High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs

Evaluation of ferritin and TfR level in plasma neural-derived exosomes as potential markers of Parkinson’s disease

IntroductionEarly diagnosis of Parkinson’s disease (PD) remains challenging. It has been suggested that abnormal brain iron metabolism leads to excessive iron accumulation in PD, although the mechanism of iron deposition is not yet fully understood. Ferritin and transferrin receptor (TfR) are involved in iron metabolism, and the exosome pathway is one mechanism by which ferritin is transported and regulated. While the blood of healthy animals contains a plentiful supply of TfR-positive exosomes, no studies have examined ferritin and TfR in plasma neural-derived exosomes.MethodsPlasma exosomes were obtained from 43 patients with PD and 34 healthy controls. Neural-derived exosomes were isolated with anti-human L1CAM antibody immunoabsorption. Transmission electron microscopy and western blotting were used to identify the exosomes. ELISAs were used to quantify ferritin and TfR levels in plasma neural-derived exosomes of patients with PD and controls. Receivers operating characteristic (ROC) curves were applied to map the diagnostic accuracy of ferritin and TfR. Independent predictors of the disease were identified using logistic regression models.ResultsNeural-derived exosomes exhibited the typical exosomal morphology and expressed the specific exosome marker CD63. Ferritin and TfR levels in plasma neural-derived exosomes were significantly higher in patients with PD than controls (406.46 ± 241.86 vs. 245.62 ± 165.47 ng/μg, P = 0.001 and 1728.94 ± 766.71 vs. 1153.92 ± 539.30 ng/μg, P < 0.001, respectively). There were significant positive correlations between ferritin and TfR levels in plasma neural-derived exosomes in control group, PD group and all the individuals (rs = 0.744, 0.700, and 0.752, respectively). The level of TfR was independently associated with the disease (adjusted odds ratio 1.002; 95% CI 1.000–1.003). ROC performances of ferritin, TfR, and their combination were moderate (0.730, 0.812, and 0.808, respectively). However, no relationship was found between the biomarkers and disease progression.ConclusionIt is hypothesized that ferritin and TfR in plasma neural-derived exosomes may be potential biomarkers for PD, and that they may participate in the mechanism of excessive iron deposition in PD

FEATURES: Real-time Adaptive Feature Learning and Document Learning for Web Search

In this paper we report our research on building FEATURES - an intelligent web search engine that is able to perform real-time adaptive feature (i.e., keyword) and document learning. Not only does FEATURES learn from the user's document relevance feedback, but also automatically extracts and suggests indexing keywords relevant to a search query and learns from the user's keyword relevance feedback so that it is able to speed up its search process and to enhance its search performance. We design two efficient and mutual-benefiting learning algorithms that work concurrently, one for feature learning and the other for document learning. FEATURES employs these algorithms together with an internal index database and a real-time meta-searcher so to perform adaptive real-time learning to find desired documents with as little relevance feedback from the user as possible. The architecture and performance of FEATURES are also discussed

Effects of electrospun membrane surface morphology on cellular behaviours and osteogenesis of bone marrow mesenchymal stem cells

Electrospun membranes are widely used in bone tissue engineering because of their similar bone extracellular matrix. The morphological characteristics of electrospun membranes, which include fibre diameter and alignment, play crucial roles in determining cellular behaviour and osteogenesis. Therefore, to investigate the effects of these two parameters on bone marrow mesenchymal stem cells (BMSCs), we prepared electrospun poly-L-lactic acid membranes using different diameters (nanoscale and microscale) and alignments (aligned and random) to investigate the effects of different surface morphologies on the proliferation, adhesion, migration, cell morphology, and osteogenesis of BMSCs. Our results showed that electrospun membranes with different surface morphologies have good biocompatibility and can regulate cell morphology, and the parallel aligned fibre orientation can promote cell migration. More importantly, BMSCs cultured on aligned nanofibres have a higher osteogenic potential than aligned microfibres and random fibres. Furthermore, our study shows that the surface morphology of electrospun membranes, which is one of the characteristics of biomaterials, can regulate the cellular behaviour of BMSCs, and that aligned nanofibre electrospun membranes can contribute to promoting osteogenesis, which can be used as the surface morphology of bone repair materials

<i>JAK2^V617F</i> Drives Mcl-1 Expression and Sensitizes Hematologic Cell Lines to Dual Inhibition of JAK2 and Bcl-xL

<div><p>Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major <i>JAK2</i> mutation observed in these diseases (<i>JAK2^V617F</i>) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in <i>JAK2^V617F</i>-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the <i>JAK2^V617F</i> mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating <i>JAK2</i> mutations.</p></div

Regulation of Mcl-1 and Bcl-XL by <i>JAK2</i>^<i>V617F</i>.

<p>(A) JAKi-I was evaluated in a panel of 66 human protein kinases as detailed in the Methods section, and Ki values determined. Red, <0.01 μM; black, 0.01–1.67 μM, green, >1.67 μM. (B) UKE-1 (<i>JAK2</i>^<i>V617F</i>) AML cells were treated for 10 min with JAKi-I as indicated. Tyrosine phoshorylation of STAT3 and STAT5 was determined by immunoblotting. (C) The <i>JAK2</i>^<i>V617F</i>-positive AML cell lines, SET-2, UKE-1, and HEL, the chronic myelogenous leukemia line, K562 (<i>JAK2</i>^<i>V617F</i>-negative), and the AML cell line, MV4;11 (<i>JAK2</i>^<i>V617F</i>-negative), were cultured in the absence of serum for 2 hr, then treated with 1 μM JAKi-I for 1 hr. Constitutive tyrosine phosphorylation of STAT3 and STAT5 was determined by immunoblotting. (D and E) Cells were treated for 6 hr with JAKi-I, and the abundance of Mcl-1 and Bcl-XL mRNA was determined by qPCR. Data represent means +/- standard deviation for two independent determinations each performed in triplicate. (F) Cells were treated with JAKi-I or Ruxolitinib over a 24-hr time course, and Mcl-1 and Bcl-XL levels were determined by immunoblotting (similar results were observed for 2 separate immuoblots). (G) Quantification of the data shown in (F). Data are expressed as the ratio of intensity of Mcl-1/β-actin for each time point. (H and I) HEL or K562 cells were transfected with either non-targeting (siNT-1) or Mcl-1-specific (siMcl1–1–4) siRNAs, treated for 72 hr with ABT-263, then lysates were prepared, and cell viability was determined. Data are means of duplicate samples and are representative of two independent experiments. (J) Cells were treated for 6 hr with or without 1 μM JAKi-I then subjected chromatin immunoprecipitation assays using normal mouse IgG, anti-acetylated histone H3, or anti-STAT3. Mcl-1 promoter binding was determined by PCR on chromatin immunoprecipitates (for <u>immunoblots</u>, similar results were obtained twice).</p

Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in <i>JAK2</i>^<i>V617F</i>-harboring AML cell lines.

<p>(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity¹⁶ to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.</p

Smoking, acute mountain sickness and altitude acclimatisation: a cohort study

RATIONALE: The relationship between cigarette smoking and acute mountain sickness (AMS) is not clear. OBJECTIVE: To assess AMS risk and altitude acclimatisation in relation to smoking. METHODS: 200 healthy non-smokers and 182 cigarette smokers were recruited from Han lowland workers. These were men without prior altitude exposure, matched for age, health status and occupation, who were transported to an altitude of 4525 masl. MEASUREMENTS: AMS, smoking habits, arterial saturation (SpO2), haemoglobin (Hb), lung function and mean pulmonary artery pressure (PAPm) were assessed upon arrival and after 3 and 6 months. MAIN RESULTS: Compared with non-smokers, smokers had a lower incidence of AMS and lower AMS scores than non-smokers upon arrival; higher Hb and PAPm associated with lower SpO2 at 3 and 6 months at altitude; and lower forced expiratory volume in 1 s and maximal voluntary ventilation at 3 and 6 months. CONCLUSIONS: Smoking slightly decreases the risk of AMS but impairs long-term altitude acclimatisation and lung function during a prolonged stay at high altitude

Reduced incidence and severity of acute mountain sickness in Qinghai-Tibet railroad construction workers after repeated 7-month exposures despite 5-month low altitude periods

The construction of the Qinghai-Tibet railroad provided a unique opportunity to study the relation between intermittent altitude exposure and acute mountain sickness (AMS). For 5 yr, workers spent 7-month periods at altitude interspaced with 5-month periods at sea level; the incidence, severity, and risk factors of AMS were prospectively investigated. Six hundred lowlanders commuted for 5 yr between near sea level and approximately 4500 m and were compared to 600 other lowland workers, recruited each year upon their first ascent to high altitude as newcomers, and to 200 Tibetan workers native to approximately 4500 m. AMS was assessed with the Lake Louise Scoring System. The incidence and severity of AMS in commuters were lower upon each subsequent exposure, whereas they remained similar in newcomers each year. AMS susceptibility was thus lowered by repeated exposure to altitude. Repeated exposure increased resting Sao(2) and decreased resting heart rate. Tibetans had no AMS, higher Sao(2), and lower heart rates. In conclusion, repetitive 7-month exposures increasingly protect lowlanders against AMS, even when interspaced with 5-month periods spent at low altitude, but do not allow attaining the level of adaptation of altitude natives