Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein
kinase kinase kinase (MAP3K) family that activates downstream MAP kinases (MAPKs), c-Jun
N-terminal kinases (JNKs) and p38 MAPKs, in response to various stresses, such as reactive
oxygen species (ROS), endoplasmic reticulum (ER) stress, lipopolysaccharide, and calcium
overload. Activation of the JNK and p38 pathways induces stress responses such as cell death,
differentiation, and the production of inflammatory cytokines. A series of studies using
ASK1-deficient mice have indicated that ASK1 plays important roles in many stress-related
diseases, including cardiovascular and neurodegenerative diseases, suggesting that small
compounds that inhibit ASK1 activity could possibly be used for the amelioration of the
development and/or progression of these diseases. In this review, we provide an overview of the
pathophysiological roles of ASK1-dependent signaling pathways and discuss the mechanistic basis
for how these could serve as potential therapeutic targets.
ASK1; inhibitors; MAP kinase; signal transduction; stress
AIMS—To clarify the
features of the background electroencephalographic (EEG) activities in
clinically well preterm infants born at less than 27 weeks gestation
and to outline their chronological changes with increasing
postconceptional age (PCA).
clinically well premature infants born at less than 27 weeks gestation
were recorded during the early postnatal period. The infants were
separated into three groups according to their PCA at the time of EEG
recording (21-22 weeks PCA, 23-24 weeks PCA, and 25-26 weeks PCA).
The mean and maximum duration of interburst intervals (IBIs), the mean
duration of bursts, and the percentage of continuous and discontinuous
patterns in each PCA group were evaluated.
three infants at 21-22 weeks PCA, seven at 23-24 weeks PCA, and five
at 25-26 weeks PCA. Eighteen EEG recordings were obtained. The mean
and maximum IBI duration decreased with increasing PCA. The percentage
of continuous patterns increased with increasing PCA. Conversely, the
percentage of discontinuous patterns decreased with increasing PCA.
premature infants born at less than 27 weeks gestation, the
characteristics of the background EEG activities were similar to those
of older premature infants. These changes reflect the development of
the central nervous system in this period.
At less than 27 weeks gestational age, the
characteristics of background EEG activities were found to be as
influenza; antibodies; neuraminidase inhibitor; click chemistry; conjugation
The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its physiological roles in T cell development remain poorly understood. Here, we generated Zfatf/f-LckCre mice and demonstrated that T cell-specific Zfat-deletion in Zfatf/f-LckCre mice resulted in a reduction in the number of CD4+CD8+double-positive (DP) cells, CD4+single positive cells and CD8+single positive cells. Indeed, in Zfatf/f-LckCre DP cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of CD3ζ phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection through ERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide deeper insight into T cell development and immune regulation.
ATP-citrate lyase catalyzes the production of acetyl-CoA from citrate, CoA and ATP. Crystals were grown of the amino-terminal portion of the human enzyme in the presence of tartrate, ATP and magnesium ions. The crystal structure shows the inhibitor tartrate and the product ADP–Mg2+ bound to the protein.
Human ATP-citrate lyase (EC 184.108.40.206) is the cytoplasmic enzyme that catalyzes the production of acetyl-CoA from citrate, CoA and ATP. The amino-terminal portion of the enzyme, containing residues 1–817, was crystallized in the presence of tartrate, ATP and magnesium ions. The crystals diffracted to 2.3 Å resolution. The structure shows ADP–Mg2+ bound to the domain that possesses the ATP-grasp fold. The structure demonstrates that this crystal form could be used to investigate the structures of complexes with inhibitors of ATP-citrate lyase that bind at either the citrate- or ATP-binding site.
ATP-citrate lyase; ATP-grasp domain
It is well known that manganese (Mn) exposure is involved in parkinsonism. The aim of our study was to test the hypotheses that Mn affects nicotinamide N-methyltransferase (NNMT) activity, increases the metabolism of nicotinamide (NA) to 1-methylnicotinamide (MNA), and leads to neurocytotoxicity.
Following demonstration of the effects of Mn concentrations on the survival rate of Mouse CD1 brain striatum neuronal cells (MS cells), the effect of Mn on NNMT activity was investigated by comparing the difference in the amount of MNA produced after various Mn concentrations were added to mouse brain cytosol fractions as an enzyme solution. Toxicity induced by MNA and its precursor NA on MS cells was measured.
The survival rate of MS cells decreased significantly with increasing concentrations of Mn in the culture medium. With respect to the influence of Mn on NNMT activity, NNMT activity increased significantly at Mn concentrations of 1 μmol/mg protein. MNA and NA neurotoxicity were compared by comparing cell survival rate. Cell survival rate dropped significantly when the cells were cultivated with 10 mM of MNA. There was also a tendency for the survival rate to fall following the addition of 10 mM NA; however, the difference with the control was not significant.
Our study suggests the possibility that Mn causes increased NNMT activity, thereby increasing MNA levels in the brain and bringing about neuron death. Daily absorption of Mn and NA may thus contribute to idiopathic Parkinson’s disease.
Parkinson’s disease; Nicotinamide N-methyltransferase; Manganese; Mouse CD1 brain striatum neuronal cells; Cytotoxicity
Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.
DNA methylation; histone H1; Oocyte-specific; nuclease-sensitivity; differentiation
This study aimed to discriminate between enamel and composite resins by differences in Hounsfield units shown on 16 section multidetector CT (MDCT) images taken of unidentified bodies.
First, we determined the Hounsfield units of composite resins in 15 extracted human teeth. We then filled a single cavity prepared in each of the teeth with one of five different types of composite resins, and scanned the teeth using our routine post-mortem CT protocol for the head and neck. Obtained data were transferred to a radiological workstation and reconstructed. Furthermore, post-mortem CT images of the head of three unidentified bodies were reconstructed in the same manner.
Four types of composite resins containing radio-opaque fillers showed a constant value of 4000 HU, and one radiolucent composite resin showed values in the range of 660–800 HU in the extracted teeth. Pixels at 4000 HU indicated that the composite resins were selected and visualized as three-dimensional colour images. Composite resins could be visualized on reconstructed images of the three unidentified bodies, and the sites visualized matched those noted on the forensic dental charts.
Discriminating enamel and composite resins containing radio-opaque materials was difficult because of their similar Hounsfield unit values. However, we did succeed in visualizing the composite resins despite limitations of the CT scale. CT reconstructed images can contribute to dental identification, particularly in cases where it is difficult to detect composite resins on external investigation, and these images can be prepared during routine dental identification work.
identification; X-ray computed; tomography scanners; dental restoration; permanent; forensic dentistry
We previously reported a human-specific gene conversion of
SIGLEC11 by an adjacent paralogous pseudogene
(SIGLEC16P), generating a uniquely human form of the Siglec-11 protein,
which is expressed in the human brain. Here, we show that Siglec-11 is expressed
exclusively in microglia in all human brains studied—a finding of potential
relevance to brain evolution, as microglia modulate neuronal survival, and Siglec-11
recruits SHP-1, a tyrosine phosphatase that modulates microglial biology. Following the
recent finding of a functional SIGLEC16 allele in human populations,
further analysis of the human SIGLEC11 and
SIGLEC16/P sequences revealed an unusual series of
gene conversion events between two loci. Two tandem and likely simultaneous gene
conversions occurred from SIGLEC16P to SIGLEC11 with a
potentially deleterious intervening short segment happening to be excluded. One of the
conversion events also changed the 5′ untranslated sequence, altering predicted
transcription factor binding sites. Both of the gene conversions have been dated to
∼1–1.2 Ma, after the emergence of the genus Homo, but prior to
the emergence of the common ancestor of Denisovans and modern humans about 800,000 years
ago, thus suggesting involvement in later stages of hominin brain evolution. In keeping
with this, recombinant soluble Siglec-11 binds ligands in the human brain. We also address
a second-round more recent gene conversion from SIGLEC11 to
SIGLEC16, with the latter showing an allele frequency of
∼0.1–0.3 in a worldwide population study. Initial pseudogenization of
SIGLEC16 was estimated to occur at least 3 Ma, which thus preceded the
gene conversion of SIGLEC11 by SIGLEC16P. As gene
conversion usually disrupts the converted gene, the fact that ORFs of
hSIGLEC11 and hSIGLEC16 have been maintained after an
unusual series of very complex gene conversion events suggests that these events may have
been subject to hominin-specific selection forces.
pseudogene; gene conversion; human evolution; human brain; microglia
To investigate four different contrast protocols to detect hypervascular hepatocellular carcinoma (HCC) most adaptable for patients at any body weight (BW) in clinical practice.
Materials and methods
A post-marketing surveillance of liver dynamic CT was prospectively performed by four different protocols in 415 patients: Protocol-A, BW-tailored dose of contrast media (CM: iohexol 300 mgI/mL), fixed injection duration (30s), fixed scan timing at arterial phase (AP); Protocol-B, BW-tailored dose of CM, fixed injection duration (30s), by bolus tracking; Protocol-C, BW-tailored dose of CM, fixed injection flow rate, by bolus tracking; Protocol-D, 100 mL constant of CM at any BW, fixed scan timing. Scan timing and tumor conspicuity at AP was scored qualitatively. The quantitative CT values of aorta and tumor liver contrast (TLC) were obtained.
The qualitative rate assessed “good” as scan timing of AP in Protocol-C was significantly lower than those in Protocols A and D (difference:16.6%, 17.4%, P = 0.0069, P = 0.0140, respectively). Scatter plot of Protocol-D (R2 = 0.1283) at AP showed significant inverse relationship between TLC and BW (P =0.0053), although not significant in Protocols A, B, C.
In patients with higher BW, protocols of BW-tailored dose of CM and/or fixed injection duration have no dependence on BW to diagnose hypervascular HCCs.
CT; Liver; Contrast media; Hepatocellular carcinoma; Injection method
Background and Purpose
Based on an experimental model of warfarin-associated intracerebral hemorrhage, we investigated whether the rapid reversal of anticoagulation using prothrombin complex concentrates (PCC) or recombinant activated coagulation factor VII (rFVIIa) reduces hematoma volume.
Mice were orally pretreated with warfarin (2 mg/kg). Intracerebral hemorrhage was induced by collagenase injection into the right striatum. Forty-five minutes later, PCC (100 IE/kg), rFVIIa (1 mg/kg), or an equal volume of saline was administered intravenously. Hematoma volume after 24 hours was quantified using a photometric hemoglobin assay.
International normalized ratio was 4.3 ± 0.4 in saline-treated mice, 0.9 ± 0.1 in rFVIIa mice, and 1.4 ± 0.2 in PCC mice. Intracerebral hemorrhage volume was 29.0 ± 19.7 µL in the saline group (n = 7), 8.6 ± 4.3 µL in the rFVIIa group (n = 6), and 6.1 ± 1.8 µL in the PCC group (n = 7; analysis of variance between-group differences P = 0.004; post hoc rFVIIa versus saline P = 0.021; PCC versus saline P = 0.007). No significant difference was found between PCC- and rFVIIa-treated animals.
Our results suggest that PCC and rFVIIa are equally effective in restoring coagulation and preventing excessive hematoma growth in acute warfarin-associated intracerebral hemorrhage.
animal models; anticoagulation; ICH; intracerebral hemorrhage; warfarin
To evaluate changes in retinal and choroidal thickness changes after three intravitreal ranibizumab (IVR) injections for polypoidal choroidal vasculopathy (PCV) using enhanced depth-imaging-optical coherence tomography (EDI-OCT).
In this retrospective, observational case series, EDI-OCT was used to measure changes in choroidal thickness at nine points in a lattice shape in the macula before and after introductory-stage IVR.
Choroidal thickness was decreased at all nine points in the lattice shape, but was significantly decreased only at the fovea.
The subfoveal choroidal thickness may be reduced by introductory-stage IVR in patients with PCV. In particular, choroidal thickness at the fovea was reduced during the early stage of treatment.
Choroidal thickness; Polypoidal choroidal vasculopathy; Enhanced depth imaging; Optical coherence tomography; Ranibizumab
Stroke induces a complex web of pathophysiology that may evolve over hours to days and weeks after onset. It is now recognized that inflammation is an important phenomenon that can dramatically influence outcomes after stroke. In this minireview, we explore the hypothesis that inflammatory signals after stroke are biphasic in nature. The high-mobility group box 1 (HMGB1) protein is discussed as an example of this idea. HMGB1 is normally present in the nucleus. Under ischemic conditions, it is released extracellularly from many types of cells. During the acute phase poststroke, HMGB1 promotes necrosis and influx of damaging inflammatory cells. However, during the delayed phase poststroke, HMGB1 can mediate beneficial plasticity and recovery in many cells of the neurovascular unit. These emerging findings support the hypothesis that inflammation after stroke can be both detrimental and beneficial, depending on the cellular situations involved.
stroke; inflammation; stroke recovery; HMGB1
The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic-GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here we identify natural variants of human STING that are poorly responsive to cGAMP, yet unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a non-canonical cyclic-di-nucleotide, cyclic[G(2′ -5′)pA(3′ -5′)p], which contains a single 2′ -5′ phosphodiester bond. Cyclic[G(2′ -5′)pA(3′ -5′)p] potently activates diverse human STING receptors and may therefore be a useful adjuvant or immunotherapeutic. Our results indicate that human STING variants have evolved that can distinguish conventional (3′ -5′) cyclic-di-nucleotides, known only to be produced by bacteria, from the non-canonical cyclic-di-nucleotide produced by mammalian cGAS.
Succinyl-CoA synthetase catalyzes the reaction succinyl-CoA + NDP + Pi ⇌ succinate + CoA + NTP, where N denotes adenosine or guanosine. The enzyme from T. aquaticus was characterized biochemically and its structure was determined in complex with GDP-Mn2+, the preferred nucleotide.
Succinyl-CoA synthetase (SCS) from Thermus aquaticus was characterized biochemically via measurements of the activity of the enzyme and determination of its quaternary structure as well as its stability and refolding properties. The enzyme is most active between pH 8.0 and 8.4 and its activity increases with temperature to about 339 K. Gel-filtration chromatography and sedimentation equilibrium under native conditions demonstrated that the enzyme is a heterotetramer of two α-subunits and two β-subunits. The activity assays showed that the enzyme uses either ADP/ATP or GDP/GTP, but prefers GDP/GTP. This contrasts with Escherichia coli SCS, which uses GDP/GTP but prefers ADP/ATP. To understand the nucleotide preference, T. aquaticus SCS was crystallized in the presence of GDP, leading to the determination of the structure in complex with GDP-Mn2+. A water molecule and Pro20β in T. aquaticus take the place of Gln20β in pig GTP-specific SCS, interacting well with the guanine base and other residues of the nucleotide-binding site. This leads to the preference for GDP/GTP, but does not hinder the binding of ADP/ATP.
thermostability; denaturation; nucleotide specificity; ATP-grasp fold; enzyme kinetics
Although much is known about vancomycin-resistant (VR) Enterococcus faecium, little is known about the epidemiology of VR Enterococcus faecalis. The predilection of VR E. faecalis to transfer the vancomycin resistance determinant to Staphylococcus aureus is much greater than that of VR E. faecium. The epidemiology of VR E. faecalis has important implications regarding the emergence of vancomycin-resistant S. aureus (VRSA); 8 of 13 reported VRSA cases have been from Michigan. A retrospective case-case-control study was conducted at the Detroit Medical Center, located in southeastern Michigan. Unique patients with VR E. faecalis infection were matched to patients with strains of vancomycin-susceptible (VS) E. faecalis and to uninfected controls at a 1:1:1 ratio. Five hundred thirty-two VR E. faecalis cases were identified and were matched to 532 VS E. faecalis cases and 532 uninfected controls. The overall mean age of the study cohort (n = 1,596) was 63.0 ± 17.4 years, and 747 (46.8%) individuals were male. Independent predictors for the isolation of VR E. faecalis (but not VS E. faecalis) compared to uninfected controls were an age of ≥65 years, nonhome residence, diabetes mellitus, peripheral vascular disease, exposure to cephalosporins and fluoroquinolones in the prior 3 months, and immunosuppressive status. Invasive procedures and/or surgery, chronic skin ulcers, and indwelling devices were risk factors for both VR E. faecalis and VS E. faecalis isolation. Cephalosporin and fluoroquinolone exposures were unique, independent predictors for isolation of VR E. faecalis. A majority of case patients had VR E. faecalis present at the time of admission. Control of VR E. faecalis, and ultimately VRSA, will likely require regional efforts focusing on infection prevention and antimicrobial stewardship.
Skeletonization is an advanced technique of graft harvesting for coronary artery bypass grafting (CABG), and while it requires meticulous attention, it has many advantages. For example, skeletonization of internal thoracic artery (ITA) can minimize sternal ischemia and lower the risk of mediastinitis, and is longer and larger than pedicled ITA. In this article we describe the surgical techniques demonstrated in our video, which details our techniques of skeletonization of arterial grafts and off-pump coronary artery bypass (OPCAB) exclusively using these in situ grafts. Our method of right gastroepiploic artery (GEA) skeletonization has only three technical steps. The first step is to pass thin vessel loops under the GEA. The second step is to unroof the tissue surrounding the GEA. The last step is to seal and sever all the branches. Skeletonization of the GEA not only prevents vasospasm but also leads to GEA dilatation, and facilitates inspection and makes sequential anastomosis easier. Bilateral use of the skeletonized ITA and use of the skeletonized GEA can cover most coronary artery target sites without any manipulation of the ascending aorta. In our consecutive series of over 1,000 patients, the stroke rate was 0.5%. Our method helps to make the technique simple and secure in this technically demanding operation, and we believe that OPCAB with these grafts provides the best possible coronary revascularization.
Coronary revascularization; coronary artery bypass; off-pump coronary artery bypass (OPCAB); internal thoracic artery (ITA); gastroepiploic artery (GEA)
Berberine (BBR) has been used for the treatment of bacterial and fungal infections and also for cancer-associated symptoms such as diarrhea. Furthermore, it has been reported that BBR may have direct antitumor effects. Although evidence supports the theory that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for treating cancer, its usage may be limited due to the resistance to the TRAIL-induced apoptosis of cancer cells. In the present study, the effect of BBR on TRAIL-induced antitumor effects was investigated in vitro using recombinant TRAIL and in vivo using a 4T1 murine breast cancer model in combination with anti-DR5 (death-inducing TRAIL receptor) monoclonal antibody therapy. BBR sensitized human breast cancer cell lines to TRAIL-mediated apoptosis in vitro. The combination of BBR and recombinant TRAIL significantly activated caspase-3 and PARP cleavage in TRAIL-resistant MDA-MB-468 cells. Furthermore, BBR in combination with TRAIL more effectively induced apoptosis compared with coptisine (COP), which is structurally related to BBR. In a murine 4T1 breast cancer model, BBR treatment enhanced the efficacy of anti-DR5 antibody therapy against primary tumor growth and lung metastasis. Thus, BBR may become a new adjuvant for overcoming the resistance of cancer cells to TRAIL/DR5-mediated therapy.
breast cancer; berberine; coptisine; TNF-related apoptosis-inducing ligand; apoptosis
The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−.
Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice.
Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.
Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
Closure of cranial sutures progresses with age; therefore, macroscopic assessment of cranial sutures has been used as one method of age estimation. Postmortem computed tomography (PMCT), which many forensic medical departments and institutes have begun to adopt, has the potential to simplify the gathering of information from cranial sutures for both surface and cross-sectional evaluation. To examine the feasibility of age estimation by cross-sectional multidetector computed tomography images of the sagittal suture, PMCT findings of 125 subjects of known age and sex were retrospectively reviewed. The sagittal suture was divided into four segments, and 20 cross-sectional slices from each segment were analyzed. These slices were each categorized by visual evaluation into one of the seven stages defined by Harth et al. according to the degree of closure. The mean stage value of 20 slices was calculated for each segment. We were able to evaluate cross-sectional images of the sagittal suture by PMCT, and a positive correlation between age and closure degree was observed. Despite the prediction interval achieved with this method not being superior to traditional macroscopic or flat-panel CT assessment, multidetector CT is a potentially useful tool, in conjunction with other methods, for age estimation, particularly in adult females and in cases where only a skull is the sole remain.
Age determination; Forensic anthropology; Multidetector computed tomography; Cranial suture
Genetic modification of human adipose tissue–derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.
Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.
hypertension; awareness; control; national survey; public health
After stroke and brain injury, cortical gray matter recovery involves mechanisms of neurovascular matrix remodeling. In white matter however, the mechanisms of recovery remain unclear. In this present study, we demonstrate that oligodendrocytes secrete matrix metalloproteinase-9 (MMP-9), which accelerates the angiogenic response after white matter injury. In primary oligodendrocyte cultures, treatment with the pro-inflammatory cytokine interleukin-1β (IL-1β) induced an upregulation and secretion of MMP-9. Conditioned media from IL-1β-stimulated oligodendrocytes significantly amplified matrigel tube formation in brain endothelial cells, indicating that MMP-9 from oligodendrocytes can promote angiogenesis in vitro. Next we asked whether similar signals and substrates operate after white matter injury in vivo. Focal white matter injury and demyelination was induced in mice via stereotactic injection of lysophosphatidylcholine (LPC) into corpus callosum. Western blot analysis showed that IL-1β expression was increased in damaged white matter. Immunostaining demonstrated MMP-9 signals in MOBP (myelin-associated oligodendrocytic basic protein)-positive oligodendrocytes. Treatment with an IL-1β-neutralizing antibody suppressed the MMP-9 response in oligodendrocytes. Finally, we confirmed that the broad spectrum MMP inhibitor GM6001 inhibited angiogenesis around the injury area in this white matter injury model. In gray matter, a neurovascular niche promotes cortical recovery after brain injury. Our study suggests that an analogous oligovascular niche may mediate recovery in white matter.
oligodendrocyte; white matter injury; matrix metalloproteinase-9; vascular remodeling; cerebral endothelial cell
A growing number of epidemiological studies have demonstrated that the consumption of green tea inhibits the growth of a variety of cancers. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has been shown to have an anti-cancer effect against many cancers. Most cancers are believed to be initiated from and maintained by a small population of tumor-initiating cells (TICs) that are responsible for chemotherapeutic resistance and tumor relapse. In neuroblastoma, an aggressive pediatric tumor that often relapses and has a poor prognosis, TICs were recently identified as spheres grown in a serum-free non-adherent culture used for neural crest stem cell growth. Although EGCG has been reported to induce growth arrest and apoptosis in neuroblastoma cells, its effect on neuroblastoma TICs remains to be defined.
Gene expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of EGCG on cell proliferation, apoptosis, and sphere formation were determined by cell counting, propidium iodide staining, and sphere (>100 μm in diameter) counting, respectively.
Neuroblastoma BE(2)-C cells showed increased expression of stem cell markers (nanog homeobox [NANOG] and octamer-binding transcription factor 4 [OCT4]), as well as decreased expression of neuronal differentiation markers (Cu2+-transporting ATPase alpha polypeptide [ATP7A] and dickkopf homolog 2 [DKK2]) in spheres grown in serum-free non-adherent culture, compared to parental cells grown in conventional culture. Although EGCG induced growth arrest and apoptosis in the parental cells in a dose-dependent manner, it was not effective against spheres. However, EGCG potently inhibited sphere formation in the BE(2)-C cells.
The present results suggest that EGCG may inhibit the development of TICs in BE(2)-C cells.
Epigallocatechin gallate; Neuroblastoma; Tumor-initiating cell; Sphere