Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.

EGCG inactivates a pore-forming toxin by promoting its oligomerization and decreasing its solvent-exposed hydrophobicity.

Natural proteinaceous pore-forming agents can bind and permeabilize cell membranes, leading to ion dyshomeostasis and cell death. In the search for antidotes that can protect cells from peptide toxins, we discovered that the polyphenol epigallocatechin gallate (EGCG) interacts directly with melittin from honeybee venom, resulting in the elimination of its binding to the cell membrane and toxicity by markedly lowering the extent of its solvent-exposed hydrophobicity and promoting its oligomerization into larger species. These physicochemical parameters have also been shown to play a key role in the binding to cells of misfolded protein oligomers in a host of neurodegenerative diseases, where oligomer-membrane binding and associated toxicity have been shown to correlate negatively with oligomer size and positively with solvent-exposed hydrophobicity. For melittin, which is not an amyloid-forming protein and has a very distinct mechanism of toxicity compared to misfolded oligomers, we find that the size-hydrophobicity-toxicity relationship also rationalizes the pharmacological attenuation of melittin toxicity by EGCG. These results highlight the importance of the physicochemical properties of pore forming agents in mediating their interactions with cell membranes and suggest a possible therapeutic approach based on compounds with a similar mechanism of action as EGCG.

Translation factor eIF5a is essential for IFNγ production and cell cycle regulation in primary CD8+ T lymphocytes.

Control of mRNA translation adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Traditionally initiation of translation is considered to be a major translational control point, however, control of peptide elongation is also important. Here we show that the function of the elongation factor, eIF5a, is regulated dynamically in naïve CD8+ T cells upon activation by post-translational modification, whereupon it facilitates translation of specific subsets of proteins. eIF5a is essential for long-term survival of effector CD8+ T cells and sequencing of nascent polypeptides indicates that the production of proteins which regulate proliferation and key effector functions, particularly the production of IFNγ and less acutely TNF production and cytotoxicity, is dependent on the presence of functional eIF5a. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation contributes to post-transcriptional regulons important for the control of inflammation.

Spinal Extradural Cyst: Case Report and Review of Literature.

BACKGROUND: Spinal extradural cyst (SEDC) accounts for <1% of spinal epidural lesions. It is commonly asymptomatic but can give rise to back pain and compressive neurologic symptoms. CASE DESCRIPTION: We report the case of a 51-year-old male who presented with gait difficulties over 5 months associated with occasional urge incontinence. Clinical examination revealed signs suggestive of thoracic myelopathy with bilateral lower limbs spasticity, decreased proprioception, and pinprick sensation. Magnetic resonance imaging showed a thoracic (T) 7-T9 extradural cystic lesion with an area of flow void on the right side between T8 and T9. A right hemilaminotomy was initially performed, and the dural defect was identified and repaired primarily. Unfortunately, there was a recurrence of the SEDC 2 weeks post operation and a T7-T9 laminoplasty with a complete excision was performed. CONCLUSIONS: Computed tomography myelography or magnetic resonance imaging flow study best visualizes the communication between the epidural cyst and subarachnoid space. The ideal surgical management for SEDC remains controversial. Our case suggests that there may be higher recurrence associated with fenestration of the SEDC and closure of the dural defect, but perhaps higher complications associated with complete excision. We present a case report and literature review of the terminology, presentation, recommended investigations, management, and outcomes of patients with SEDC.

Early Decompressive Hemicraniectomy for Malignant Middle Cerebral Artery Infarction in Asian Patients: A Single-Center Study.

INTRODUCTION: Early decompression craniectomy (within 48 hours of stroke onset) in acute and malignant middle cerebral artery (MCA) ischemic stroke (IS) reduces mortality and increases the proportion of patients with favorable functional outcome. Various cultural and social issues among Asians lead to some differences in clinical practice, especially when surgical interventions are involved. Accordingly, decompressive craniectomy in Asian patients with stroke is often delayed. MATERIALS AND METHODS: Data for all patients with acute IS hospitalized in our center were entered into a prospectively maintained registry. In this retrospective analysis, data for all patients with malignant MCA IS who underwent decompressive craniectomy were extracted. Various demographic, clinical, and neuroimaging factors were analyzed for identifying independent predictors of favorable functional outcome at 6 months, which was defined as modified Rankin Scale score of 0-3 points. RESULTS: From January 2005 to December 2014, a total of 75 patients with acute MCA IS underwent decompressive craniectomy. Median age was 55 years (interquartile range 44-64) with male preponderance (66%) and median National Institute of Health Stroke Scale score 21 points (interquartile range 18-24). A considerable proportion of these patients (38.7%) received intravenous thrombolysis. The majority (70%) of patients suffered right MCA IS, and decompressive surgery was performed within 48 hours of symptom onset in 50 (67%) of the patients. Favorable functional outcome was achieved in 25 (33.3%) patients at 6 months. Right MCA stroke (odds ratio 9.158; 95% confidence interval 1.881-44.596, P = 0.006) and early decompression surgery (odds ratio 4.011; 95% confidence interval 1.058-15.208, P = 0.041) were independent predictors of favorable functional outcome at 6 months. CONCLUSIONS: Early decompression craniectomy, especially in right MCA ischemic stroke, is associated with better favorable functional outcome.

HLA class I downregulation is associated with enhanced NK-cell killing of melanoma cells with acquired drug resistance to BRAF inhibitors.

The frequent development of drug resistance to targeted therapies in cancer patients has stimulated interest in strategies counteracting resistance. Combining immunotherapies with targeted therapies is one such strategy. In this context, we asked whether human NK cells can target melanoma cells that have acquired resistance to selective inhibitors targeting activating mutants of the B-Raf kinase (BRAF inhibitors, BRAFi). We generated drug-resistant cell variants in vitro from human BRAF-mutant melanoma cell lines MEL-HO, COLO-38, SK-MEL-37, 1520 and from primary melanoma cells freshly isolated from two patients. All drug-resistant cell variants remained susceptible to lysis by IL-2-activated NK cells; and two BRAFi-resistant lines (BRAFi-R) became significantly more susceptible to NK-cell lysis than their parental lines. This was associated with significant HLA class I antigen downregulation and PD-L1 upregulation on the drug-resistant lines. Although blocking HLA class I enhanced the extent of lysis of both BRAFi-R and parental cells to NK-cell-mediated lysis, antibody-mediated inhibition of PD1-PD-L1 interactions had no detectable effect. HLA class I antigen expression on BRAFi-R melanoma variants thus appears to play a major role in their susceptibility to NK-cell cytotoxicity. These findings suggest that NK-cell-based immunotherapy may be a viable approach to treat melanoma patients with acquired resistance to BRAF inhibitors.

Krüppel-like factor 15 activates hepatitis B virus gene expression and replication.

UNLABELLED: Hepatitis B virus (HBV) is a small DNA virus that requires cellular transcription factors for the expression of its genes. To understand the molecular mechanisms that regulate HBV gene expression, we conducted a yeast one-hybrid screen to identify novel cellular transcription factors that may control HBV gene expression. Here, we demonstrate that Krüppel-like factor 15 (KLF15), a liver-enriched transcription factor, can robustly activate HBV surface and core promoters. Mutations in the putative KLF15 binding site in the HBV core promoter abolished the ability of KLF15 to activate the core promoter in luciferase assays. Furthermore, the overexpression of KLF15 stimulated the expression of HBV surface antigen (HBsAg) and the core protein and enhanced viral replication. Conversely, small interfering RNA knockdown of the endogenous KLF15 in Huh7 cells resulted in a reduction in HBV surface- and core-promoter activities. In electrophoretic mobility shift and chromatin immunoprecipitation assays, KLF15 binds to DNA probes derived from the core promoter and the surface promoter. Introduction of an expression vector for KLF15 short hairpin RNA, together with the HBV genome into the mouse liver using hydrodynamic injection, resulted in a significant reduction in viral gene expression and DNA replication. Additionally, mutations in the KLF15 response element in the HBV core promoter significantly reduced viral DNA levels in the mouse serum. CONCLUSION: KLF15 is a novel transcriptional activator for HBV core and surface promoters. It is possible that KLF15 may serve as a potential therapeutic target to reduce HBV gene expression and viral replication.

Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling.

AIMS: Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. METHODS AND RESULTS: CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. CONCLUSION: We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: implications for prevention of skin cancer.

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component (280-320 nm), to human skin is the major cause of skin cancers. UV exposure also leads to the development of precancerous conditions such as actinic keratosis and elicits a variety of other adverse effects such as sunburn, inflammation, hyperplasia, immunosuppression and skin aging. Therefore, there is a need to intensify our efforts towards the development of novel mechanism-based approaches/agents for the protection of UVB-mediated damages. Chemoprevention is being investigated as a potential approach for the management of UV damages including skin cancer. We have earlier shown that sanguinarine, a benzophenanthridine alkaloid, inhibits UVB exposure-mediated damages in HaCaT keratinocytes. In this study, to determine the relevance of our in vitro findings to in vivo situations, we assessed the effects of sanguinarine on UVB-mediated damages in SKH-1 hairless mice. Our data demonstrated that a topical application of sanguinarine (5 micromol 0.3 mL(-1) ethanol per mouse), either as a pretreatment (30 min prior to UVB) or posttreatment (5 min after UVB), resulted in a significant decrease in UVB-mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Further, sanguinarine treatments (pre and post) also resulted in a significant decrease in UVB mediated (1) generation of H2O2 and (2) increases in the protein levels of markers of tumor promotion/proliferation viz. ornithine decarboxylase (ODC), proliferating cell nuclear antigen (PCNA) and Kiel antigen-67. Based on this data, we suggest that sanguinarine could be developed as an agent for the management of conditions elicited by UV exposure including skin cancer. However, further detailed studies are needed to support this suggestion.

Patch-test reactions to formaldehydes, bioban, and other formaldehyde releasers.

BACKGROUND: Contact allergy to formaldehyde, Bioban, and other formaldehyde releasers and cross-reactivity between them have been reported in the literature; however, not many studies have data on this cross-reactivity. OBJECTIVE: To study (1) the rates of allergy to formaldehyde and to Bioban and other formaldehyde releasers and (2) the rates of cross-reactivity between them. METHODS: We present a retrospective chart analysis of patch-test results for all patients referred for allergic contact dermatitis testing at the Milton S. Hershey Medical Center from June 2004 to September 2005. Anyone allergic to formaldehyde, Bioban, or other formaldehyde releasers was included. Cross-reactivity between the agents was then analyzed. RESULTS: The charts of 210 patients were analyzed. Of these patients, 24 (11%) were allergic to formaldehyde, Bioban, or other formaldehyde-releasing agents. Seventeen (8.1%) of the patients were allergic to formaldehyde, 15 (7.1%) were allergic to Bioban, and 20 (9.5%) were allergic to other formaldehyde-releasing agents. Eleven (65%) of the 17 formaldehyde-allergic patients were also allergic to Bioban. Of the 20 patients allergic to formaldehyde-releasing agents, 14 (70%) were also allergic to one of the three Bioban products tested. Of the 15 patients allergic to Bioban, 11 (73%) were allergic to formaldehyde, 14 (93%) were allergic to formaldehyde-releasing agents, and 11 (73%) were allergic to both formaldehyde and formaldehyde-releasing agents. CONCLUSION: A high cross-reactivity rate between formaldehyde, Bioban, and other formaldehyde-releasing agents was found.

Activation of hepatitis B virus S promoter by a cell type-restricted IRE1-dependent pathway induced by endoplasmic reticulum stress.

IRE1-alpha is an integral membrane protein of the endoplasmic reticulum (ER) that is a key sensor in the cellular transcriptional response to stress in the ER. Upon induction of ER stress, IRE1-alpha is activated, resulting in the synthesis of the active form of the transcription factor XBP1 via IRE1-mediated splicing of its mRNA. In this report, we have examined the role of IRE1-alpha and XBP1 in activation of the hepatitis B virus S promoter by ER stress. Cotransfection experiments revealed that overexpression of either IRE1-alpha or XBP1 activated this promoter. Conversely, cotransfected dominant-negative IRE1-alpha or small interfering RNA directed against XBP1 decreased the activation of the S promoter by ER stress, confirming an important role for the IRE1-alpha/XBP1 signaling pathway in activation of the S promoter. However, XBP1 does not bind directly to the S promoter; rather, a novel S promoter-binding complex that does not contain XBP1 is induced in cells undergoing ER stress in an XBP1-dependent manner. This complex, as well as transcriptional activation of the S promoter, is induced by ER stress in hepatocytes but not in fibroblasts, despite the presence of active XBP1 in the latter. Thus, the hepatitis B virus S promoter responds to a novel, cell type-restricted transcriptional pathway downstream of IRE1-alpha and XBP1.

Comparison of the effect of two different doses of 0.75% glucose-free ropivacaine for spinal anesthesia for lower limb and lower abdominal surgery.

We compared the clinical efficacy and safety of two doses of ropivacaine for spinal anesthesia in Chinese patients undergoing lower limb and lower abdominal surgery. In this randomized, open-label study, 40 patients were divided into two groups: group A received 3.5 mL (26.25 mg) of 0.75% glucose-free ropivacaine, and group B received 4.5 mL (33.75 mg). Sensory and motor blocks were assessed during and after surgery through to complete recovery. Seven standard measurements were taken: time to onset of sensory blocks; maximum sensory cephalad spread; time to maximum sensory block; maximum number of blocked segments; duration of sensory block at L3; time to onset of complete motor block; and duration until complete motor block recovery. Vital signs and any adverse effects related to spinal anesthesia were also recorded. No significant differences were found between the two groups: time to onset of sensory block at L3 in group A vs B (2.1 +/- 9.6 vs 1.7 +/- 7.3 minutes), maximum cephalad spread [T4-5 (C3-T11) vs T4 (C3-T8)], maximum number of blocked segments (18.0 +/- 3.4 vs 19.8 +/- 3.7), time to maximum sensory block (34.0 +/- 22.9 vs 26.8 +/- 17.9 minutes), duration of sensory block at L3 (251.2 +/- 34.7 vs 277.3 +/- 51.1 minutes), time to onset of complete motor block (13.4 +/- 6.4 vs 10.3 +/- 3.4 minutes), and time for complete recovery from motor block (264 +/- 52.1 vs 292.5 +/- 64.5 minutes). No significant differences in global hemodynamic changes were found during and after the operation. While shivering was more frequent in group B during the operation, the difference was not significant. Otherwise, there were no differences in adverse effects during and after surgery. We conclude that both doses of 0.75% glucose-free ropivacaine, 26.25 mg (3.5 mL) and 33.75 mg (4.5 mL), have the same efficacy and safety in Chinese patients undergoing spinal anesthesia for lower limb and lower abdominal surgery.

CCAAT/enhancer-binding protein beta (nuclear factor for interleukin 6) transactivates the human MDR1 gene by interaction with an inverted CCAAT box in human cancer cells.

We investigated the mechanisms of MDR1 gene activation by CCAAT/enhancer binding protein beta (C/EBPbeta, or nuclear factor for interleukin 6) in human cancer cells. Transfection of the breast cancer cell line MCF-7 and its doxorubicin-selected variant MCF-7/ADR by either C/EBPbeta or C/EBPbeta-LIP (a dominant-negative form of C/EBPbeta) confirmed their roles in the activation or repression of the endogenous, chromosomally embedded MDR1 gene. Cotransfection experiments with promoter constructs revealed a C/EBPbeta interaction on the MDR1 promoter via the region within -128 to -75. Deletions within the putative AP-1 box (-123 to -111) increased MDR1 promoter activity when stimulated by C/EBPbeta, suggesting that the AP-1 site negatively regulates MDR1 activation by C/EBPbeta. Mutations within the inverted CCAAT box (Y box) (-82 to -73) abolished the C/EBPbeta-stimulated MDR1 promoter activity, indicating that the Y box is required for MDR1 activation by C/EBPbeta. Chromatin immunoprecipitation (ChIP) revealed that C/EBPbeta precipitates a transcription complex containing C/EBPbeta, the MDR1 promoter sequences (-250 to +54), and the hBrm protein. In conclusion, alteration of expression or function of C/EBPbeta plays an important role in MDR1 gene regulation. C/EBPbeta activates the endogenous MDR1 gene of MCF-7 cells, and this activation was associated with a novel C/EBPbeta interaction region within the proximal MDR1 promoter (-128 to -75). The mechanisms of MDR1 activation by C/EBPbeta include C/EBPbeta binding of the chromatin of the MDR1 gene and interactions of C/EBPbeta with the Y box and Y box-associated proteins.

Spinal anesthesia with two different dosages of 0.75% glucose-free ropivacaine: a comparison of efficacy and safety in Chinese parturients undergoing cesarean section.

BACKGROUND: We compared the clinical efficacy and safety between 2 doses of 2.5 ml (18.75 mg) and 3 ml (22.5 mg) of 0.75% glucose free spinal ropivacaine in Chinese parturients undergoing Cesarean section. METHODS: In this randomized, open-label study, 40 parturients enrolled were divided into two groups: Group A received a 2.5 ml 0.75% ropivacaine as opposed to 3 ml in Group B. Sensory and motor blocks were assessed during and after surgery until complete recovery. Eight standard measurements were taken: time at onset of sensory block; maximum cephalic sensory spread; maximum number of blocked segments; time to maximum sensory block; duration of sensory block at L3; time at onset of complete motor block and duration until complete recovery. Vital signs and any adverse effects related to spinal anesthesia were also recorded. RESULTS: Five of the 6 variables showed no significant difference between groups A and B: onset time of sensory block at L3 was 1.8 +/- 6.7 min vs. 2.3 +/- 9.8 min; maximum cephalic spread was T3-4 (C3-T7) vs. T3 (C2-T8); maximum number of blocked segments was 20.7 +/- 3.5 vs. 20.2 +/- 3.4; time to maximum sensory block 15.4 +/- 5.5 min vs. 20.3 +/- 15.1 min; time for regression to L3 was 200.8 +/- 59.5 min vs. 215.0 +/- 37.6 min and time for complete recovery of motor block 208.5 +/- 55.5 min vs. 226.5 +/- 461. min. Group B had a significantly faster onset time for complete motor block (P < 0.05) 15.4 +/- 5.6 min vs. 10.4 +/- 4.7 min. Moreover, there were no significant differences in global hemodynamic changes during and after the operation. Transient hypotension attacks were more frequent in group A at the beginning of anesthesia, perhaps due to inadequate prehydration. Otherwise, there were no differences in adverse effects during or after surgery. CONCLUSIONS: We conclude that for Cesarean section in Chinese parturients either 18.75 mg (2.5 ml) or 22.5 mg (3 ml) 0.75% glucose-free ropivacaine can provide a spinal anesthesia of the same efficacy and safety.

p53 Binds and activates the xeroderma pigmentosum DDB2 gene in humans but not mice.

The DDB2 gene, which is mutated in xeroderma pigmentosum group E, enhances global genomic repair of cyclobutane pyrimidine dimers and suppresses UV-induced mutagenesis. Because DDB2 transcription increases after DNA damage in a p53-dependent manner, we searched for and found a region in the human DDB2 gene that binds and responds transcriptionally to p53. The corresponding region in the mouse DDB2 gene shared significant sequence identity with the human gene but was deficient for p53 binding and transcriptional activation. Furthermore, when mouse cells were exposed to UV, DDB2 transcription remained unchanged, despite the accumulation of p53 protein. These results demonstrate direct activation of the human DDB2 gene by p53. They also explain an important difference in DNA repair between humans and mice and show how mouse models can be improved to better reflect cancer susceptibility in humans.