Oxygen (O2), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O2 during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O2 concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O2 concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.
acute lung injury; Apoptosis; autophagy; caveolin-1; Fas; hyperoxia; LC3B
We describe the first reported case of endocarditis due to Neisseria skkuensis. The organism from the blood cultures taken on admission day was identified initially as unidentified Gram-negative cocci by Vitek2. Finally, it was identified as Neisseria skkuensis by 16 rRNA gene sequence analysis.
Vancomycin-intermediate resistance has not been previously reported among sequence type 72 (ST72) methicillin-resistant Staphylococcus aureus (MRSA) isolates of SCCmec type IV (ST72-MRSA-IV), which are distinctive community genotype strains in Korea. We report the first case of vancomycin treatment failure due to development of vancomycin-intermediate resistance in infection caused by an ST72-MRSA-IV isolate.
Endothelial cell (EC) apoptosis and apoptosis resistant proliferation have been proposed to play crucial roles in the development of featured plexiform lesions in the pathogenesis of pulmonary hypertension (PH). Subsequently, EC injury associated smooth muscle cell (SMC) proliferation facilitates vascular remodeling and eventually leads to narrowed vascular lumen, increased pulmonary vascular resistance, increased pulmonary arterial pressure, and right heart failure. The imbalance between cell death and proliferation occurs in every stage of pulmonary vascular remodeling and pathogenesis of PH, and involves every cell type in the vasculature including, but not limited to ECs, SMCs, and fibroblasts. Despite extensive studies, the detailed cellular and molecular mechanisms on how the transition from initial apoptosis of ECs to apoptosis resistant proliferation on ECs and SMCs remains unclear. Recent knowledge on autophagy, a conservative and powerful regulatory machinery existing in almost all mammalian cells, has shed light on the complex and delicate control on cell fate in the development of vascular remodeling in PH. In this review, we will discuss the recent understandings on how the cross-talk between apoptosis and autophagy regulates cell death or proliferation in PH pathogenesis, particularly in pulmonary vascular remodeling involving ECs and SMCs.
apoptosis; autophagy; beclin-1; LC3; pulmonary hypertension
Vascular endothelial growth factor (VEGF) plays a critical role in tumor progression, angiogenesis and metastasis. Cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)2, MMP9 and wild-type (WT) p53 has been found to regulate the production of VEGF. Whether VEGF regulates the production of COX-2, MMP2, MMP9 and WTp53, however, has yet to be determined. This study examined the influence of the overexpression or knockdown of VEGF on the protein levels of COX-2, MMP2, MMP9 and WTp53 as well as cell growth and cell cycle progression in Lewis lung carcinoma (LLC) cells. LLC cells were transfected with pIRES2-VEGF-GFP in the VEGF-overexpressing group (LLC-VEGF), pIRES2-GFP in the mock group (LLC-GFP) or pSUPER-VEGF-GFP in the VEGF knockdown group (LLC-RNAi). Protein levels were detected by western blot analysis. LLC cell growth exhibited no marked change in the LLC-VEGF group, but was significantly retarded in the LLC-RNAi group. Further examination revealed that more cells entered the S stage in the LLC-VEGF group than in the control (or mock) group (45.3 vs. 29.1%, P<0.05), and that cell growth was retarded in the LLC-RNAi group. Moreover, COX-2 and MMP2 and MMP9 proteins were significantly increased in the LLC-VEGF group (approximately 1.84-, 1.89- and 1.83-fold, respectively, vs. control, P<0.05), but significantly decreased in the LLC-RNAi group, whereas the expression of WTp53 exhibited the opposite pattern of change. VEGF expression was positively correlated with COX-2, MMP2 and MMP9 expression (r=0.984, r=0.978, r=0.969, respectively, P<0.01) and negatively correlated with WTp53 (r=−0.833, p<0.01). The activities of MMP2 and MMP9 were increased in the LLC-VEGF group. In conclusion, VEGF overexpression may promote the expression of COX-2 and MMPs, but inhibits WTp53 production in LLC cells; VEGF underexpression may have an inverse effect. These changes are closely correlated with the infiltration and metastasis of lung cancer.
lung neoplasm; vascular endothelial growth factor; cyclo oxygenase-2; metalloproteinases
Antimicrobial resistance in Streptococcus pneumoniae remains a serious concern worldwide, particularly in Asian countries, despite the introduction of heptavalent pneumococcal conjugate vaccine (PCV7). The Asian Network for Surveillance of Resistant Pathogens (ANSORP) performed a prospective surveillance study of 2,184 S. pneumoniae isolates collected from patients with pneumococcal infections from 60 hospitals in 11 Asian countries from 2008 to 2009. Among nonmeningeal isolates, the prevalence rate of penicillin-nonsusceptible pneumococci (MIC, ≥4 μg/ml) was 4.6% and penicillin resistance (MIC, ≥8 μg/ml) was extremely rare (0.7%). Resistance to erythromycin was very prevalent in the region (72.7%); the highest rates were in China (96.4%), Taiwan (84.9%), and Vietnam (80.7%). Multidrug resistance (MDR) was observed in 59.3% of isolates from Asian countries. Major serotypes were 19F (23.5%), 23F (10.0%), 19A (8.2%), 14 (7.3%), and 6B (7.3%). Overall, 52.5% of isolates showed PCV7 serotypes, ranging from 16.1% in Philippines to 75.1% in Vietnam. Serotypes 19A (8.2%), 3 (6.2%), and 6A (4.2%) were the most prominent non-PCV7 serotypes in the Asian region. Among isolates with serotype 19A, 86.0% and 79.8% showed erythromycin resistance and MDR, respectively. The most remarkable findings about the epidemiology of S. pneumoniae in Asian countries after the introduction of PCV7 were the high prevalence of macrolide resistance and MDR and distinctive increases in serotype 19A.
To investigate the characteristics of main Streptococcus pneumoniae clones of serotype 6D (ST282 and ST3171) in South Korea, antimicrobial susceptibility testing was performed, and 11 genes around the cps locus were sequenced on ST2826D, ST31716D, and ST816A isolates. The antimicrobial susceptibility patterns were very similar between clones belonging to the same clonal complex, ST816A and ST2826D; nonsusceptibilities to penicillin and cefuroxime, high MICs of ceftriaxone, and high resistance rates to trimethoprim-sulfamethoxazole. However, ST31716D isolates showed resistance to only macrolides and clindamycin. The sequences of 11 genes around the cps locus indicated the same genetic backgrounds between the ST816A and ST2826D isolates. On the other hand, ST31716D isolates showed nucleotide and amino acid differences from ST816A and ST2826D isolates in most genes, indicating a different genetic background. The mosaic structure of dexB gene in ST2826D isolates indicated that recombination might occur in the dexB gene. Our results suggest that the multidrug-resistant ST2826D pneumococcal clone has emerged by serial genetic recombination, including capsular switch.
AIM: To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure.
METHODS: Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected.
RESULTS: Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 μmol/L vs 24.2 ± 6.45 μmol/L, 12.47 ± 3.62 μmol/L, 3.77 ± 1.83 μmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative.
CONCLUSION: The HBAL showed great efﬁciency and safety in the treatment of acute liver failure.
Hybrid bioartificial liver; Acute liver failure; Flat plate bioreactor; Co-culture; Nanofiber scaffold
Our institute has developed a novel bio-artificial liver (BAL) support system, based on a multi-layer radial-flow bioreactor carrying porcine hepatocytes and mesenchymal stem cells. It has been shown that porcine hepatocytes are capable of carrying infectious porcine endogenous retroviruses (PERVs) into human cells, thus the microbiological safety of any such system must be confirmed before clinical trials can be performed. In this study, we focused on assessing the status of PERV infection in beagles treated with the novel BAL.
Five normal beagles were treated with the novel BAL for 6 hours. The study was conducted for 6 months, during which plasma was collected from the BAL and whole blood from the beagles at regular intervals. DNA and RNA in both the collected peripheral blood mononuclear cells (PBMCs) and plasma samples were extracted for conventional PCR and reverse transcriptase (RT)-PCR with PERV-specific primers and the porcine-specific primer Sus scrofa cytochrome B. Meanwhile, the RT activity and the in vitro infectivity of the plasma were measured.
Positive PERV RNA and RT activity were detected only in the plasma samples taken from the third circuit of the BAL system. All other samples including PBMCs and other plasma samples were negative for PERV RNA, PERV DNA, and RT activity. In the in vitro infection experiment, no infection was found in HEK293 cells treated with plasma.
No infective PERV was detected in the experimental animals, thus the novel BAL had a reliable microbiological safety profile.
Microbiological safety; Porcine endogenous retrovirus; Bio-artificial liver; Porcine hepatocyte; Mesenchymal stem cells
Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.
Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.
Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics.
Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.
Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
autophagy; hypoxia; hypertension, pulmonary
Panton-Valentine leukocidin (PVL)-positive USA300 clone has been the most successful community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clone spreading in North America. In contrast, PVL-negative ST72-CA-MRSA has been predominant in Korea, and there has been no report of infections by the USA300 strain except only one case report of perianal infection. Here, we describe the first case of pneumonia caused by the USA300 strain following pandemic influenza A (H1N1) in Korea. A 50-year-old man was admitted with fever and cough and chest radiograph showed pneumonic consolidation at the right lower lung zone. He received a ventilator support because of respiratory failure. PCR for pandemic influenza A (H1N1) in nasopharyngeal swab was positive, and culture of sputum and endotracheal aspirate grew MRSA. Typing of the isolate revealed that it was PVL-positive, ST 8-MRSA-SCCmec type IV. The analysis of the PFGE patterns showed that this isolate was the same pulsotype as the USA300 strain.
Influenza, Human; Methicillin-Resistant Staphylococcus aureus; Pneumonia
Recently, Streptococcus pneumoniae serotypes 6C and 6D have been identified. It is thought that they emerged by the replacement of wciNβ in the capsular loci of serotypes 6A and 6B, respectively. However, their evolution has not been unveiled yet. To investigate the evolution of four serotypes of S. pneumoniae serogroup 6, four genes of the capsular polysaccharide synthesis (cps) locus, wchA, wciN, wciO, and wciP, of isolates of S. pneumoniae serotypes 6A, 6B, 6C, and 6D were sequenced. Multilocus sequence typing (MLST) was performed to investigate their genetic backgrounds. The wchA gene of serotype 6C and 6D isolates was distinct from that of serotype 6A and 6B isolates, which may suggest cotransfer of wchA with wciNβ. Otherwise, serotypes 6C and 6D displayed different genetic backgrounds from serotypes 6A and 6B, which was suggested by MLST analysis. In addition, serotype 6C isolates showed distinct wciP polymorphisms from other serotypes, which also indicated that serotype 6C had not recently originated from serotype 6A. Although serotype 6D shared the same amino acid polymorphisms of wciO with serotype 6B, wciP of serotype 6D differed from that of serotype 6B. The data indicate the implausibility of the scenario of a recent emergence of the cps locus of serotype 6D by genetic recombination between serotypes 6B and 6C. In addition, five serotype 6A and 6B isolates (6X group) displayed cps loci distinct from those of other isolates. The cps locus homogeneity and similar sequence types in MLST analysis suggest that most of the 6X group of isolates originated from the same ancestor and that the entire cps locus might have recently been transferred from an unknown origin. Serotype 6B isolates showed two or more cps locus subtypes, indicating a recombination-mediated mosaic structure of the cps locus of serotype 6B. The collective data favor the emergence of cps loci of serotypes 6A, 6B, 6C, and 6D by complicated recombination.
Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1+/−) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1+/− mice relative to wild-type mice. Endothelial cells from Becn1+/− mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1+/− cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1+/− endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.
angiogenesis; autophagy; beclin 1; hypoxia-inducible factor
The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.
AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes.
METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells.
RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant.
CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.
Chitosan nanofiber scaffold; Porcine hepatocyte; Porcine endogenous retrovirus; Bioartificial liver
AIM: To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs) could be maintained in serum from acute-on-chronic liver failure (ACLF) patients.
METHODS: Hepatocyte supportive functions and cytotoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evaluated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemokine profile was also examined for the normal serum and liver failure serum.
RESULTS: Hepatocyte growth factor (HGF) and Tumor necrosis factor; tumor necrosis factor (TNF)-α were remarkably elevated in response to ACLF while epidermal growth factor (EGF) and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver support functions in the homo-hepatocyte culture. Hepatocytes co-cultured with MSCs could tolerate the cytotoxicity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cultured with healthy human serum in vitro. In addition, co-cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.
CONCLUSION: ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.
Acute-on-chronic liver failure serum; Primary hepatocytes; Bone marrow marrow mesenchymal stem cells; Co-culture; Hepatocyte-based modality
Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABAA channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocamal slices that insulin (1 nM) “turns on” new extrasynaptic GABAA channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC50) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABAA antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.
Due to longer life spans, patients newly diagnosed with unruptured intracranial aneurysms (UIAs) are increasing in number. This study aimed to evaluate how management of UIAs in patients age 65 years and older affects the clinical outcomes and post-procedural morbidity rates in these patients.
We retrospectively reviewed 109 patients harboring 136 aneurysms across 12 years, between 1997 and 2009, at our institute. We obtained the following data from all patients : age, sex, location and size of the aneurysm(s), presence of symptoms, risk factors for stroke, treatment modality, and postoperative 1-year morbidity and mortality. We classified these patients into three groups : Group A (surgical clipping), Group B (coil embolization), and Group C (observation only).
Among the 109 patients, 56 (51.4%) underwent clipping treatment, 25 (23%) patients were treated with coiling, and 28 observation only. The overall morbidity and mortality rates were 2.46% and 0%, respectively. The morbidity rate was 1.78% for clipping and 4% for coiling. Factors such as hypertension, diabetes mellitus, hypercholesterolemia, smoking, and family history of stroke were correlated with unfavorable outcomes. Two in the observation group refused follow-up and died of intracranial ruptured aneurysms. The observation group had a 7% mortality rate.
Our results show acceptable favorable outcome of treatment-related morbidity comparing with the natural history of unruptured cerebral aneurysm. Surgical clipping did not lead to inferior outcomes in our study, although coil embolization is generally more popular for treating elderly patients. In the treatment of patients more than 65 years old, age is not the limiting factor.
Unruptured intracranial aneurysms; Elderly patients; Risk factors; Clipping; Coil embolization; Treatment-related morbidity
Vinyl chloride (VC) is a toxic groundwater pollutant associated with plastic manufacture and chlorinated solvent use. Aerobic bacteria that grow on VC as a carbon and energy source can evolve in the laboratory from bacteria that grow on ethene, but the genetic changes involved are unknown. We investigated VC adaptation in two variants (JS623-E and JS623-T) of the ethene-oxidizing Mycobacterium strain JS623. Missense mutations in the EtnE gene developed at two positions (W243 and R257) in cultures exposed to VC but not in cultures maintained on ethene. Epoxyalkane-coenzyme M transferase (EaCoMT) activities in cell extracts of JS623-E and JS623-T (150 and 645 nmol/min/mg protein, respectively) were higher than that of wild-type JS623 (74 nmol/min/mg protein), and in both variant cultures epoxyethane no longer accumulated during growth on ethene. The heterologous expression of two variant etnE alleles (W243G [etnE1] and R257L [etnE2]) from strain JS623 in Mycobacterium smegmatis showed that they had 42 to 59% higher activities than the wild type. Recombinant JS623 cultures containing mutant EtnE genes cloned in the vector pMV261 adapted to growth on VC more rapidly than the wild-type JS623 strain, with incubation times of 60 days (wild type), 1 day (pMVetnE1), and 35 days (pMVetnE2). The JS623(pMVetnE) culture did not adapt to VC after more than 60 days of incubation. Adaptation to VC in strain JS623 is consistently associated with two particular missense mutations in the etnE gene that lead to higher EaCoMT activity. This is the first report to pinpoint a genetic change associated with the transition from cometabolic to growth-linked VC oxidation in bacteria.
It has been reported that hepatitis B virus (HBV) core protein (HBc) can inhibit the transcription of human interferon-induced MxA gene. In this study, we investigated whether HBc protein mutations at hot spots (L60V, S87G and I97L) could still inhibit MxA transcription and the potential significance of this inhibition in virus replication in vitro. Our data indicated that the IFN-induced MxA mRNA expression level and MxA promoter activity was significantly down-regulated by mutant protein of HBc(I97L), compared to WT and the other two mutated HBc proteins(L60V or S87G). However, in Huh7 cells stably expressing WT or the mutated HBc proteins (L60V, S87G or I97L), IFN-α could inhibit the extra- and intracellular HBV DNA level and HBsAg secretion to a similar level compared to that in cells transfected with control plasmids. In conclusion, HBc protein with I97L mutation may play an especial role in suppressing the transcription of MxA gene. Moreover, the inhibitory effect on MxA gene transcription by the WT or mutated HBc proteins (L60V, S87G and I97L) has no impact on inhibition of HBV replication by IFN-α in Huh7 cells. The clinical significance of the inhibitory effect of MxA gene transcription by HBc protein requires further study.
The aim of this study was to analyze the correlation between thromboembolic complications and antiplatelet drugs before and after neurointervention.
Blood samples and radiographic data of patients who received a neurointervention (coil embolization, stent placement or both) were collected prospectively. Rapid platelet function assay-aspirin (RPFA-ASA) was used to calculate aspirin resistance in aspirin reaction units (ARU). For clopidogrel resistance, a P2Y12 assay was used to analyze the percentage of platelet inhibition. ARU > 550 and platelet inhibition < 40% were defined as aspirin and clopidogrel resistance, respectively.
Both aspirin and clopidogrel oral pills were administered in fifty-three patients before and after neurointerventional procedures. The mean resistance values of all patients were 484 ARU and < 39%. Ten (17.0%) of 53 patients showed resistance to aspirin with an average of 597 ARU, and 33 (62.3%) of 53 patients showed resistance to clopidogrel with an average of < 26%. Ten patients demonstrated resistance to both drugs, 5 of which suffered a thromboembolic complication after neurointervention (mean values : 640 ARU and platelet inhibition < 23%). Diabetic patients and patients with hypercholesterolemia displayed mean aspirin resistances of 513.7 and 501.8 ARU, and mean clopidogrel resistances of < 33.8% and < 40.7%, respectively.
Identifying individuals with poor platelet inhibition using standard regimens is of great clinical importance and may help prevent cerebral ischemic events in the future. Neurointerventional research should focus on ideal doses, timing, choices, safety, and reliable measurements of antiplatelet drug therapy, as well as confirming the clinical relevance of aggregometry in cerebrovascular patients.
Aspirin; Clopidogrel; Resistance; Neurointervention
Residual aneurysm from incomplete clipping or slowly recurrent aneurysm is associated with high risk of subarachnoid hemorrhage. We describe complete treatment of the lesions by surgical clipping or endovascular treatment.
We analyzed 11 patients of residual or recurrent aneurysms who had undergone surgical clipping from 1998 to 2009. Among them, 5 cases were initially clipped at our hospital. The others were referred from other hospitals after clipping. The radiologic and medical records were retrospectively analyzed.
All patients presented with subarachnoid hemorrhage at first time, and the most frequent location of the ruptured residual or recurrent aneurysm was in the anterior communicating artery to posterior-superior direction. Distal anterior cerebral artery, posterior communicating artery, and middle cerebral artery was followed. Repositioning of clipping in eleven cases, and one endovascular treatment were performed. No residual aneurysm was found in postoperative angiography, and no complication was noted in related to the operations.
These results indicate the importance of postoperative or follow up angiography and that reoperation of residual or slowly recurrent aneurysm should be tried if such lesions being found. Precise evaluation and appropriate planning including endovascular treatment should be performed for complete obliteration of the residual or recurrent aneurysm.
Intracranial aneurysm; Subarachnoid hemorrhage; Reoperation
AIM: To evaluate tracking of magnetically labeled mesenchymal stem cells (MSCs) after intraportal transplantation.
METHODS: Mononuclear cells were isolated from bone marrow aspirates of pigs by density gradient centrifugation, cultured and expanded, after which, they were incubated with super paramagnetic iron oxide (SPIO). Prussian blue staining was performed to highlight intracellular iron. To establish swine models of acute liver injury, 0.5 g/kg D-galactosamine was administrated to 10 pigs, six of which were injected via their portal veins with SPIO-labeled MSCs, while the remaining four were injected with unlabeled cells. Magnetic resonance imaging (MRI) was performed with a clinical 1.5T MR scanner immediately before transplantation and 6 h, 3 d, 7 d and 14 d after transplantation. Prussian blue staining was again performed with the tissue slices at the endpoint.
RESULTS: Prussian blue staining of SPIO-labeled MSCs had a labeling efficiency of almost 100%. Signal intensity loss in the liver by SPIO labeling on the FFE (T2*WI) sequence persisted until 14 d after transplantation. Histological analysis by Prussian blue staining confirmed homing of labeled MSCs in the liver after 14 d; primarily distributed in hepatic sinusoids and liver parenchyma.
CONCLUSION: MSCs were successfully labeled with SPIO in vitro. MRI can monitor magnetically labeled MSCs transplanted into the liver.
Magnetic resonance imaging; Mesenchymal stem cells; Super paramagnetic iron oxide; Stem cell transplantation
TARDBP mutations have been reported in patients with amyotrophic lateral sclerosis (ALS) in different populations except Chinese. The present aim is to investigate the association between TARDBP mutations and Chinese patients with ALS.
71 SALS patients and 5 FALS families with non-SOD1 mutations were screened for TARDBP mutations via direct sequencing.
A novel heterozygous variation, Ser292Asn (875G>A), was identified in the proband and 4 asymptomatic relatives including the children of the dead patient from a FALS family. Thus the dead patient, the proband's brother, was speculated to carry Ser292Asn though his sample was unavailable to the detection. This variation was not found in 200 unrelated control subjects. A homology search of the TDP-43 protein in different species demonstrated that it was highly conserved. Also, it was predicted to be deleterious to protein function with SIFT-calculated probabilities of 0.00. Therefore, Ser292Asn is predicted to be a pathogenic mutation. In addition, we have found two silent mutations (Gly40Gly and Ala366Ala) and one novel polymorphism (239-18t>c).
The present data have extended the spectrum of TARDBP mutations and polymorphisms, and supported the pathological role of TDP-43 in Chinese ALS patients.