The objective of this study was to determine the prevalence, species and subtypes of Cryptosporidium infecting yaks in the Qinghai Province of Northwestern China. The prevalence of Cryptosporidium spp. was detected by microscopy and nested-PCR. A total of 586 fecal samples were collected from yaks in 6 counties, of which 142 (24.2%) samples tested positive for Cryptosporidium. The small subunit (SSU) rRNA gene of fifty-five samples were amplified and sequenced successfully and demonstrated that Cryptosporidium bovis (31/55, 56.4%) was the most common species, followed by C. parvum (16/55, 29.1%) and C. ryanae (5/55, 9.0%). Mixed infections of C. parvum and C. bovis (n = 2), C. ryanae and C. bovis (n = 1) were also detected. All three species were found in yaks ranging in age from <1 year, 1–2 years, to >2 years. Cryptosporidium was most commonly detected in spring (28.4%), followed by summer (20.9%), then winter (17.5%). Cryptosporidium parvum positive samples were subtyped using the 60 kDa glycoprotein (gp60) gene. Subtypes IIaA15G2R1 (n = 8), IIaA16G2R1 (n = 2), IIaA14G1R1 (n = 1), IIaA14G2R1 (n = 1) and IIaA16G3R1 (n = 1) were detected. All of these subtypes are zoonotic, and may pose a potential threat to human health.
It is well known that novel environments can enhance learning and memory. However, the underlying mechanisms remain poorly understood. Here, we report that, in freely moving rats, novelty exploration facilitates the production of hippocampal CA1 long-term depression (LTD), a well characterized form of synaptic plasticity believed to be a cellular substrate of spatial learning, and thereby converts short-term memory (STM) into long-term memory (LTM) in an inhibitory avoidance learning procedure. Blocking the induction or the expression of CA1 LTD with two mechanistically and structurally distinct inhibitors prevents not only novelty acquisition but also the novelty exploration-promoted conversion of STM into LTM. Moreover, production of LTD with a strong electrical stimulation induction protocol or facilitation of hippocampal LTD by pharmacological inhibition of glutamate transporter activity mimics the behavioral effects of novelty exploration, sufficiently promoting the conversion of STM into LTM. Together, our findings suggest that induction of LTD may play an essential role not only in novelty acquisition but also in novelty-mediated memory enhancement.
The herbicide alachlor (2-chloro-2′6′-diethyl-N-methoxymethylacetanilide) has been known as a probable human carcinogen, and the MCL (minimum contamination level) for drinking water has been set at 2 μg L-1. Therefore, the advanced methods for effectively removing it from water are a matter of interest. Catalyzed ozonation is a promising method for refractory organics degradation. Cu/Al2O3 catalyzed ozonation for degrading an endocrine disruptor (alachlor) in water was investigated.
Experimental results showed that the ozonation of alachlor can be effectively catalyzed and enhanced by Cu/Al2O3-honeycomb. The main intermediate products formed (aliphatic carboxylic acids) were mineralized to a large extent in the catalytic process.
This study has shown that Cu/Al2O3-honeycomb is a feasible and efficient catalyst in the ozonation of alachlor in water. Less intermediate oxidation product was produced in the catalytic process than in the uncatalytic one. Furthermore, the mineralization of alachlor could be enhanced by increasing the pH of the reaction solution.
Catalytic ozonation; Alachlor; Degradation
Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase in eukaryotes and plays a critical role in various aspects of the cell cycle. Plk1 exerts its multiple functions by phosphorylating its substrates. In this study, we found that Plk1 can interact with cyclin T1/Cdk9 complex-the main form of the positive transcription elongation complex b (P-TEFb), and its C-terminal polo-box domain is responsible for the binding. Further analysis indicated that Plk1 could phosphorylate cyclin T1 at Ser564 and inhibit the kinase activity of cyclin T1/Cdk9 complex on phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. By taking the approach of luciferase assay, we demonstrated that over-expression of both wild type Plk1 and constitutively active form of Plk1 inhibits the P-TEFb dependent HIV-1 LTR transcription, while knockdown of Plk1 increases the HIV-1 LTR transcription. Consistently, the data from the HIV-1 pseudovirus reporter assay indicated that Plk1 blocks the gene expression of HIV-1 pseudovirus. Taken together, our results revealed that Plk1 negatively regulates the RNA polymerase II-dependent transcription through inhibiting the activity of cyclin T1/Cdk9 complex.
This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.
Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.
To determine whether 5-fluorouracil (5-FU) sensitivity is associated with the mRNA expressions of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) in patients with hepatocellular carcinoma (HCC) treated with 5-FU-based transarterial chemoembolization (TACE).
Formalin-fixed, paraffin-embedded tumor specimens from 40 patients treated with 5-FU-based TACE were selected for the examination of TS, DPD, and TP expression level by a quantitative real-time reverse transcription- polymerase chain reaction (PCR) technique. Patients were categorized into high and low expression groups according to the median expression level of each enzyme. Associations between the mRNA expression levels of TS, DPD, and TP and clinical parameters including treatment efficacies, clinicopathological factors, and prognosis were assessed.
High DPD expression was associated with worse treatment outcome, including intrahepatic disease progression rate (hazard ratio [HR] for high DPD versus low DPD, 2.212; 95% confidence interval [CI], 1.030–4.753; P = 0.042), extrahepatic disease progression rate (HR for high versus low DPD, 3.171; 95% CI, 1.003–10.023; P = 0.049), and progression-free survival (HR for high versus low DPD, 2.308; 95% CI, 1.102–4.836; P = 0.027). No correlation was found between the mRNA expression of TS/TP and treatment outcome.
DPD mRNA expression level was negatively correlated with the clinical outcomes of HCC patients treated with 5-FU-based TACE. These results provide indirect evidence that high DPD mRNA expression is a predictive marker of treatment resistance for 5-FU.
dihydropyrimidine dehydrogenase; 5-fluorouracil; hepatocellular carcinoma; thymidylate synthase; thymidine phosphorylase; transarterial chemoembolization
In several models of aging, microglia become more inflammatory and reactive to immune challenges. For example, peripheral LPS injection causes exaggerated microglial activation associated with prolonged sickness and depressive-like behavior in aged BALB/c mice. Therefore, the purpose of this study was to determine the extent to which age-related amplified microglial activation was associated with reduced sensitivity to the anti-inflammatory and M2 promoting cytokines interleukin (IL)-10 and IL-4. In initial studies with adult mice, LPS induced a time-dependent increase in M1 and M2 mRNA profiles in microglia. Furthermore, peripheral LPS injection markedly increased surface expression of IL-4 receptor-alpha (IL-4Rα), but not IL-10 receptor-1 (IL-10R1) on microglia. In BV-2 cells, IL-4, but not IL-10, re-directed LPS-activated microglia towards an M2 phenotype. Based on these findings, comparisons of M1 and M2 activation profiles, induction of IL-4Rα, and sensitivity to IL-4 were determined in microglia from adult (3–4 mo) and aged (18–22 mo) mice. In aged microglia, LPS promoted an exaggerated and prolonged M1 and M2 profile compared to adults. Moreover, IL-4Rα protein was not increased on aged microglia following LPS injection. To determine the consequence of impaired IL-4Rα upregulation, adult and aged mice were injected with LPS and activated microglia were then isolated and treated ex vivo with IL-4. While ex vivo IL-4 induced an M2 profile in activated microglia from adult mice, activated microglia from aged mice retained a prominent M1 profile. These data indicate that activated microglia from aged mice are less sensitive to the anti-inflammatory and M2-promoting effects of IL-4.
Microglia; Aging; IL-4; IL-10; M1 and M2 activation profiles
It is known that toll-like receptor (TLR)4 plays an important role in atherosclerosis. Since both microvascular (MIC) and macrovascular (MAC) endothelial cells (ECs) are present in atherosclerotic lesions, this study compared TLR4-triggered inflammatory response and crosstalk with mononuclear cells between MIC and MAC ECs.
METHODS AND RESULTS
ELISA, real-time PCR and gene expression profiling showed that TLR4 activation by LPS stimulated a much higher expression of inflammatory genes including cytokines, chemokines, growth factors and adhesion molecules in MIC ECs than MAC ECs. Furthermore, co-culture studies showed that TLR4 activation in MIC ECs, but not MAC ECs, induced a crosstalk with U937 mononuclear cells through MIC EC-released IL-6 to upregulate matrix metalloproteinase (MMP)-1 expression in U937 cells. To explore molecular mechanisms underlying the different responses to TLR4 activation between MIC and MAC ECs, we showed that MIC ECs had a higher expression of TLR4 and CD14 and a higher TLR4-mediated NFκB activity than MAC ECs.
This study showed that TLR4 activation triggers a more robust inflammatory response in MIC ECs than MAC ECs. Given the importance of inflammatory cytokines and MMPs in plaque rupture, MIC ECs may play a key role in plaque destabilization through a TLR4-dependent mechanism.
Toll-like receptor; Inflammation; Endothelial Cells; Gene Expression; Interleukin 6
Background and Purpose
Given the high risk of stroke after TIA (transient ischemia attack) or stroke and the adverse reaction of bleeding of antiplatelets, we undertook a meta-analysis, reviewed randomized controlled trials (RCTs) comparing aspirin plus clopidogrel with aspirin alone to determine the efficacy and adverse reaction of bleeding of the two protocols in the prevention of stroke.
We analyzed the incidences of stroke, bleeding and severe bleeding by using fixed-effect model or random-effect model on the basis of the result of heterogeneity test.
Five qualified RCTs satisfied the inclusion criteria. We found that treatment with aspirin plus clopidogrel was associated with lower incidence of stroke (Risk Ratio (RR), 0.66, 95% confidence interval (CI), 0.47 to 0.93), higher incidence of bleeding (RR, 1.75, 95% CI, 1.48 to 2.05) as compared with aspirin-alone treatment. In terms of severe bleeding, no statistical difference existed between them (RR, 2.21, 95% CI, 0.25 to 19.52).
The combined use of aspirin and clopidogrel is more effective than aspirin alone for patients with previous TIA or stroke for the prevention of stroke, with risk of bleeding being higher. No statistical difference was found in severe bleeding between the two treatment protocols.
Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.
Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.
Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.
One of the remarkable features of networks is module that can provide useful insights into not only network organizations but also functional behaviors between their components. Comprehensive efforts have been devoted to investigating cohesive modules in the past decade. However, it is still not clear whether there are important structural characteristics of the nodes that do not belong to any cohesive module. In order to answer this question, we performed a large-scale analysis on 25 complex networks with different types and scales using our recently developed BTS (bintree seeking) algorithm, which is able to detect both cohesive and sparse modules in the network. Our results reveal that the sparse modules composed by the cohesively isolated nodes widely co-exist with the cohesive modules. Detailed analysis shows that both types of modules provide better characterization for the division of a network into functional units than merely cohesive modules, because the sparse modules possibly re-organize the nodes in the so-called cohesive modules, which lack obvious modular significance, into meaningful groups. Compared with cohesive modules, the sizes of sparse ones are generally smaller. Sparse modules are also found to have preferences in social and biological networks than others.
Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.
Genetically modified mesenchymal stem cells (MSCs) have great potential in the application of regenerative medicine and molecular therapy. In the present manuscript, we introduce a nanopolymer, polyethylenimine600-β-cyclodextrin (PEI600-β-CyD), as an efficient polyplex-forming plasmid delivery agent with low toxicity and ideal transfection efficiency on primary MSCs. PEI600-β-CyD causes significantly less cytotoxicity and apoptosis on MSCs than 25 kDa high-molecular-weight PEI (PEI25kDa). PEI600-β-CyD also exhibits similar transfection efficiency as PEI25kDa on MSCs, which is higher than that of PEI600Da. Quantum dot-labeled plasmids show that PEI600-β-CyD or PEI25kDa delivers the plasmids in a more scattered manner than PEI600Da does. Further study shows that PEI600-β-CyD and PEI25kDa are more capable of delivering plasmids into the cell lysosome and nucleus than PEI600Da, which correlates well with the results of their transfection-efficiency assay. Moreover, among the three vectors, PEI600-β-CyD has the most capacity of enhancing the alkaline phosphatase activity of MSCs by transfecting bone morphogenetic protein 2, 7, or special AT-rich sequence-binding protein 2. These results clearly indicate that PEI600-β-CyD is a safe and effective candidate for the nonviral gene delivery of MSCs because of its ideal inclusion ability and proton sponge effect, and the application of this nanopolymer warrants further investigation.
polyethylenimine; cyclodextrin; gene delivery; mesenchymal stem cells
The neuropeptide galanin and its receptors (GalR) are found to be up-regulated in brains suffering from nerve injury, but the specific role played by galanin remains unclear. This study aimed to explore the neuroprotective role of galanin after shear stress induced nerve injury in the primary cultured cortical neurons of rats. Our results demonstrated that no significant changes in cell death and viability were found after galanin treatment when subjected to a shear stress of 5 dyn/cm2 for 12 h, after increasing magnitude of shear stress to 10 dyn/cm2 for 12 h, cell death was significantly increased, while galanin can inhibit the nerve injury induced by shear stress with 10 dyn/cm2 for 12 h. Moreover, Gal2-11 (an agonist of GalR2/3) could also effectively inhibit shear stress-induced nerve injury of primary cultured cortical neurons in rats. Although GalR2 is involved in the galanin protection mechanism, there was no GalR3 expression in this system. Moreover, galanin increased the excitatory postsynaptic currents (EPSCs), which can effectively inhibit the physiological effects of shear stress. Galanin was also found to inhibit the activation of p53 and Bax, and further reversed the down regulation of Bcl-2 induced by shear stress. Our results strongly demonstrated that galanin plays a neuroprotective role in injured cortical neurons of rats.
To reduce connective tissue IL-6 level stimulated by LPS, it is essential to control IL-6 expression in both mononuclear cells and fibroblasts. However, it is unclear whether the regulatory mechanisms for both cells are similar or not. In this study, we found that signaling pathways mediating LPS-stimulated IL-6 in mononuclear U937 cells and fibroblasts were different. Furthermore, our studies showed that while LPS activated AP-1 and NFκB in U937 cells, it only activated NFκB in fibroblasts. Analysis of nuclear AP-1 subunits showed that LPS stimulated c-Fos, Fra-1 and Jun D activities in U937 cells, but not fibroblasts. The lack of ERK involvement in LPS-stimulated IL-6 in fibroblasts was further supported by the observations that simvastatin, which is known to target ERK-AP-1, failed to inhibit LPS-stimulated IL-6 by fibroblasts. Finally, we showed that targeting NFκB pathway was highly effective in inhibition of LPS-stimulated IL-6 in coculture of U937 cells and fibroblasts.
Inflammation; LPS; Cytokine; Gene expression; Signal transduction; Transcription
Somitogenesis is a fundamental characteristic feature of development in various animal embryos. Molecular evidence has proved that the Notch and Wnt pathways play important roles in regulating the process of somitogenesis and there is crosstalk between these two pathways. However, it is difficult to investigate the detailed mechanism of these two pathways and their interactions in somitogenesis through biological experiments. In recent years some mathematical models have been proposed for the purpose of studying the dynamics of the Notch and Wnt pathways in somitogenesis. Unfortunately, only a few of these models have explored the interactions between them.
In this study, we have proposed three mathematical models for the Notch signalling pathway alone, the Wnt signalling pathway alone, and the interactions between them. These models can simulate the dynamics of the Notch and Wnt pathways in somitogenesis, and are capable of reproducing the observations derived from wet experiments. They were used to investigate the molecular mechanisms of the Notch and Wnt pathways and their crosstalk in somitogenesis through the model simulations.
Three mathematical models are proposed for the Notch and Wnt pathways and their interaction during somitogenesis. The simulations demonstrate that the extracellular Notch and Wnt signals are essential for the oscillating expressions of both Notch and Wnt target genes. Moreover, the internal negative feedback loops and the three levels of crosstalk between these pathways play important but distinct roles in maintaining the system oscillation. In addition, the results of the parameter sensitivity analysis of the models indicate that the Notch pathway is more sensitive to perturbation in somitogenesis.
The phospholipid, cardiolipin, is essential for maintaining mitochondrial structure and optimal function. Cardiolipin-deficiency in humans, Barth syndrome, is characterized by exercise intolerance, dilated cardiomyopathy, neutropenia, and 3-methyl-glutaconic aciduria. The causative gene is the mitochondrial acyl-transferase, tafazzin, that is essential for remodeling acyl chains of cardiolipin. We sought to determine metabolic rates in tafazzin-deficient mice during resting and exercise, and investigate the impact of cardiolipin-deficiency on mitochondrial respiratory chain activities. Tafazzin-knockdown in mice markedly impaired oxygen consumption rates during an exercise, without any significant effect on resting metabolic rates. CL-deficiency resulted in significant reduction of mitochondrial respiratory reserve capacity in neonatal cardiomyocytes that is likely to be caused by diminished activity of complex-III, which requires CL for its assembly and optimal activity. Our results may provide mechanistic insights of Barth syndrome pathogenesis.
Barth syndrome; tafazzin; complex-III; cardiolipins; exercise intolerance; mouse models
In this study we assess exome sequencing (ES) as a diagnostic alternative for genetically heterogeneous disorders. Since ES readily identified a previously reported homozygous mutation in the CAPN3 gene for an individual with an undiagnosed limb girdle muscular dystrophy, we evaluated ES as a generalizable clinical diagnostic tool by assessing the targeting efficiency and sequencing-coverage of 88 genes associated with muscle disease (MD) and spastic paraplegia (SPG). We used three exome-capture kits on 125 individuals. Exons constituting each gene were defined using the UCSC and CCDS databases. The three exome-capture kits targeted 47–92% of bases within the UCSC-defined exons, and 97%–99% of bases within the CCDS-defined exons. An average of 61.2–99.5% and 19.1–99.5% of targeted bases per gene were sequenced to 20X coverage within the CCDS-defined MD and SPG coding exons, respectively. Greater than 95–99% of targeted known mutation positions were sequenced to ≥1X coverage and 55–87% to ≥20X coverage in every exome. We conclude therefore that ES is a rapid and efficient first tier method to screen for mutations, particularly within the CCDS annotated exons, although its application requires disclosure of the extent of coverage for each targeted gene and supplementation with second tier Sanger sequencing for full coverage.
CAPN3; exome; LGMD; HSP; neuromuscular disorders; clinical genetic testing
AIM: To investigate the normal hepatic magnetic resonance spectroscopy findings choline/lipid2 (Cho/Lip2) associated with age and body mass index (BMI).
METHODS: A total of 58 single-voxel proton spectra of the liver were acquired at 3.0 T using the eight-channel phased array abdominal coil as the receiver coil. Consecutive stacks of breath-hold spectra were acquired using the point resolved spectroscopy technique at a short echo time of 30 ms and a repetition time of 1500 ms. The spectra were processed with the SAGE software package. Areas and heights for metabolite resonance were obtained. Student’s t test for unpaired data was used for comparisons of shimming, Cho/Lip2, and lipid content.
RESULTS: There were significant negative correlations between the Cho/Lip2 peak height ratios and BMI (r = -0.615) and age (r = -0.398) (all P < 0.01). Compared with the high-BMI group, the low-BMI group was younger (39.1 ± 13.0 years vs 47.6 ± 8.5 years, t = -2.954, P = 0.005); had better water suppression (93.4% ± 1.4% vs 85.6% ± 11.6%, t = 2.741, P = 0.014); had higher Cho/Lip2 peak heights ratio (0.2 ± 0.14 vs 0.05 ± 0.04, t = 6.033, P < 0.000); and had lower lipid content (0.03 ± 0.08 vs 0.29 ± 0.31, t = -3.309, P = 0.004). Compared with the older group, the younger group had better shimming effects (17.1 ± 3.6 Hz vs 22.0 ± 6.8 Hz, t = -2.919, P = 0.008); higher Cho/Lip2 peak heights ratios (0.03 ± 0.05 vs 0.09 ± 0.12, t = 2.4, P = 0.020); and lower lipid content (0.05 ± 0.11 vs 0.23 ± 0.32, t = -2.337, P = 0.031). Compared with the low-choline peak group, the high-choline peak group had lower lipid content (0.005 ± 0.002 vs 0.13 ± 0.23, t = -3.796, P < 0.000); lower BMI (19.6 ± 2.4 vs 23.9 ± 3.0, t = -4.410, P < 0.000); and younger age (34.7 ± 10.0 years vs 43.2 ± 12.5 years, t = -2.088, P = 0.041).
CONCLUSION: Lipid accumulation could result from the increased fat in the body depending on age and BMI. Lipid can mask the resonance signal of choline.
Magnetic resonance spectroscopy; High-field imaging; Choline
Although it is known that periodontal MMP-8 expression is associated with periodontal disease, the information concerning the periodontal MMP-8 expression in diabetic patients with periodontal disease is insufficient.
Materials and Methods
Periodontal tissue specimens were collected from 7 patients without periodontal disease and diabetes (Group 1), 15 patients with periodontal disease alone (Group 2) and 10 patients with both periodontal disease and diabetes (Group 3). The frozen sections were prepared and MMP-8 protein expression was detected using immunohistochemistry and quantified. For in vitro study, human U937 mononuclear cells were pre-exposed to normal or high glucose and then treated with LPS.
The nonparametric Kruskal-Wallis test showed that the difference in MMP-8 protein levels among the three groups were statistically significant (p = 0.003). Nonparametric analysis using Jonckheere-Terpstra test showed a tendency of increase in periodontal MMP-8 levels across Group 1 to Group 2 to Group 3 (p = 0.0002). In vitro studies showed that high glucose and LPS had a synergistic effect on MMP-8 expression.
Our current study showed an increasing trend in MMP-8 protein expression levels across patients without both periodontal disease and diabetes, patients with periodontal disease alone and patients with both diseases.
Periodontal diseases; Diabetes mellitus; MMP-8; Gene expression
The rol (designated for resorcinol) gene cluster rolRHMD is involved in resorcinol catabolism in Corynebacterium glutamicum, and RolR is the TetR-type regulator. In this study, we investigated how RolR regulated the transcription of the rol genes in C. glutamicum. The transcription start sites and promoters of rolR and rolHMD were identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that RolR negatively regulated the transcription of rolHMD and of its own gene. Further, a 29-bp operator rolO was located at the intergenic region of rolR and rolHMD and was identified as the sole binding site for RolR. It contained two overlapping inverted repeats and they were essential for RolR-binding. The binding of RolR to rolO was affected by resorcinol and hydroxyquinol, which are the starting compounds of resorcinol catabolic pathway. These two compounds were able to dissociate RolR-rolO complex, thus releasing RolR from the complex and derepressing the transcription of rol genes in C. glutamicum. It is proposed that the binding of RolR to its operator rolO blocks the transcription of rolHMD and of its own gene, thus negatively regulated resorcinol degradation in C. glutamicum.
Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.