Adaptive Prefetching for Device-Independent File I/O

Device independent I/O has been a holy grail to operating system designers since the early days of UNIX. Unfortunately, existing operating systems fall short of this goal for multimedia applications. Techniques such as caching and sequential read-ahead can help mask I/O latency in some cases, but in others they increase latency and add substantial jitter. Multimedia applications, such as video players, are sensitive to vagaries in performance since I/O latency and jitter affect the quality of presentation. Our solution uses adaptive prefetching to reduce both latency and jitter. Applications submit file access plans to the prefetcher, which then generates I/O requests to the operating system and manages the buffer cache to isolate the application from variations in device performance. Our experiments show device independence can be achieved: an MPEG video player sees the same latency when reading from a local disk or an NFS server. Moreover, our approach reduces jitter substantially

Predictable File Access Latency for Multimedia

Multimedia applications are sensitive to I/O latency and jitter when accessing data in secondary storage. Transparent adaptive prefetching (TAP) uses software feedback to provide multimedia applications with file system quality of service (QoS) guarantees. We are investigating how QoS requirements can be communicated and how they can be met by adaptive resource management. A preliminary test of adaptive prefetching is presented

Synthetic Files: Enabling Low-Latency File I/O for QoSAdaptive Applications

Files are a tried and true operating system abstraction. They offer a clean and simpl

Feedback-based Dynamic Proportion Allocation for Disk I/O

In this paper we propose to use feedback control to automatically allocate disk bandwidth in order to match the rate of disk I/O to the real-rate [13] needs of applications. We describe a model for adaptive resource management based on measuring the relative progress of stages in a producer-consumer pipeline. We show how to use prefetching to transform a passive disk into an active data producer whose progress can be controlled via feedback. Our progress-based framework allows the integrated control of multiple resources. The resulting system automatically adapts to varying application rates as well as to varying device latencies. 1 Introduction Real-rate applications [13] have specific disk I/O rate and throughput requirements that are driven by real-world demands. Examples of applications with real-rate disk I/O requirements include multimedia applications, real-time databases, and Internet servers. Real-rate applications suffer from uneven I/O throughput on conventional systems. To..

2009

ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n For instance, forced expression of the Fas regulating miR146a caused an immune disorder similar to ALPS in transgenic mice. Methods Study cohorts and DNA isolation Twenty-six ALPS patients, relatives and healthy controls were enrolled in the study. Written informed consent was obtained from all participants. Experiments were approved by the Ethical Review Boards of Hadassah, the Israeli Ministry of Health and the local Ethics committee of the University of Düsseldorf. Mononuclear cells were derived from peripheral blood by Ficoll (Biochrom, Berlin, Germany) density centrifugation. DNT cells were magnetically selected employing the double-negative T-cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). Genomic DNA was isolated from whole blood or DNT cells using the DNA blood kit (Qiagen, Hilden, Germany). Whole-exome sequencing and data analysis After exclusion of mutations in known ALPS-associated genes by targeted Sanger sequencing (Online Supplementary Methods, Online Supplementary Table S1) whole-exome sequencing was carried out as described elsewhere. Sequencing data were aligned against the human reference genome hg19 (GRCh37, statistics provided in Online 39 Single nucleotide variations, small insertions and deletions were annotated using Variant Effect Predictor 40 (based on Ensemble database v70). Variations were imported into a proprietary MySQL database driven workbench (termed Single Nucleotide Polymorphism Database, SNuPy). STRING 9.1 41 was used to identify high confidence (≥0.900) Fas pathway interaction partners (Online Primary T-cell culture Primary T cells were cultured in RPMI1640 (Life Technologies, Darmstadt, Germany) and Panserin 401 (PAN-Biotech, Aidenbach, Germany) mixed 1:1, supplemented with 10% fetal calf serum, 100 mg gentamycin (Life Technologies) and 30 U/mL IL2 (Miltenyi). They were activated with 7 mg/mL phytohemagglutinin (Life Technologies) for 4 days. Immunophenotyping and enzyme-linked immunosorbent assays DNT cells in peripheral blood were measured using a FACSCalibur equipped with CellQuestPro software (Becton Dickinson, BD, Heidelberg, Germany) employing anti-CD3, anti-TCRαβ (both from BD), anti-CD4 and anti-CD8 (both from Miltenyi) antibodies. Immunophenotyping was performed using: anti-B220, anti-HLA-DR, anti-CD27, anti-CD19, anti-CD25 (all from BD) and anti-CD45R (Beckman Coulter, Krefeld, Germany). Expression of Fas, FasL and IL12RB1 was measured using anti-CD95 (BD), anti-CD178/FasL (Miltenyi) and anti-CD212 antibodies (BD). FasL, IL10 and IFNγ levels in plasma and cell culture supernatants of activated T cells were measured by enzymelinked immunosorbent assays (R&D-Systems, Wiesbaden, Germany) employing an Infinite M200 microplate reader equipped with Magellan software (Tecan, Maennedorf, Switzerland). T cells were stimulated with IL12, IL23 and IL2/IL27 (Miltenyi) as indicated. Measurement of apoptosis Activated primary T cells were stimulated with recombinant SuperFasL (100 ng/mL, Enzo Life Sciences, Loerrach, Germany), 1 mM staurosporine (LC Laboratories, Woburn, MA, USA), IL12 Results Interleukin-12 induces upregulation of FasL and FasL-dependent apoptosis in healthy T cells, whereas FasL-deficient T cells from patients with autoimmune lymphoproliferative syndrome lack this response Of 26 analyzed ALPS cases, 20 had no known ALPSassociated mutation. Four patients harbored heterozygous germline mutations in the Fas receptor gene. Two siblings had a homozygous truncating FASLG mutation (g.172628545insT, p.P69Afs*75) that led to loss of FasL surface expression ( 1190 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n IL12 signaling may be protected against this physiological apoptosis trigger. Identification of a homozygous c.698G>A, p.R212* mutation in the IL12RB1 gene To analyze whether mutations in the IL12 pathways or related genes may cause a phenotype similar to ALPS, we sequenced the exomes of the remaining 20 ALPS-U patients who had classical ALPS symptoms without a known genetic cause, and their relatives. Sanger sequencing of all exons and exon/intron boundaries of FAS, FASLG and CASP10 was used to exclude classical disease-causing germline or somatic mutations. By whole-exome sequencing we identified an IL12RB1 mutation in one of the ALPS families. A KEGG-based protein interaction analysis interface of our in-house developed proprietary MySQL database driven workbench (termed Single Nucleotide Polymorphism Database, SNuPy) gave this candidate disease-causing mutation highest priority because of its predicted interaction with FasL ( As the mutated sequence seemed to encode a truncated protein that lacks the transmembrane domain necessary for membrane anchorage, we carried out FACS analyses of the patient's lymphocytes to test for IL12RB1 expression on the cell surface. To this end, primary lymphocytes from the patient and healthy individuals were stimulated for 4 days with phytohemagglutinin/IL2. Whereas lympho- + cells specifically induced in IL12-treated compared to untreated cells is shown. (C) Cultivated patient's T cells are resistant to apoptosis induced by stimulation with IL12. T cells were activated as in (A). Apoptosis was induced by incubation with 100 ng/mL IL12 for 2 days and measured employing flow cytometric detection of annexin V-FITC and propidium iodide. The difference in the apoptosis rate compared to that of an untreated control is depicted. Specific apoptosis ranged from 4-12% in the healthy controls between comparable experiments and was absent in the patient's cells. (D) T cells were activated by phytohemagglutinin /IL2 treatment as described in (A). Fas receptor-mediated apoptosis was triggered by application of 100 ng/mL optimized and preoligomerized recombinant FasL for 16 h and apoptosis was measured as in (C). In (B-D) mean values and standard deviations of representative experiments repeated at least three times and carried out in duplicate are shown. Similar results were obtained using samples from two individuals with homozygous FASLG (g.172628545insT, p.P69Afs*75) mutation and five wild-type controls. A B C D © F e r r a t a S t o r t i F o u n d a t i o n cytes from healthy individuals upregulated IL12RB1 surface expression upon activation, IL12RB1 remained absent in the patient's lymphocytes ( The homozygous c.698G>A, p.R212* mutation in the IL12RB1 gene was associated with an autoimmune lymphoploliferative syndrome-like phenotype The male patient harboring the homozygous IL12RB1 c.698G>A, p.R212* mutation originated from a consanguineous family of Palestinian descent. He was referred in 1996 at the age of 4 years because of the suspicion of lymphoma. In the follow up of this patient for more than 16 years he presented with classical clinical features for the diagnosis of ALPS (Table 1, The heterozygous parents and siblings appeared clinically normal, although immune phenotyping revealed increased DNT cell counts in two of them In vitro the apoptotic response of the patient's lymphocytes to treatment with recombinant FasL and a classical apoptosis-inducing agent (staurosporine) was similar to that of age-and gender-matched healthy blood donors [FasL-induced apoptosis: 58% ± 5% (patient), 57% ± 4% (healthy control); staurosporine-induced apoptosis: 86% ± 6% (patient), 86% ± 7% (healthy control)]. This demon- S. Nabhani et al. 1192 haematologica | 2015; 100(9) To analyze whether the defect in IL12RB1 affects FasL signaling, we first tested protein expression of FasL. Activation and expansion of T cells usually leads to upregulation of Fas signaling pathway components. However, in the absence of IL12RB1 expression the patient's T lymphocytes showed a significantly lower expression of both membrane-bound and soluble FasL protein compared to that of healthy controls ( The IL12RB1 c.698G>A, p.R212* mutation abrogates responsiveness of T cells to interleukin-12 To test whether lack of IL12RB1 expression affects IL12 signaling we analyzed phosphorylation of STAT4, a crucial downstream component of the pathway STAT4 activation eventually leads to transcription and production of IFNg. To test whether this is deficient in the patient we measured the induction of IFNG transcription by quantitative real-time polymerase chain reaction after 24 and 48 h of treatment with IL12 ( Finally, we tested the responsiveness of the patient's primary lymphocytes to apoptosis mediated by prolonged IL12 treatment ( Discussion Although ALPS is frequently caused by mutations in known genes, such as FAS, FASLG or CASP10, in 20-30% of cases the defect is still unknown. It is highly likely that defects in or overexpression of regulators of these genes such as miR-146a It might be reasoned that the observed autoimmunity and lymphoproliferation are likely a side-effect of recurrent infections, because IL12RB1 mutations have previously been associated with a predisposition to mycobacterial infections. However, the patient experienced only one non-recurrent episode of infection with Salmonella in 16 years of follow-up arguing against a secondary effect. Consistently, it was recently demonstrated, employing an IL12RB2 knockout mice model, that lack of IL12 signaling predisposes to spontaneous lymphoproliferation, autoimmunity and B-cell lymphoma. 45 A similar phenotype is described for targeted IL12RB1 knockout mice. Heterozygous human carriers appear clinically normal with normal IL12/IL23 signaling and IFNg production. Our study demonstrates for the first time that loss of IL12RB1 expression in a patient leads to reduced upregu- S. Nabhani et al. 1194 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n Deregulation of FasL as a cause of ALPS-like disease haematologica | 2015; 100(9) 1195 Densitometric measurement of signals derived by the western blot in the left panel carried out on a LAS-3000 equipped with LAS-3000 Image Reader software (Fujifilm, Düsseldorf, Germany). The difference of relative arbitrary units of pSTAT4 expression related to STAT4 expression is shown. The β-actin control was used to compensate differences due to loading. A representative result of two independent experiments is shown. A B C D E © F e r r a t a S t o r t i F o u n d a t i o n lation of FasL and loss of the apoptotic response of T cells to prolonged treatment with IL12. In addition, the general level of FasL expression in activated T cells and the level of secreted FasL in the plasma or cell culture supernatant were much lower in the patient than in controls. Lower levels of FasL expression in the patient are probably attributable to a lack of IFNg, because transcription of the FASLG promoter is positively regulated by the interferonregulatory factors IRF-1 and IRF-2. 48 IL12 is known as a factor that can stimulate growth and function of T cells and the differentiation of naive T cells into Th1 cells. However, prolonged stimulation of T cells with IL12 leads to apoptosis and stimulation of T cells with IL12 during activation enhances the tendency to undergo Fas-mediated activation induced cell death due to upregulation of FasL and downregulation of the inhibitor FLIPs. 27 Therefore, similar to defective Fas/FasL signaling, absence of IL12 signaling could lead to decreased death of T cells and accumulation of autoreactive T cells. In addition, it has been shown that low levels of IL12 drive the differentiation of activated T cells to long-lived selfrenewing memory CD8 + T cells rather than to short-lived effector cells when acute infections resolve. 49 This is dependent on IL12-controlled expression of T-bet and reflected in the phenotype of T-bet knockout mice. Acknowledgment

Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20–30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease