Metformin protects against pericyte apoptosis and promotes neurogenesis through suppressing JNK p38 MAPK signalling activation in ischemia/reperfusion injury.

Metformin (MET) has been the subject of many classic studies in possessing antiapoptotic, anti-inflammatory, antioxidation activities and antiviral. Recently investigators have examined the anti-apoptosis effects of MET in acute myocardial infarction and Intracerebral hemorrhage, but very little is currently known about how it regulates ischemic stroke-induced pericytes apoptosis and neural stem cells (NSCs) proliferation. The present research explored the potential neuroprotective mechanisms of MET using transient middle cerebral artery occlusion(tMCAO) mice. The experimental work presented that tMCAO mice treated by metformin had better neurologic outcomes on days 1, 3, and 7 after operation, and alleviated blood-brain barrier (BBB) destruction, brain water content and infarct volume on 72 h after surgery. The data showed that MET alleviated BBB disruption by reducing PDGFRß/ matrix metalloproteinase-9 (MMP9) positive cells, relieving zonula occludens-1 (ZO-1) drop away and increasing pericyte coverage through remarkably reducing the percentage of PDGFRß/caspase-3 positive cells. In addition, MET induced antiapoptotic activity followed by downregulating cleaved caspase-3 and Bax expression. Moreover, JNK signaling pathway has been proved to be pivotal in mediating apoptosis in cerebral ischemia/reperfusion (I/R) injury. The results of this research illustrated that MET treatment downregulated the levels of phosphorylated JNK and P38 in vivo, however the use of JNK activator anisomycin (ANI) could reverse the neuroprotection effect of MET, demonstrating that the JNK pathway is associated with the anti-apoptosis mechanisms of MET. Finally, metformin remarkably increased the percentage of BrdU/DCX-positive cells in subventricular zone (SVZ) and up-regulated BDNF、VEGF and NGF expression after ischemia/reperfusion(I/R) injury on day 7. Our data illustrated that metformin provides an effective therapy for I/R injury.

DNA Hypomethylation of DOCK1 Leading to High Expression Correlates with Neurologic Deterioration and Poor Function Outcomes after Spontaneous Intracerebral Hemorrhage.

OBJECTIVE: Spontaneous intracerebral hemorrhage (ICH) is a blood clot arising in the brain parenchyma in the absence of trauma or surgery and accounts for 10% to 15% of all strokes, leading to higher rates of mortality and morbidity than either ischemic stroke or subarachnoid hemorrhage. We sought to investigate the potential association of DOCK1 with neurological deficits and outcomes in patients with spontaneous ICH. METHODS: Identification of methylation-regulated differentially expressed genes (MeDEGs) between ICH patients and matched controls was performed by analyzing the raw data from the GSE179759 and GSE125512 datasets deposited in the Gene Expression Omnibus. A total of 114 patients who were admitted to our hospital for spontaneous ICH were retrospectively analyzed, with 108 healthy volunteers who had received physical examinations at the same period as controls. The mRNA expression of DOCK1 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The hematoma volume was calculated according to the Coniglobus formula. The severity of neurological deficits was evaluated using National Institutes of Health Stroke Scale (NIHSS) scores and function outcomes were evaluated by modified Rankin Scale (mRS) scores. RESULTS: A total of 15 MeDEGs between ICH patients and matched controls were identified. The mRNA expression of DOCK1 was remarkably higher in the serum samples of patients with spontaneous ICH than in the healthy controls. According to hematoma volume after ICH attack, small (<10 mL), medium (10 to 30 mL), and large (>30 mL) groups were arranged. The proportions of male patients and patients aged ≥60 years were significantly higher in the large group than in the small and medium groups (P < 0.05). The mRNA expression of DOCK1 was significantly higher in the large group than in the small and medium groups (P < 0.05). According to NIHSS scores, mild (NIHSS scores ≤15), moderate (NIHSS scores from 16 to 30), and severe (NIHSS scores from 31 to 45) groups were classified. It was observed that the severe group had higher proportions of male patients and patients aged ≥60 years than the mild and moderate groups (P < 0.05). The severe group exhibited a higher mRNA expression of DOCK1 than the mild and moderate groups (P < 0.05). According to mRS scores, higher proportions of male patients and patients aged ≥60 years were observed in the unfavorable group than the favorable group (P < 0.05). The patients in the unfavorable group showed an elevated DOCK1 mRNA expression compared to those in the favorable group (P < 0.05). CONCLUSION: The study provided evidence that male gender, older age, and higher DOCK1 mRNA expression were related to higher admission hematoma volume, neurologic deterioration, and poor function outcomes in patients with spontaneous ICH.

Protective effects of Dimethyl malonate on neuroinflammation and blood-brain barrier after ischemic stroke.

OBJECTIVES: After ischemic stroke, microglia will be activated and play a key role in neuroinflammation and the destruction of the blood-brain barrier (BBB), and activated microglia could polarize into pro-inflammation M1 phenotype and anti-inflammation M2 phenotype. Dimethyl malonate (DMM) could reduce reactive oxygen species and we speculate DMM could regulate microglia to protect ischemic brain. METHODS: We used transient middle cerebral artery occlusion (tMCAO) mouse model to simulate ischemic stroke and adult male C57BL/6 mice were used in our study. 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct volume. Evans Blue and Brain water content were used to evaluate the destruction of BBB. We used a five-point scale to assess the neurologic function of mice. Western blot and Immunofluorescence were used to measure microglia, pericytes and the expression of related proteins. RESULTS: DMM reduced cerebral infarct volume, Evans blue leakage, brain water content and improved neurologic deficits after tMCAO. The number of activated microglia and M1 microglia were decreased and the number of M2 microglia and pericytes were increased after DMM treatment. The expression of tumor necrosis factor-α was reduced while protein levels of IL-10 and ZO-1 were increased through DMM treatment. CONCLUSIONS: DMM could regulate activation and polarization of microglia to inhibit neuroinflammation and protect BBB.

Donepezil promotes neurogenesis via Src signaling pathway in a rat model of chronic cerebral hypoperfusion.

Donepezil, a selective acetylcholinesterase (AchE) inhibitor, enhances stroke-induced neurogenesis within subventricular zone (SVZ). Src/Pyk-2 is one of the downstream pathways of acetylcholine receptors (AchRs), and has been shown to participate in the activation of fibroblast growth factor receptor (FGFR)/epidermal growth factor receptor (EGFR) signaling in cancer cells. In this study, we investigated whether donepezil could promote SVZ neurogenesis in chronic cerebral hypoperfusion (CCH) injury via Src signaling pathway. In the bilateral carotid artery occlusion (2VO) rat model, we observed more nestin/5-bromo-2'-deoxyuridine (BrdU)-positive cells and doublecortin (DCX)/BrdU-positive cells in the SVZ than that in the sham group. Further, donepezil obviously improved neurologic function after 2VO, induced the greater number of SVZ proliferative NSCs and neuroblasts, and elevated levels of Src, p-FGFR1, p-EGFR, p-Akt and p-Raf in ipsilateral SVZ. Lastly, Src inhibitor KX-01 abolished the beneficial effects of donepezil in 2VO rats. These results suggest that donepezil could upregulate Src signaling pathway to enhance CCH-induced SVZ neurogenesis.

Effects of RO27-3225 on neurogenesis, PDGFRß+ cells and neuroinflammation after cerebral infarction.

Cerebral infarction causes severe social and economic burdens to patients due to its high morbidity and mortality rates, and the available treatments are limited. RO27-3225 is a highly selective melanocortin receptor 4 agonist that alleviates damage in many nervous system diseases, such as cerebral hemorrhage, traumatic brain injury and chronic neurodegenerative diseases. However, the effect of RO27-3225 on cerebral infarction remains unclear. In this study, we used a mouse model of transient middle cerebral artery occlusion (tMCAO) and administered RO27-3225 or saline to the mice through intraperitoneal injection. RO27-3225 increased the number of Nestin+/BrdU+ cells and doublecortin (DCX)+/BrdU+ cells in the subventricular zone (SVZ) and the number of DCX+/BrdU+ cells in the peri-infarct area on day 7 after tMCAO. Furthermore, RO27-3225 decreased the number of activated microglia (Iba1+ cells with a specific morphology) and the expression levels of Iba1, TNFα, IL6, and iNOS proteins and increased the number of PDGFRß+ cells in the peri-infarct region on day 3 after tMCAO. Finally, RO27-3225-treated mice exhibited significantly decreased infarct volumes, brain water contents, and neurological deficits after cerebral infarction. Thus, RO27-3225 can improve the outcomes following cerebral infarction, partially by regulating neurogenesis in the SVZ, PDGFRß+ cell survival and neuroinflammation in the peri-infarct zone. Our research reveals that RO27-3225 is a potential new treatment for cerebral infarction.

Neuroprotective Action of Teriflunomide in a Mouse Model of Transient Middle Cerebral Artery Occlusion.

Teriflunomide has been reported to inhibit microglial activation in experimental models of traumatic brain injury. However, its roles in ischemic stroke and underlying mechanisms of action are still undiscovered. In this study, we investigated the effects of teriflunomide on brain edema, neurologic deficits, infarct volume, neuroinflammation, blood-brain barrier (BBB) permeability, and neurogenesis in a mouse model of transient middle cerebral artery occlusion (tMCAO). tMCAO mice treated with teriflunomide showed lower brain water content on day 3, milder neurologic deficits and smaller infarct volume on day 7 than those treated with vehicle. Additionally, mice received teriflunomide had fewer activated Iba-1-positive microglia and lower protein levels of interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and 3-Nitrotyrosine (3-NT) compared with those received vehicle on day 3. Further, teriflunomide alleviated Evans blue dye leakage, increased pericyte coverage and protein levels of platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor ß (PDGFRß) and Bcl2, and decreased the number of PDGFRß/matrix metalloproteinase 9 (MMP9)-positive cells. Moreover, teriflunomide reduced the loss of zonula occludens-1 (ZO-1) and occludin. Finally, teriflunomide significantly upregulated the number of 5-bromo-20-deoxyuridine (BrdU)/doublecortin (DCX)-positive cells and expression of mammalian achaete-scute homolog 1 (Mash1), DCX and Pbx1 in subventricular zone (SVZ) on day 7 after stroke. Our results indicate that teriflunomide exhibits protective roles in ischemic stroke by inhibiting neuroinflammation, alleviating BBB disruption and enhancing neurogenesis.

A likely protective effect of dimethyl itaconate on cerebral ischemia/reperfusion injury.

As a membrane-permeable derivative of itaconate, dimethyl itaconate (DMI) was recently showed to limit inflammatory response of activated macrophages, and to decrease the generation of reactive oxygen species and reduce cardiac ischemia/reperfusion injury. However, the effect of DMI in the context of cerebral ischemia/reperfusion injury remains unclear. Here, we treated the transient middle cerebral artery occlusion (tMCAO) mice with DMI or saline at the beginning of occlusion, and allowed them to recover for 3 days. We found that DMI obviously decreased the neurologic deficit score. Further, DMI significantly inhibited the toxic conversion of the peri-infarct microglia, and decreased the protein level of interleukin 1ß. The present findings suggest that DMI might be recognized as a promising candidate for the treatment of ischemic stroke.

EPO promotes axonal sprouting via upregulating GDF10.

Erythropoietin (EPO) has an exact neuroprotective effect on stroke. However, it remains unknown whether it participates in axonal sprouting after neuron damage. Growth and differentiation factor 10 (GDF10) has been shown to be a trigger of axonal sprouting after stroke. Hence, it was hypothesized that EPO promotes axonal sprouting mainly through GDF10. In the present in vitro experiment, it was found that EPO could promote axonal sprouting and GDF10 expression in a dose-dependent manner. The knockdown of GDF10 using siRNA abolished the effect of EPO-mediated axonal sprouting, indicating that GDF10 is the executor of EPO-mediated axonal sprouting. The treatment of neurons with nuclear factor-kappaB (NF-κB) inhibitor JSH-23 could inhibit the accumulation of NF-κB phospho-p65 (p-p65) in the nucleus, the upregualtion of GDF10 and extending of axonal length. Furthermore, the addition of Janus kinase 2 (JAK2) inhibitor CEP-33779 or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 to the culture medium also blocked the nuclear translocation of p-p65, the expression of GDF10, and axonal sprouting, suggesting that EPO induces axonal sprouting via activating cellular JAK2 and PI3K signaling. Impeding JAK2 signaling with CEP-33779 can suppress the phosphorylation of PI3K, and this confirms that the upstream of PI3K signaling is JAK2. These present results provide a novel insight into the role of EPO and the molecular mechanism of axonal sprouting, which is beneficial for the development of novel approaches for neurological recovery after brain injury, including stroke.

Wnt-3a alleviates neuroinflammation after ischemic stroke by modulating the responses of microglia/macrophages and astrocytes.

Neuroinflammation crucially influences functional recovery after ischemic stroke. Wnt-3a, a novel Wnt protein that specifically promotes Wnt/ß-catenin signaling pathway, has been shown to regulate apoptosis and cell proliferation, but how it affects ischemic stroke-induced toxic brain inflammation remains unknown. Using a transient middle cerebral artery occlusion (tMCAO) mouse model in this study, we found that intranasal Wnt-3a-treated tMCAO mice had apparently reduced infarct volume and decreased brain water content after being allowed to recover for 72 h, as well as better neurologic outcomes on days 3, 7, and 14. Mice received Wnt-3a had significantly fewer tMCAO-induced peri-infarct TUNEL-positive cells compared with those received vehicle. Further, Wnt-3a-delivered tMCAO mice had notably fewer peri-infarct CD68-positive cells and lower ionized calcium-binding adapter molecule (Iba)-1 protein level. Wnt-3a significantly downregulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α, and upregulated the expression of arginase 1 (Arg1) and CD206. Finally, Wnt-3a obviously decreased the number of tMCAO-induced peri-infarct glial fibrillary acidic protein (GFAP)/C3-positive cells, increased the number of GFAP/S100A10-positive cells, attenuated the protein levels of GFAP and interleukin 15 (IL15), and elevated IL33 protein level. Our findings suggest that intranasal Wnt-3a could ameliorate toxic responses of microglia/macrophages and astrocytes in ischemic brain injury, supporting that Wnt-3a might be potentially appropriate for ischemic stroke treatment functioning as an immunomodulatory agent.

Carbon monoxide-releasing molecule-3 protects against ischemic stroke by suppressing neuroinflammation and alleviating blood-brain barrier disruption.

BACKGROUND: At low levels, carbon monoxide (CO) has been shown to have beneficial effects on multiple organs and tissues through its potential anti-inflammatory, anti-apoptotic, and anti-proliferative properties. However, the effect of CO-releasing molecule (CORM)-3, a water-soluble CORM, on ischemic stroke and its mechanism of action are still unclear. METHODS: We investigated the role of CORM-3 in the mouse model of transient middle cerebral artery occlusion (tMCAO). CORM-3 or saline was administered to mice by retro-orbital injection at the time of reperfusion after 1-h tMCAO or at 1 h after sham surgery. We assessed infarct volume and brain water content at 24 and 72 h after ischemia, blood-brain barrier permeability at 6 and 72 h after ischemia, and neurologic deficits on days 1, 3, 7, and 14. RESULTS: Among mice that underwent tMCAO, those that received CORM-3 had significantly smaller infarct volume and greater expression of neuronal nuclear antigen (NeuN) and microtubule-associated protein 2 than did saline-treated mice. CORM-3-treated mice had significantly fewer activated microglia in the peri-infarction zone than did control mice and exhibited downregulated expression of ionized calcium-binding adapter molecule (Iba)-1, tumor necrosis factor-α, and interleukin 1ß. CORM-3-treated mice had significantly lower brain water content and enhanced neurologic outcomes on days 3, 7, and 14 post-tMCAO. Lastly, CORM-3 treatment reduced Evans blue leakage; increased expression of platelet-derived growth factor receptor-ß, tight junction protein ZO-1, and matrix protein laminin; and decreased protein level of matrix metalloproteinase-9. CONCLUSION: CORM-3 treatment at the time of reperfusion reduces ischemia-reperfusion-induced brain injury by suppressing neuroinflammation and alleviating blood-brain barrier disruption. Our data suggest that CORM-3 may provide an effective therapy for ischemic stroke.

Effects of crenolanib, a nonselective inhibitor of PDGFR, in a mouse model of transient middle cerebral artery occlusion.

Neurogenesis in the subventricular zone (SVZ) plays a vital role in neurologic recovery after stroke. However, only a small fraction of newly generated neuroblasts from the SVZ will survive long-term. Successful migration and survival of neuroblasts requires angiogenesis, lesion-derived chemo-attractants, and appropriate local microenvironments, which are partly regulated by the platelet-derived growth factor receptor (PDGFR) signaling pathway. In this study, we investigated the effects of PDGFR inhibition in a mouse model of transient middle cerebral artery occlusion (MCAO). We blocked the pathway using a nonselective PDGFR inhibitor, crenolanib, during the acute post-MCAO phase (days 1-3) or during the sub-acute phase (days 7-9). Downregulating the PDGFR signaling pathway with crenolanib from day 1 to day 3 after MCAO significantly decreased the migration of neuroblasts from the SVZ to the peri-infarct region, decreased angiogenesis, and lowered expression of vascular endothelial growth factor, stromal cell-derived factor-1, and monocyte chemotactic protein-1. Downregulation of the PDGFR signaling pathway on days 7-9 with crenolanib significantly increased apoptosis of the neuroblasts that had migrated to the peri-infarct region, increased the number of activated microglia, and decreased the expression of brain-derived neurotrophic factor, neurotrophin-3, and interleukin-10. Crenolanib treatment increased the apoptosis of pericytes and decreased the pericyte/vascular coverage, but had no effects on apoptosis of astrocytes. We conclude that the PDGFR signaling pathway plays a vital role in the SVZ neurogenesis after stroke. It can also affect angiogenesis, lesion-derived chemo-attractants, and the local microenvironment, which are all important to stroke-induced neurogenesis.

Alpha-7 Nicotinic Receptor Signaling Pathway Participates in the Neurogenesis Induced by ChAT-Positive Neurons in the Subventricular Zone.

Choline acetyltransferase-positive (ChAT+) neurons within the subventricular zone (SVZ) have been shown to promote neurogenesis after stroke in mice by secreting acetylcholine (ACh); however, the mechanisms remain unclear. Receptors known to bind ACh include the nicotinic ACh receptors (nAChRs), which are present in the SVZ and have been shown to be important for cell proliferation, differentiation, and survival. In this study, we investigated the neurogenic role of the alpha-7 nAChR (α7 nAChR) in a mouse model of middle cerebral artery occlusion (MCAO) by using α7 nAChR inhibitor methyllycaconitine. Mice subjected to MCAO exhibited elevated expression of cytomembrane and nuclear fibroblast growth factor receptor 1 (FGFR1), as well as increased expression of PI3K, pAkt, doublecortin (DCX), polysialylated - neuronal cell adhesion molecule (PSA-NCAM), and mammalian achaete-scute homolog 1 (Mash1). MCAO mice also had more glial fibrillary acidic protein (GFAP)/5-bromo-2'-deoxyuridine (BrdU)-positive cells and DCX-positive cells in the SVZ than did the sham-operated group. Methyllycaconitine treatment increased cytomembrane FGFR1 expression and GFAP/BrdU-positive cells, upregulated the levels of phosphoinositide 3-kinase (PI3K) and phospho-Akt (pAkt), decreased nuclear FGFR1 expression, decreased the number of DCX-positive cells, and reduced the levels of DCX, PSA-NCAM, and Mash1 in the SVZ of MCAO mice compared with levels in vehicle-treated MCAO mice. MCAO mice treated with α7 nAChR agonist PNU-282987 exhibited the opposite effects. Our data show that α7 nAChR may decrease the proliferation of neural stem cells and promote differentiation of existing neural stem cells after stroke. These results identify a new mechanism of SVZ ChAT+ neuron-induced neurogenesis.

ChAT-positive neurons participate in subventricular zone neurogenesis after middle cerebral artery occlusion in mice.

The mechanisms of post-stroke neurogenesis in the subventricular zone (SVZ) are unclear. However, neural stem cell-intrinsic and neurogenic niche mechanisms, as well as neurotransmitters, have been shown to play important roles in SVZ neurogenesis. Recently, a previously unknown population of choline acetyltransferase (ChAT)+ neurons residing in rodent SVZ were identified to have direct control over neural stem cell proliferation by indirectly activating fibroblast growth factor receptor (FGFR). This finding revealed possible neuronal control over SVZ neurogenesis. In this study, we assessed whether these ChAT+ neurons also participate in stroke-induced neurogenesis. We used a permanent middle cerebral artery occlusion (MCAO) model produced by transcranial electrocoagulation in mice, atropine (muscarinic cholinergic receptor [mAchR] antagonist), and donepezil (acetylcholinesterase inhibitor) to investigate the role of ChAT+ neurons in stroke-induced neurogenesis. We found that mAchRs, phosphorylated protein kinase C (p-PKC), and p-38 levels in the SVZ were upregulated in mice on day 7 after MCAO. MCAO also significantly increased the number of BrdU/doublecortin-positive cells and protein levels of phosphorylated-neural cell adhesion molecule and mammalian achaete scute homolog-1. FGFR was activated in the SVZ, and doublecortin-positive cells increased in the peri-infarction region. These post-stroke neurogenic effects were enhanced by donepezil and partially decreased by atropine. Neither atropine nor donepezil affected peri-infarct microglial activation or serum concentrations of TNF-α, IFN-γ, or TGF-ß on day 7 after MCAO. We conclude that ChAT+ neurons in the SVZ may participate in stroke-induced neurogenesis, suggesting a new mechanism for neurogenesis after stroke.

Membrane order parameters for interdigitated lipid bilayers measured via polarized total-internal-reflection fluorescence microscopy.

Incorporating ethanol in lipid membranes leads to changes in bilayer structure, including the formation of an interdigitated phase. We have used polarized total-internal-reflection fluorescence microscopy (pTIRFM) to measure the order parameter for Texas Red DHPE incorporated in the ethanol-induced interdigitated phase (LßI) formed from ternary lipid mixtures comprising dioleoylphosphatidylcholine, cholesterol and egg sphingomyelin or dipalmitoylphosphatidylcholine. These lipid mixtures have 3 co-existing phases in the presence of ethanol: liquid-ordered, liquid-disordered and LßI. pTIRFM using Texas Red DHPE shows a reversal in fluorescence contrast between the LßI phase and the surrounding disordered phase with changes in the polarization angle. The contrast reversal is due to changes in the orientation of the dye, and provides a rapid method to identify the LßI phase. The measured order parameters for the LßI phase are consistent with a highly ordered membrane environment, similar to a gel phase. An acyl-chain labeled BODIPY-FL-PC was also tested for pTIRFM studies of ethanol-treated bilayers; however, this probe is less useful since the order parameters of the interdigitated phase are consistent with orientations that are close to random, either due to local membrane disorder or to a mixture of extended and looping conformations in which the fluorophore is localized in the polar headgroup region of the bilayer. In summary, we demonstrate that order parameter measurements via pTIRFM using Texas Red-DHPE can rapidly identify the interdigitated phase in supported bilayers. We anticipate that this technique will aid further research in the effects of alcohols and other additives on membranes.

Changes in order parameters associated with ceramide-mediated membrane reorganization measured using pTIRFM.

The enzymatic generation of ceramide has significant effects on the biophysical properties of lipid bilayers and can lead to the extensive reorganization of cell membranes. We have synthesized and characterized a headgroup-labeled fluorescent lipid probe (NBD-ceramide, NBD-Cer) and demonstrated that it can be used for polarized total internal reflection fluorescence microscopy experiments to probe changes in membrane order that result from ceramide incorporation. NBD-Cer measures significantly higher order parameters for the liquid-ordered (Lo) domains ([P2] = 0.40 ± 0.03) than for the liquid-disordered phase (Ld, fluid, [P2] = 0.22 ± 0.02) of phase-separated bilayers prepared from egg sphingomyelin, dioleolyphosphatidylcholine, and cholesterol mixtures. The probe also responds to changes in packing induced by the direct incorporation of ceramide or the variation in the ionic strength of the aqueous medium. Order parameter maps obtained after enzyme treatment of bilayers with coexisting Lo and Ld phases show two distinct types of behavior. In regions of high enzyme activity, the initial Lo/Ld domains are replaced by large, dark features that have high membrane order corroborating previous hypotheses that these are ceramide-enriched regions of the membrane. In areas of low enzyme activity, the size and shape of the Lo domains are conserved, but there is an increase in the order parameter for the initial Ld phase ([P2] = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the Lo domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.

[Analysis of pathogen isolated from lower respiratory tract in coalminer's pneumoconiosis patients complicated with infection].

OBJECTIVE: To investigate the composition and resistance of main pathogens isolated form Lower respiratory tract in coalminer's pneumoconiosis patients complicated with infection to provide the basis for clinical treatment. METHOD: Coalminer's pneumoconiosis patients complicated with infection during 2009 to 2010 were divided into mechanical ventilation group and non mechanical ventilation group. Specimens were obtained from lower respiratory tract by fibrobronchoscopy with protected specimen brush in patients of both groups to perform isolation, culture, identification and susceptibility test of pathogen. RESULT: Total 111 patients were enrolled, 36 of them in mechanical ventilation group and 75 patients in non mechanical ventilation group. The pathogenic bacteria detection rate of patients in mechanical ventilation group was significantly higher than that of patients in non mechanical ventilation group (88.9% vs. 46.7%, P < 0.01). In non mechanical ventilation group, Mycobacterium tuberculosis was detected in 3 patients, and 27 strains of G- bacilli, 3 strains of G+ coccus, and 2 strains of fungus; and 26 strains of G- bacilli, 3 strains of G+ coccus, and 3 strains of fungus were detected in mechanical ventilation group. There was no significant difference in term of strains between the two groups (P > 0.05). Rate of resistance to main antibiotics of patients in mechanical ventilation group was higher than that of patients in non mechanical ventilation group. CONCLUSION: Resistance of pathogenic bacteria isolated from lower respiratory tract was severe in coalminer's pneumoconiosis patients complicated with infection, which was higher in patients treated with mechanical ventilation than patients without mechanical ventilation. Mycobacterium tuberculosis and fungal infection and increasing resistance prompted that clinicians must attach importance to rational drug use and keep to monitoring bacterial resistance.

Nanoscale imaging of epidermal growth factor receptor clustering: effects of inhibitors.

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.