expressions of pro-caspase-12, pro-caspase-3 and pro-caspase-9 were decreased. These data indicate that Trx-1 mediated endoplasmic reticulum and mitochondria pathways are involved in brain damage in MG. Trx-1 may be a pivotal target for brain protective treatment in MG.
The SNF1/AMPK pathway has a central role in response to nutrient stress in yeast and mammals. Previous studies on SNF1 function in phytopathogenic fungi mostly focused on the catalytic subunit Snf1 and its contribution to the derepression of cell wall degrading enzymes (CWDEs). However, the MoSnf1 in Magnaporthe oryzae was reported not to be involved in CWDEs regulation. The mechanism how MoSnf1 functions as a virulence determinant remains unclear. In this report, we demonstrate that MoSnf1 retains the ability to respond to nutrient-free environment via its participation in peroxisomal maintenance and lipid metabolism. Observation of GFP-tagged peroxisomal targeting signal-1 (PTS1) revealed that the peroxisomes of ?Mosnf1 were enlarged in mycelia and tended to be degraded before conidial germination, leading to the sharp decline of peroxisomal amount during appressorial development, which might impart the mutant great retard in lipid droplets mobilization and degradation. Consequently, ?Mosnf1 exhibited inability to maintain normal appressorial cell wall porosity and turgor pressure, which are key players in epidermal infection process. Exogenous glucose could partially restore the appressorial function and virulence of ?Mosnf1. Toward a further understanding of SNF1 pathway, the ?-subunit MoSip2, ?-subunit MoSnf4, and two putative Snf1-activating kinases, MoSak1 and MoTos3, were additionally identified and characterized. Here we show the null mutants ?Mosip2 and ?Mosnf4 performed multiple disorders as ?Mosnf1 did, suggesting the complex integrity is essential for M. oryzae SNF1 kinase function. And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3. Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ?Mosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3. Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.
The linear discrete-time descriptor noncausal multirate system is considered for the presentation of a new design approach for optimal preview control. First, according to the characteristics of causal controllability and causal observability, the descriptor noncausal system is constructed into a descriptor causal closed-loop system. Second, by using the characteristics of the causal system and elementary transformation, the descriptor causal closed-loop system is transformed into a normal system. Then, taking advantage of the discrete lifting technique, the normal multirate system is converted to a single-rate system. By making use of the standard preview control method, we construct the descriptor augmented error system. The quadratic performance index for the multirate system is given, which can be changed into one for the single-rate system. In addition, a new single-rate system is obtained, the optimal control law of which is given. Returning to the original system, the optimal preview controller for linear discrete-time descriptor noncausal multirate systems is derived. The stabilizability and detectability of the lifted single-rate system are discussed in detail. The optimal preview control design techniques are illustrated by simulation results for a simple example.
Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched (469)KTPAEAR(475) of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.
Uterine leiomyomas are benign tumors that develop from smooth muscle cells (SMCs). The reactive oxygen species (ROS) have been shown to be involved in the signaling pathways that stimulate proliferation of a variety of cell types. Thioredoxin-1 (TRX-1) is a redox-regulating protein, which is overexpressed in various tumors. In the present study, we investigated the expressions of TRX-1 and its related molecules in uterine leiomyomas and matched adjacent myometrium. Our results showed the expression of TRX-1 was increased in leiomyomas compared with the matched adjacent myometrium by quantitative RT-PCR and western blotting. FOXO3A expression was increased in leiomyomas compared with myometrium by western blotting. The mRNA levels of hypoxia-inducible factor-1?, cyclooxygenase-2 and cyclin D1 were increased in leiomyomas compared with the adjacent myometrium. The mRNA level of (thioredoxin-1-binding protein) TBP-2 in leiomyomas was not altered when compared with the matched adjacent myometrium. These results suggest that TRX-1 and some of its related molecules are associated with the pathogenesis of uterine leiomyomas. The identification of TRX-1 signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.
An improved pseudotargeted method using gas chromatography/mass spectrometry (GC/MS) was developed to investigate the metabolic profile of tobacco leaves from three planting regions (Yunnan, Guizhou, and Henan provinces). The analytical characteristics of the method with regard to reproducibility, precision, linearity, and stability were satisfactory for metabolic profiling study. Partial least-squares-discriminant analysis and hierarchical cluster analysis demonstrated that the metabolic profiles of tobacco from the Yunnan and Guizhou regions were different from that from the Henan province. The amino acid (e.g., phenylalanine, leucine, and tyrosine) and carbohydrate (e.g., fructose, trehalose, and sucrose) contents were the highest in Henan tobacco. The highest contents of organic acids (e.g., isocitrate, citrate, and fumarate) of the TCA cycle and antioxidants (e.g., quinate, chlorogenic acid, and ascorbate) were found in Guizhou tobacco. The correlation coefficients between metabolite content and climate factors (rainfall, sunshine, and temperature) demonstrated that drought facilitated the accumulation of sugars and amino acids. The content of TCA cycle intermediates could be influenced by multiple climate factors. This study demonstrates that the pseudotargeted method with GC/MS is suitable for the investigation of the metabolic profiling of tobacco leaves and the assessment of differential metabolite levels related to the growing regions.
Acetaminophen (APAP) is widely used as an antipyretic agent which is safe at therapeutic doses. However, overdose of APAP induces fatal and non-fatal hepatic necroses. The chemical reactive metabolites of APAP initiate toxicity and inflammatory response within the liver and lead to acute liver failure. However, the mechanism underlying APAP-induced liver injury is unknown. Thioredoxin-1 (TRX-1) is an important redox regulator, which plays roles in resisting oxidative stress, regulating inflammation and inhibiting apoptosis. Panaxatriol saponin (PTS) is one of the biologically active fractions of Panax notoginseng which is a traditional Chinese medicine. The aim of this study was to investigate the mechanism on PTS protecting liver from APAP hepatotoxicity.
A rapid, specific and sensitive ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of four trace alkaloids, including aconitine (AC), hypaconitine (HA), mesaconitine (MA) and yunaconitine (YA) in human urine samples. UPLC-MS-MS system coupled with an electrospray ionization (ESI) source was performed in multiple reaction monitoring (MRM) mode. The sample preparation was performed with hollow fiber microextraction (HF-LPME) prior to the analysis. The enrichment factors of the four alkaloids were 102 - 301. The separation was applied on a Waters ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 micro m) with a gradient elution of acetonitrile and 10 mmol/L NH4HCO3, as mobile phase. The retention times , were less than 3 min. This method significantly improved the detection sensitivity, and the limits of quantitation were from 0. 01 to 0.1 ng/L. The calibration curves were linear over the ranges of 0.01 - 10 ng/L for AC, MA and YA, 0.1 - 100 ng/L for HA in human urine samples, and the correlation coefficients were 0. 998 1, 0.998 4, 0.999 5 and 0.998 6, respectively. The method was proved to be rapid and sensitive for aconitum alkaloid analysis in urine samples.
Species of Diaporthe (anamorph Phomopsis) comprise a diverse and widely distributed group of phytopathogens, saprophytes and endophytes. However, the degree of genetic diversity of endophytic Diaporthe has not yet been fully investigated. In this paper, a survey of endophytes from 28 plants in southeast China yielded 116 Diaporthe isolates, out of which 64 haplotypes were determined using DnaSP ver. 5.1 based on alignment result of internal transcribed spacer of ribosomal nucleotide sequences (ITS rDNA). Many haplotypes turned out to be quite different from known species and displayed high diversity. Among them, 14 strains from 5 discriminating terminal clades were selected to go through further analysis according to partial sequence of translation elongation factor 1-? (tef1-?) and again they got separated from others. The following multi-gene phylogenetic analysis based on ITS rDNA, tef1-?, ? tubulin and calmodulin grouped eight most discrepant strains into three distinctive clusters, cluster 1 (Rc001, Eu004 and Eu009), cluster 2 (ZJWCF252, Sjm001 and Ac001) and cluster 3 (Pcs013 and Sfp005) respectively with high support values. These clusters above represent three potentially novel species. This research provides strong evidence of high biodiversity and novelty of Diaporthe endophytes from southeast China, which is thus important not only for better resolving the taxonomy in this genus, but also for further utilization due to their multiple application.
The aim of this study was to explore the association of (99m)Tc-HYNIC-octreotide SPECT/CT somatostatin receptor scintigraphy (SRS) with the pathological grading and expression of somatostatin receptor 2 (SSTR2) for meningioma, and to define possible roles of SRS in the pathological grading of meningioma. Thirty patients with meningiomas diagnosed by MRI and treated with (99m)Tc-HYNIC-octreotide SPECT/CT SRS. Meningioma tissues were obtained from analyzing pathological grading and measuring the expression of SSTR2 with immunohistochemical staining. The meningioma side (T) to the contralateral side (NT) ratios (T/TN) of radioactive counts were calculated to investigate their association with the pathological grading of meningioma and the expression of SSTR2. All 30 cases showed high meningioma radioactivity accumulation using SRS with a sensitivity of 100 %, while CT scans only detected 25 cases with a sensitivity of 83 %. Twenty cases with grade I meningioma had a T/NT ratio of 3.80 ± 1.67, which was significantly lower than the other 10 cases (9.57 ± 3.78) with a grade II meningioma (P < 0.01). All meningiomas expressed SSTR2 as detected by immunohistochemical staining, and the T/NT ratio was positively associated with the pathological grading of meningioma and the expression of SSTR2 (with r of 0.784 and 0.805, respectively). (99m)Tc-HYNIC-octreotide SPECT/CT SRS is a sensitive technique for detecting meningioma, and the T/NT ratio of the SRS data closely correlates with the pathological grade of meningioma and the expression of SSTR2.
Recent discoveries of large leg feathers in some theropods have implications for our understanding of the evolution of integumentary features on the avialan leg, and particularly of their relevance for the origin of avialan flight. Here we report 11 basal avialan specimens that will greatly improve our knowledge of leg integumentary features among early birds. In particular, they provide solid evidence for the existence of enlarged leg feathers on a variety of basal birds, suggest that extensively scaled feet might have appeared secondarily at an early stage in ornithuromorph evolution, and demonstrate a distal-to-proximal reduction pattern for leg feathers in avialan evolution.
The development of oligodendrocytes, the myelinating cells of the vertebrate CNS, is regulated by a cohort of growth factors and transcription factors. Less is known about the signaling pathways that integrate extracellular signals with intracellular transcriptional regulators to control oligodendrocyte development. Cyclin dependent kinase 5 (Cdk5) and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Here we demonstrate a previously unrecognized function of Cdk5 in regulating oligodendrocyte maturation and myelination. During late embryonic development Cdk5 null animals displayed a reduction in the number of MBP+ cells in the spinal cord, but no difference in the number of OPCs. To determine whether the reduction of oligodendrocytes reflected a cell-intrinsic loss of Cdk5, it was selectively deleted from Olig1+ oligodendrocyte lineage cells. In Olig1(Cre/+); Cdk5(fl/fl) conditional mutants, reduced levels of expression of MBP and PLP mRNA were observed throughout the CNS and ultrastructural analyses demonstrated a significant reduction in the proportion of myelinated axons in the optic nerve and spinal cord. Pharmacological inhibition or RNAi knockdown of Cdk5 in vitro resulted in the reduction in oligodendrocyte maturation, but had no effect on OPC cell proliferation. Conversely, over-expression of Cdk5 promoted oligodendrocyte maturation and enhanced process outgrowth. Consistent with this data, Cdk5(-/-) oligodendrocytes developed significantly fewer primary processes and branches than control cells. Together, these findings suggest that Cdk5 function as a signaling integrator to regulate oligodendrocyte maturation and myelination.
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. ?mopex5 was unable to use lipids as sole carbon source and lost pathogenicity completely. Similar as ?mopex7, ?mopex5 exhibited significant reduction in lipid utilization and mobilization, appressorial turgor genesis and H(2)O(2) resistance. Additionally, ?mopex5 presented some distinct defects which were undetected in ?mopex7 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.
?-Melanocyte-stimulating hormone (?-MSH) functions as a mediator of inflammation and immunity; however, the short half-life and high dose needed limit the comprehensive clinical application of ?-MSH. The aim of this study was to generate human bone marrow-derived mesenchymal stem cells (MSCs) that express and secrete high levels of bioactive ?-MSH. MSCs were obtained from a normal donor and assessed for proliferation, surface markers, and adipogenic and osteogenic differentiation. A lentivirus-encoding ?-MSH was constructed. MSCs were infected with this lentivirus-encoding ?-MSH and assessed for stability and the expression and secretion of bioactive ?-MSH. The cumulative MSC expansion rates pre- and post-lentivirus infection were not significantly different (P > 0.05). The MSCs remained stable after infection with the lentivirus-encoding ?-MSH. The concentration of ?-MSH in the supernatants of MSCs infected with the lentivirus-encoding ?-MSH was 17.55 ng/ml (P < 0.001), and a melanin assay indicated that bioactive ?-MSH was secreted from MSCs infected with the lentivirus-encoding ?-MSH, with an optical absorbance at OD(405) of 0.886 (P < 0.001). These results suggested that MSCs were promising cell carriers for the expression and secretion of high levels of bioactive ?-MSH.
Granule cell migration influences the laminar structure of the cerebellum and thereby affects cerebellum function. Bergmann glia are derived from radial glial cells and aid in granule cell radial migration by providing a scaffold for migration and by mediating interactions between Bergmann glia and granule cells. In this review, we summarize Bergmann glia characteristics and the mechanisms underlying the effect of Bergmann glia on the radial migration of granule neurons in the cerebellum. Furthermore, we will focus our discussion on the important factors involved in glia-mediated radial migration so that we may elucidate the possible mechanistic pathways used by Bergmann glia to influence granule cell migration during cerebellum development.
The aim of this study was to determine whether recurrence of meningiomas could be reduced by combining somatostatin receptor scintigraphy (SRS) of Tc-HYNIC-octreotide SPECT/CT and radio guidance with a hand-held ?-probe during surgery.
Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-?1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-?1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering.
Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ?Movma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae.
We have studied the activation kinetics of zinc sulfide (ZnS) using silver as an activator by a quartz crystal microbalance with dissipation (QCM-D). The zinc sulfide coating on QCM-D sensor was shown to have similar crystallographic structure, composition, and surface properties as nature sphalerite through the characterization of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and xanthate adsorption measurement using QCM-D. The activation of ZnS sensor by silver was confirmed by the mass increase in ZnS sensor coupled with subsequent xanthate adsorption during QCM-D measurement, the change of surface wettability, and the presence of Ag(2)S on the surface. Two distinct stages on the silver uptake vs. time curve were identified and fitted well by a logarithmic function for the initial stage and a parabolic law in the later stage, which agrees with the two-stage zinc-silver reaction kinetics reported previously. Argon sputtering followed by XPS measurement on the ZnS surface demonstrated the penetration of silver into the bulk ZnS after activation. The present study is the first of its kind to apply the QCM-D technique to investigate sphalerite activation, which introduces a new in situ approach to investigate surface adsorption and activation in many mineral processes and surface modifications.
Changes in the levels or biochemistry of cerebrospinal fluid (CSF) neuropeptides with opioid-like properties have been suggested to reflect alterations in specific biological processes. We have determined various kinetic parameters for methionine-enkephalin (MET) degradation by CSF samples from nonneurological patients. Study subjects included 9 males (51-67 years of age) and 5 females (47-61 years of age). Aliquots, removed from an incubation vessel containing buffer, CSF, and peptide [tyr-3,5-H(N)MET], were analyzed for tyrosine and other degradation products. Essentially all of the labeled tyrosine from the added MET was recovered as free amino acid after 60 minutes of incubation (1:2 ratio, vol:vol; optimum pH 7.4; and temperature 37°C); other possible peptide metabolites (>3%) were not detected. Irrespective of age or gender, the peptides degradation half-life and initial velocity values were in a limited range; t1/2 26.2 ± 5.5 and 20.8-33.8 minutes, and Iv 0.03 ±0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. Km and Vmax values were 0.19 ± 0.02 and 0.17-0.21 mM, and 9.8 ± 2.2 and 7.6-12.0 ?mol·L·min, respectively. Neither CSF sample storage time (up to a year) nor repeated freezing and thawing (up to 3 times over a year) altered the kinetics or products of this reaction. These preliminary findings might serve as reference values when conducting similar studies using CSF from patients diagnosed with specific neurological conditions; significant alterations in MET degradation profile in such a population could provide valuable biological markers for diagnostic and treatment purposes.
The crop is characteristic of seed-eating birds today, yet little is known about its early history despite remarkable discoveries of many Mesozoic seed-eating birds in the past decade. Here we report the discovery of some early fossil evidence for the presence of a crop in birds. Two Early Cretaceous birds, the basal ornithurine Hongshanornis and a basal avian Sapeornis, demonstrate that an essentially modern avian digestive system formed early in avian evolution. The discovery of a crop in two phylogenetically remote lineages of Early Cretaceous birds and its absence in most intervening forms indicates that it was independently acquired as a specialized seed-eating adaptation. Finally, the reduction or loss of teeth in the forms showing seed-filled crops suggests that granivory was possibly one of the factors that resulted in the reduction of teeth in early birds.
The prepared slices of Chinese crude drugs are growing important in recent years, and faced with new developments and opportunities. The author analyzed the importance of formulate national processing procedures of prepared slices of Chinese crude drugs combined actual work, proposed the overall objectives and tasks for the formulation, and emphasized to need to correctly deal with several important factors during the process of formulate "National processing procedures of prepared slices of Chinese crude drugs", unified the national standards of prepared slices, solved the real problems that the prepared slices of Chinese crude drugs industry faced.
A simple hollow fiber based centrifuge ultrafiltration pretreatment procedure has been developed for the analysis of active components with high polarity in Chinese traditional and herbal drugs which usually contain macromolecule impurities. The procedure combined with HPLC was applied to the determination of hesperidin in Huoxiangzhengqi water. Sample solutions were purified by our patent hollow fiber centrifuge ultrafiltration device. Under the effect of the centrifgual force, micromolecules were removed from solution samples, thus it increased the service life of the column. The accuracy and repeatability of this method have also been improved. The separation was carried out on a Promosil C18 column (4.6 mm x 150 mm, 5 microm) with methanol -0.5% acetic acid solution (35:65) as mobile phase at a flow rate of 1.0 mL x min(-1). The detector wavelength was 283 nm and the column temperature was 30 degrees C. A good linear relation was obtained in the range of 4.69 - 150 mg x L(-1) (r = 0.999 7) and the average recovery was 103.0% with RSD of 1.9%. This method is simple, rapid and accurate, and it provides a simple and cheap ultrafiltration means for the analysis of the polar components in Chinese traditional and herbal drugs.
The introduction of luteinizing hormone-releasing hormone (LHRH) analogs and their antagonists is revolutionizing the treatment of prostate cancer. In this study, poly(D,L-lactideco-glycolide) (PLGA) microspheres containing a highly potent LHRH antagonist (LXT-101) of interest in the indication of prostate cancer were evaluated on release mechanisms in vitro and biological performance in vivo. LXT-101 microspheres were prepared by the water/oil/water double emulsion method and the solid/oil/oil method. The results showed that the mechanism of LXT-101 releasing from PLGA 14,000 microspheres was the cooperation of drug diffusion and polymer degradation. This clarified the relationship between the microsphere characterization and hormone level in vivo. The larger microspheres (33 ?m) could inhibit the testosterone level to castration for a longer time (35 days) than the smaller microspheres (15 ?m, 14 days). The formulation containing the hydrophilic additive (polyethylene glycol 6000) could suppress the testosterone level to castration for a longer time (> 35 days) than the formulation without polyethylene glycol (14 days). The appearance of testis, vesicular seminalis, and prostates changed after treatment. The weights of sexual organs decreased significantly. The in-vivo release of the LXT-101 PLGA 14,000 microspheres curve showed that in-vivo release started immediately after day 1 (22.7%) and was rapid during the first 5 days (40.2% release). The LXT-101 microspheres could be a promising drug delivery system candidate to treat sex hormone-dependent tumors and other related disorders.
Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.
Nudds and Dyke (Reports, 14 May 2010, p. 887) compared the rachis diameters of the primary feathers of Archaeopteryx and Confuciusornis to those of modern birds and found that the primary feathers of these two basal birds were too weak to support sustained flight. Our measurements of Confuciusornis specimens suggest that their conclusions need to be further evaluated.
In extant birds, the hand is permanently abducted towards the ulna, and the wrist joint can bend extensively in this direction to fold the wing when not in use. Anatomically, this asymmetric mobility of the wrist results from the wedge-like shape of one carpal bone, the radiale, and from the well-developed convexity of the trochlea at the proximal end of the carpometacarpus. Among the theropod precursors of birds, a strongly convex trochlea is characteristic of Coelurosauria, a clade including the highly derived Maniraptora in addition to tyrannosaurs and compsognathids. The shape of the radiale can be quantified using a radiale angle between the proximal and distal articular surfaces. Measurement of the radiale angle and reconstruction of ancestral states using squared-change parsimony shows that the angle was small (15 degrees) in primitive coelurosaurs but considerably larger (25 degrees) in primitive maniraptorans, indicating that the radiale was more wedge-shaped and the carpal joint more asymmetric. The radiale angle progressively increased still further within Maniraptora, with concurrent elongation of the forelimb feathers and the forelimb itself. Carpal asymmetry would have permitted avian-like folding of the forelimb in order to protect the plumage, an early advantage of the flexible, asymmetric wrist inherited by birds.
Spectacular fossils from the Early Cretaceous Jehol Group of northeastern China have greatly expanded our knowledge of the diversity and palaeobiology of dinosaurs and early birds, and contributed to our understanding of the origin of birds, of flight, and of feathers. Pennaceous (vaned) feathers and integumentary filaments are preserved in birds and non-avian theropod dinosaurs, but little is known of their microstructure. Here we report that melanosomes (colour-bearing organelles) are not only preserved in the pennaceous feathers of early birds, but also in an identical manner in integumentary filaments of non-avian dinosaurs, thus refuting recent claims that the filaments are partially decayed dermal collagen fibres. Examples of both eumelanosomes and phaeomelanosomes have been identified, and they are often preserved in life position within the structure of partially degraded feathers and filaments. Furthermore, the data here provide empirical evidence for reconstructing the colours and colour patterning of these extinct birds and theropod dinosaurs: for example, the dark-coloured stripes on the tail of the theropod dinosaur Sinosauropteryx can reasonably be inferred to have exhibited chestnut to reddish-brown tones.
The holotype of the theropod non-avian dinosaur Microraptor gui from the Early Cretaceous of China shows extensive preservation of feathers in a halo around the body and with flight feathers associated with both the fore and hindlimbs. It has been questioned as to whether or not the feathers did extend into the halo to reach the body, or had disassociated and moved before preservation. This taxon has important implications for the origin of flight in birds and the possibility of a four-winged gliding phase.
Hypertrophic scarring remains a major problem for patients who have suffered from surgeries or burns. Vascularization plays an important role in the early phase of hypertrophic scarring. Therefore, the inhibition of angiogenesis might be used as a preventive strategy. In this study, we assessed the effect of anti-angiogenesis resulting from adenovirus-mediated METH1 (metalloprotease and thrombospondin1) gene expression on the hypertrophic scar formation in a rabbit ear model of hypertrophic scarring. We first investigated the number of microvessel and microcirculatory perfusion in untreated scars on days 10, 30, 60, and 90 after epithelialization. Then, we examined the effect of anti-angiogenesis by adenovirus-mediated METH1 expression on hypertrophic scar formation by calculating the scar elevation index, counting the microvessel and argyrophilic nucleolar organizer region particle, and detecting the amount of collagen on days 30 and 60 after treatment. We found that untreated scar tissues at the proliferative phase (days 10-60 after epithelialization) had a significantly higher density of microvessel and microcirculatory perfusion than those at the mature phase (day 90 after epithelization) (both p<0.05). On days 30 and 60 after treatment, the hypertrophic scar formation was significantly inhibited in the treatment group. There was significantly reduced scar elevation index, microvessel count, number of argyrophilic nucleolar organizer region, and total collagen content for treated scars. Our results demonstrate that METH1 has a markedly inhibitive effect on the formation of hypertrophic scar, and may thus have a promising application in the prevention of human hyperthropic scars.
A new avian genus and species, Zhongjianornis yangi gen. et sp. nov., is reported from the Lower Cretaceous lacustrine deposits of the Jiufotang Formation in Liaoning, northeast China. The new taxon is characterized by possessing the following combination of features: upper and lower jaws toothless, snout pointed, humerus with large and robust deltopectoral crest, second phalanx of the major manual digit longer than the first phalanx, unguals of the alular and major digits of similar length and significantly shorter than the corresponding penultimate phalanges, tibiotarsus slender and more than twice the length of the tarsometatarsus, and metatarsal IV longer than the other metatarsals. Phylogenetic analysis indicates that Zhongjianornis is phylogenetically basal to Confuciusornis and the dominant Mesozoic avian groups, Enantiornithes and Ornithurae, and therefore provides significant new information regarding the diversification of birds in the Early Cretaceous. It also represents the most basal bird that completely lacks teeth, suggesting that tooth loss was more common than expected in early avian evolution and that the avian beak appeared independently in several avian lineages, most probably as a response to selective pressure for weight reduction. Finally, the presence of a significantly enlarged humeral deltopectoral crest suggests that Zhongjianornis shares with other basal birds such as Jeholornis, Sapeornis and Confuciusornis a distinctive mode of adaptation for flight contrasting with that seen in more advanced birds, which instead possess an elongated sternum and a prominent keel.
High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition.
Radiotherapy is frequently applied in the treatment of malignant gliomas, but it is unclear if radiotherapy exerts its effects via induction of apoptosis. The present study was designed to determine whether a single-fraction ?-60Co radiation can induce apoptosis.
The impact of cranberry juice was investigated with respect to the initial adhesion of three isogenic strains of the bacterium Burkholderia cepacia with different extracellular polymeric substance (EPS) producing capacities, viz. a wild-type cepacian EPS producer PC184 and its mutant strains PC184rml with reduced EPS production and PC184bceK with a deficiency in EPS production. Adhesion experiments conducted in a parallel-plate flow chamber demonstrated that, in the absence of cranberry juice, strain PC184 had a significantly higher adhesive capacity compared to the mutant strains. In the presence of cranberry juice, the adhesive capacity of the EPS-producing strain PC184 was largely reduced, while cranberry juice had little impact on the adhesion behavior of either mutant strain. Thermodynamic modeling supported the results from adhesion experiments. Surface force apparatus (SFA) and scanning electron microscope (SEM) studies demonstrated a strong association between cranberry juice components and bacterial EPS. It was concluded that cranberry juice components could impact bacterial initial adhesion by adhering to the EPS and impairing the adhesive capacity of the cells, which provides an insight into the development of novel treatment strategies to block the biofilm formation associated with bacterial infection.
A simple, accurate and sensitive ionic liquid-based headspace single-drop microextraction procedure followed by high-performance liquid chromatography was developed and validated for the determination of camphor and trans-anethole in compound liquorice tablets. The volume of the ionic liquid microdrop was increased to 12 µL by modifying the device of the suspended drop. The stability of the microdrop and the sensitivity of the method were improved. Under the optimum experimental conditions, the calculated calibration curves gave acceptable linearity for camphor and trans-anethole with correlation coefficients of 0.9990 and 0.9998, respectively. The repeatability of the proposed method, expressed as relative standard deviation, was below 4.5% (n = 5). The limits of detection for the two target analytes were found to be 9.77 and 1.95 × 10(-2) ?g/mL, respectively. In this study, the separation, purification and enrichment were achieved in one step in an airtight system, which reduced the interferences caused by other complicated constituents, increased the signal-to-noise of the method and ensured the accuracy of the results because there was no loss of volatile components. It is expected to be widely applied for sample pretreatment of volatile components with high boiling points in samples with complicated matrices such as the extractions of plants or Chinese traditional drugs.
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