[Down-regulation of miR-146b-5p promotes malignant transformation of fusion cells after co-culture of macrophages with glioma stem cells in vitro].

Objective: To observe mutual interactions between macrophages(Mφ) and glioma stem cells (GSCs)in dual-color tracing model in vitro, to identify the biological characteristics of fusion cells in multiple levels, and to analysis the relevant molecular mechanisms. Methods: Red fluorescent protein(RFP) gene was stably transfected into human GSCs cell line SU4. Mφ cells were obtained from Balb/c nude mice with enhanced green fluorescent protein (EGFP) expression. Then two cells were co-cultured in dual-color tracing platform. RFP/EGFP double positive cells with high proliferation ability were mono-cloned. The fusion cells were verified by Western blot, fluorescence in situ hybridization, immunocytochemistry and chromosome karyotype analysis.The biological characteristics of fusion cells were further analyzed, together with relevant molecular changes. Results: RFP / EGFP double positive cells were obtained through in vitro co-culture. RFP and EGFP coexpression were proved at transcriptional and translational levels in the fusion cells. They also co-expressed GSCs marker Nestin and Mφ marker CD68, and karyotype analysis showed two types of characteristic chromosomes, which confirmed that the fusion cells originated from spontaneous fusion between SU4-RFP and Mφ.Fusion cell proliferation rate and invasion ability were higher than SU4-RFP, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. Fusion cells transfected with miR-146b-5p showed a higher apoptosis rate(18.83%) and lower tumor formation(4/5). Conclusion: Mφ could fuse with GSCs spontaneously in local tumor micro-environment. The proliferation and invasion abilities of fusion cells were higher than their parent cells, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. It revealed the possible mechanisms of malignant progression of gliomas.

Determination of pseudocontact shifts of low-populated excited states by NMR chemical exchange saturation transfer.

Despite the critical roles of excited states in protein functions, they remain intractable for most structural studies because of their notably low populations. Chemical shifts for "invisible" states in slow exchange with the ground state are intuitively observed using nuclear magnetic resonance (NMR) chemical exchange saturation transfer (CEST) experiments. Here, we present a CEST NMR spectroscopy study for the observation of protein pseudocontact shifts (PCSs) of excited states, which carry valuable angular and distance information about these states. We first validate this approach, dubbed PCS-CEST, in the slow-exchange system of Abp1p SH3-Ark1p labeled with lanthanide ions, where the PCSs of the minor states resemble those of the holo-form ground state as expected. We further demonstrate that pre-existing folding transitional conformations of an FF domain exhibit remarkably lower PCS values than the ground state, which suggests that the low-populated ensemble is unfolded or largely unfolded. A higher resolution of PCSs of the minor states is achieved using our 1D selective CEST experiments. Thus, PCS-CEST provides an exquisite structural probe into the minor but functionally essential excited states.

Cloning and characterization of the SERK1 gene in triploid Pingyi Tiancha [Malus hupehensis (Pamp.) Rehd. var. pingyiensis Jiang] and a tetraploid hybrid strain.

This study aims to explore the roles of somatic embryogenesis receptor-like kinase (SERK) in Malus hupehensis (Pingyi Tiancha). The full-length sequences of SERK1 in triploid Pingyi Tiancha (3n) and a tetraploid hybrid strain 33# (4n) were cloned, sequenced, and designated as MhSERK1 and MhdSERK1, respectively. Multiple alignments of amino acid sequences were conducted to identify similarity between MhSERK1 and MhdSERK1 and SERK sequences in other species, and a neighbor-joining phylogenetic tree was constructed to elucidate their phylogenetic relations. Expression levels of MhSERK1 and MhdSERK1 in different tissues and developmental stages were investigated using quantitative real-time PCR. The coding sequence lengths of MhSERK1 and MhdSERK1 were 1899 bp (encoding 632 amino acids) and 1881 bp (encoding 626 amino acids), respectively. Sequence analysis demonstrated that MhSERK1 and MhdSERK1 display high similarity to SERKs in other species, with a conserved intron/exon structure that is unique to members of the SERK family. Additionally, the phylogenetic tree showed that MhSERK1 and MhdSERK1 clustered with orange CitSERK (93%). Furthermore, MhSERK1 and MhdSERK1 were mainly expressed in the reproductive organs, in particular the ovary. Their expression levels were highest in young flowers and they differed among different tissues and organs. Our results suggest that MhSERK1 and MhdSERK1 are related to plant reproduction, and that MhSERK1 is related to apomixis in triploid Pingyi Tiancha.

Upregulation of miR-335 reduces myocardial injury following myocardial infarction via targeting MAP3K2.

OBJECTIVE: Acute myocardial infarction (AMI) is a serious cardiovascular disease with a high incidence worldwide and the main cause of sudden cardiac death. The aim of this article was to study the protective role of miR-335 in myocardial infarction (MI) and the underlying molecular mechanism. MATERIALS AND METHODS: Thirty Sprague Dawley (SD) rats were randomly divided into sham group, MI + NC group and MI + agomiR-335 group. The expression of miR-335 in rat myocardium was detected by quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Western blot was performed to detect the expression of TNF-α, IL-6, IL-1ß, Caspase-3, Cleaved Caspase-3 (C-Caspase-3) and MAP3K2 in rat myocardium. On the 7th day of the establishment of the rat MI model, a high-resolution small animal ultrasound system was utilized to detect the cardiac function of the rats, and the heart tissues and blood samples of the rats were collected. The corresponding kits were purchased to detect the contents of LDH, CK-MB, MDA and SOD in rat serum, and HE staining was employed to observe the morphology of rat myocardial tissue. RESULTS: The expression of miR-335 in myocardial infarcted zones and border zones of MI rats decreased significantly. The upregulation of miR-335 remarkably inhibited myocardial inflammation and apoptosis after MI, thus improving the cardiac function of MI rats. Compared with the sham group, the MAP3K2 expression in the MI + NC group was observably increased, while the overexpression of miR-335 could inhibit the expression of this protein. Through the Luciferase reporter gene experiment, we found that miR-335 could directly target MAP3K2. CONCLUSIONS: The expression of miR-335 decreased in myocardial tissue after MI, and the overexpression of miR-335 reduced myocardial damage by inhibiting oxidative stress, inflammation, and apoptosis via targeting MAP3K2, thereby improving the cardiac function of MI rats.

Comparison of the effects of ischaemia on early components of the somatosensory evoked potential in brainstem, thalamus, and cerebral cortex.

In 14 ventilated, normocapnic baboons anaesthetised with alpha-chloralose, local CBF (hydrogen clearance) and the amplitude and latency of local components of the somatosensory evoked potential (SEP, median nerve stimulation) were measured bilaterally in ventrobasal thalamus (VPL), medial lemniscus (ML), and cerebral cortex before and during progressive ischaemia, produced by occlusion of the right middle cerebral artery and subsequent controlled reductions in mean systemic blood pressure (MSBP). The first significant reduction from control of the left cortical SEP amplitude occurred in the range of 30-40 mm Hg MSBP, but those of the VPL and ML responses only below 30 mm Hg; in the range of 20-30 mm Hg, the average SEP amplitudes in cortex, VPL, and ML were 8.6, 72.6, and 90.7% of control, respectively. In terms of local CBF, the cortical SEP threshold was in the range of 15-20 ml/100 g/min (as in previous work), that of VPL in the range of 10-15 ml/100 g/min, but the ML response was only markedly reduced below 10 ml/100 g/min. Thus, the differential ischaemic sensitivity of the SEP between the three regions was clearly demonstrated. These results indicate that as one descends the neuraxis, there is an increasing resistance of electrophysiological function to systemic hypotension, together with a decreasing threshold for local ischaemia.

Carotid-cavernous sinus fistula associated with a primitive trigeminal artery.

Carotid-cavernous sinus fistulas are not rare, but they have never been reported in association with persistent primitive trigeminal artery. We recently encountered such a case. The Jaeger-Hamby procedure was employed, with mandatory occlusion of the primitive trigeminal artery.

Acoustic neurinoma presenting as intratumoral bleeding.

The authors report a case of acoustic neurinoma presenting as intratumoral bleeding. This is the tenth reported occurrence. The literature is reviewed. All cases that have been reported have appeared with sudden onset of headache, vomiting, and decreased levels of consciousness. Aneurysm rupture, an arteriovenous malformation, or other vascular anomalies are suspected first. Preexisting unilateral hearing impairment is a valuable clue to differential diagnosis. Contrast-enhanced computed tomographic scans and cerebral angiograms are important tools for correct diagnosis. The tumor size (greater than 2 cm) and the thin, dilated vessels within the tumor are considered as pathogenetic factors for bleeding. When neurological status is not stable, placement of a ventriculoperitoneal shunt followed by urgent extirpation of the tumor is indicated.

Relationship between hemispheric cerebral blood flow, central conduction time, and clinical grade in aneurysmal subarachnoid hemorrhage.

The relationship between central conduction time (CCT) and hemispheric cerebral blood flow (CBF) has been examined in 20 patients presenting with subarachnoid hemorrhage. A total of 63 combined CCT/CBF recordings were performed at various times throughout the hospital course of these patients, and the findings were correlated to clinical status. The initial-slope index of the CBF (CBF isi) was found to correlate well with clinical grade, and a gradation in flow was noted between the different neurological grades. Patients in Grades I and II (Hunt and Hess classification) had the highest flows (mean CBF isi = 47.2 +/- 8.1); Grade III patients had intermediate flows (mean CBF isi = 39.6 +/- 7.8); and Grade IV patients had the lowest flows (mean CBF isi = 32.0 +/- 6.4). While CCT tended to become increasingly prolonged with worsening grade, a significant difference could not be demonstrated between Grade I, II, and III patients. Only when Grade IV status was reached was the CCT significantly prolonged. When CBF isi and CCT were examined, a threshold relationship was noted between CBF isi and CCT prolongation. At flow values above 30, little change was noted in CCT, and CCT remained in the normal range. However, at flow values below 30, CCT became increasingly prolonged as blood flow diminished. The degree of CCT prolongation appeared to be directly proportional to the degree of blood flow diminution at flows below threshold.

Somatosensory evoked potential monitoring during the management of aneurysmal SAH.

Somatosensory evoked responses to median nerve stimulation have been recorded during the management of subarachnoid hemorrhage from intracranial aneurysms. The type of measurement was central conduction time (CCT), the time interval between the N14 peak (at C-2) and the N20 peak (at the cortex). Significant differences were found between patients with aneurysms in clinical Grade 4 and normal individuals, although there was no significant difference between most aneurysm patients in Grades 1 through 3. The prolongation of CCT was found of prognostic value. Significant prolongation (mean plus two standard deviations) from the normal period of 5.4 +/- 0.4 msec was found significantly useful to predict a less than satisfactory outcome (a patient with neurological signs or disabled) at 2 months, and these differences were detectable as early as 48 hours postoperatively. Differences between conduction time in the two hemispheres could also be used prognostically, although the significance of the differences did not appear until 48 to 72 hours after surgery, and was in any event less than that of CCT.

Perioperative use of somatosensory evoked responses in aneurysm surgery.

Somatosensory evoked potentials have been recorded during 34 operations for intracranial aneurysm. The central conduction time (CCT), the time between the N14 peak (recorded at C-2) and the N20 peak (recorded at the cortex) in response to median nerve stimulation, has been found to be increased by administration of halothane, by brain retraction, and by temporary vascular occlusion in some instances. Increase of CCT to more than 10 msec, or disappearance of the response was associated in two cases with postoperative neurological deficit, neither permanent. In a further case, prolongation of CCT was used as a guide to the repositioning of an imperfectly placed clip on a middle cerebral artery aneurysm. The value of the technique as a perioperative monitoring system is discussed.