A rapid and scalable system for studying gene function in mice using conditional RNA interference.

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:

Small RNAs as guardians of the genome.

Transposons populate the landscape of all eukaryotic genomes. Often considered purely genomic parasites, transposons can also benefit their hosts, playing roles in gene regulation and in genome organization and evolution. Peaceful coexistence with mobile elements depends upon adaptive control mechanisms, since unchecked transposon activity can impact long-term fitness and acutely reduce the fertility of progeny. Here, we review the conserved roles played by small RNAs in the adaptation of eukaryotes to coexist with their genomic colonists. An understanding of transposon-defense pathways has uncovered recurring themes in the mechanisms by which genomes distinguish "self" from "non-self" and selectively silence the latter.

Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary.

In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.

piRNA-mediated regulation of transposon alternative splicing in the soma and germ line.

Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. Here we show that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, we show that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a (HP1a; encoded by Su(var)205). Furthermore, we show that this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. Our findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development.

Diagnostic Utility of Exome Sequencing for Kidney Disease.

BACKGROUND: Exome sequencing is emerging as a first-line diagnostic method in some clinical disciplines, but its usefulness has yet to be examined for most constitutional disorders in adults, including chronic kidney disease, which affects more than 1 in 10 persons globally. METHODS: We conducted exome sequencing and diagnostic analysis in two cohorts totaling 3315 patients with chronic kidney disease. We assessed the diagnostic yield and, among the patients for whom detailed clinical data were available, the clinical implications of diagnostic and other medically relevant findings. RESULTS: In all, 3037 patients (91.6%) were over 21 years of age, and 1179 (35.6%) were of self-identified non-European ancestry. We detected diagnostic variants in 307 of the 3315 patients (9.3%), encompassing 66 different monogenic disorders. Of the disorders detected, 39 (59%) were found in only a single patient. Diagnostic variants were detected across all clinically defined categories, including congenital or cystic renal disease (127 of 531 patients [23.9%]) and nephropathy of unknown origin (48 of 281 patients [17.1%]). Of the 2187 patients assessed, 34 (1.6%) had genetic findings for medically actionable disorders that, although unrelated to their nephropathy, would also lead to subspecialty referral and inform renal management. CONCLUSIONS: Exome sequencing in a combined cohort of more than 3000 patients with chronic kidney disease yielded a genetic diagnosis in just under 10% of cases. (Funded by the National Institutes of Health and others.).

The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript.

The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.

Out of reach? Correlates of cervical cancer underscreening in women with varying levels of healthcare interactions in a United States integrated delivery system.

One in five U.S. women with health insurance are underscreened for cervical cancer. We sought to identify whether underscreening correlates differed among women with different levels of health care interaction. Among women age 30-64 years who were members of an integrated U.S. health system, we used 2014-2015 electronic health record data to identify underscreened cases (≥3.4 years since last Papanicolaou (Pap) test, n=3352) and screening-adherent controls (<3.4 years since last Pap test, n=45,359) and extracted data on potential underscreening correlates (demographics, health history, and healthcare utilization). We calculated the odds of underscreening in the total population and by subgroups defined by healthcare visits and online health portal usage in the prior 12 months. Underscreening was associated with older age (50-64 vs. 30-39; odds ratio (OR)=1.6; 95%CI=1.4-1.8), current tobacco use (vs. never use; OR=2.1; 95%CI=1.8-2.2), higher BMI (≥35 kg/m2 vs <25 kg/m2, OR=2.0; 95%CI=1.8-2.3), screening non-adherence for colorectal cancer (OR=5.1; 95%CI=4.6-5.7) and breast cancer (OR=8.1, 95%CI=7.2-9.0), and having no recent visit with their primary care provider (PCP) nor recent health portal use (vs. recent PCP visit and portal use; OR=8.4, 95%CI=7.6-9.4). Underscreening correlates were similar between the total study population and within all healthcare interaction groups. Interaction with the healthcare system is associated with lower odds of underscreening, but sociodemographic and health status correlates are similar regardless of primary care visits or online portal use. These data support the need for additional interventions to reach insured women who remain underscreened for cervical cancer.

Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.

BACKGROUND: Identification of chromosomal aneuploidies and copy number variants that are associated with fetal structural anomalies has substantial value. Although whole-exome sequencing (WES) has been applied to case series of a few selected prenatal cases, its value in routine clinical settings has not been prospectively assessed in a large unselected cohort of fetuses with structural anomalies. We therefore aimed to determine the incremental diagnostic yield (ie, the added value) of WES following uninformative results of standard investigations with karyotype testing and chromosomal microarray in an unselected cohort of sequential pregnancies showing fetal structural anomalies. METHODS: In this prospective cohort study, the parents of fetuses who were found to have a structural anomaly in a prenatal ultrasound were screened for possible participation in the study. These participants were predominantly identified in or were referred to the Columbia University Carmen and John Thain Center for Prenatal Pediatrics (New York, NY, USA). Fetuses with confirmed aneuploidy or a causal pathogenic copy number variant were excluded from WES analyses. By use of WES of the fetuses and parents (parent-fetus trios), we identified genetic variants that indicated an underlying cause (diagnostic genetic variants) and genetic variants that met the criteria of bioinformatic signatures that had previously been described to be significantly enriched among diagnostic genetic variants. FINDINGS: Between April 24, 2015, and April 19, 2017, 517 sequentially identified pregnant women found to have fetuses with a structural anomaly were screened for their eligibility for inclusion in our study. 71 (14%) couples declined testing, 87 (17%) trios were missing at least one DNA sample (from either parent or the fetus), 69 (13%) trios had a clinically relevant abnormal karyotype or chromosomal microarray finding, 51 (10%) couples did not consent to WES or withdrew consent, and five (1%) samples were not of good enough quality for analysis. DNA samples from 234 (45%) eligible trios were therefore used for analysis of the primary outcome. By use of trio sequence data, we identified diagnostic genetic variants in 24 (10%) families. Mutations with bioinformatic signatures that were indicative of pathogenicity but with insufficient evidence to be considered diagnostic were also evaluated; 46 (20%) of the 234 fetuses assessed were found to have such signatures. INTERPRETATION: Our analysis of WES data in a prospective cohort of unselected fetuses with structural anomalies shows the value added by WES following the use of routine genetic tests. Our findings suggest that, in cases of fetal anomalies in which assessment with karyotype testing and chromosomal microarray fail to determine the underlying cause of a structural anomaly, WES can add clinically relevant information that could assist current management of a pregnancy. The unique challenges of WES-based prenatal diagnostics require analysis by a multidisciplinary team of perinatal practitioners and laboratory specialists. FUNDING: Institute for Genomic Medicine (Columbia University Irving Medical Center).

Cost-effectiveness studies of HPV self-sampling: A systematic review.

HPV self-sampling (HPV-SS) can increase cervical cancer screening participation by addressing barriers in high- and low- and middle-income settings. Successful implementation of HPV-SS programs will depend on understanding potential costs and health effects. Our objectives were to summarize the methods and results of published HPV-SS cost and cost-effectiveness studies, present implications of these results for HPV-SS program implementation, and identify knowledge gaps. We followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. One reviewer searched online databases for articles published through June 12, 2019, identified eligible studies, and extracted data; a second reviewer checked extracted data for accuracy. Eligible studies used an economic model to compare HPV-SS outreach strategies to standard-of-care tests. Of 16 eligible studies, 14 reported HPV-SS could be a cost-effective strategy. Studies differed in model type, HPV-SS delivery methods, triage strategies for positive results, and target populations. Most (9/16) modeled HPV-SS in European screening programs, 6/16 targeted women who were underscreened for cervical cancer, and 5/16 modeled HPV-SS in low- and middle-income countries. The most commonly identified driver of HPV-SS cost-effectiveness was the level of increase in cervical cancer screening attendance. Lower HPV-SS material and testing costs, higher sensitivity to detect cervical precancer, and longer duration of underscreening among HPV-SS users were also associated with increased cost-effectiveness. Future HPV-SS models in high-income settings should explore the effect of widespread vaccination and new triage strategies such as partial HPV genotyping. Knowledge gaps remain about the cost-effectiveness of HPV-SS in low- and middle-income settings.

Somatic SLC35A2 variants in the brain are associated with intractable neocortical epilepsy.

OBJECTIVE: Somatic variants are a recognized cause of epilepsy-associated focal malformations of cortical development (MCD). We hypothesized that somatic variants may underlie a wider range of focal epilepsy, including nonlesional focal epilepsy (NLFE). Through genetic analysis of brain tissue, we evaluated the role of somatic variation in focal epilepsy with and without MCD. METHODS: We identified somatic variants through high-depth exome and ultra-high-depth candidate gene sequencing of DNA from epilepsy surgery specimens and leukocytes from 18 individuals with NLFE and 38 with focal MCD. RESULTS: We observed somatic variants in 5 cases in SLC35A2, a gene associated with glycosylation defects and rare X-linked epileptic encephalopathies. Nonsynonymous variants in SLC35A2 were detected in resected brain, and absent from leukocytes, in 3 of 18 individuals (17%) with NLFE, 1 female and 2 males, with variant allele frequencies (VAFs) in brain-derived DNA of 2 to 14%. Pathologic evaluation revealed focal cortical dysplasia type Ia (FCD1a) in 2 of the 3 NLFE cases. In the MCD cohort, nonsynonymous variants in SCL35A2 were detected in the brains of 2 males with intractable epilepsy, developmental delay, and magnetic resonance imaging suggesting FCD, with VAFs of 19 to 53%; Evidence for FCD was not observed in either brain tissue specimen. INTERPRETATION: We report somatic variants in SLC35A2 as an explanation for a substantial fraction of NLFE, a largely unexplained condition, as well as focal MCD, previously shown to result from somatic mutation but until now only in PI3K-AKT-mTOR pathway genes. Collectively, our findings suggest a larger role than previously recognized for glycosylation defects in the intractable epilepsies. Ann Neurol 2018.

Functional identification of optimized RNAi triggers using a massively parallel sensor assay.

Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in nine mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides insights into sequence requirements of effective RNAi.

An Exome Sequencing Study to Assess the Role of Rare Genetic Variation in Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an increasingly recognized, often fatal lung disease of unknown etiology. OBJECTIVES: The aim of this study was to use whole-exome sequencing to improve understanding of the genetic architecture of pulmonary fibrosis. METHODS: We performed a case-control exome-wide collapsing analysis including 262 unrelated individuals with pulmonary fibrosis clinically classified as IPF according to American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association guidelines (81.3%), usual interstitial pneumonia secondary to autoimmune conditions (11.5%), or fibrosing nonspecific interstitial pneumonia (7.2%). The majority (87%) of case subjects reported no family history of pulmonary fibrosis. MEASUREMENTS AND MAIN RESULTS: We searched 18,668 protein-coding genes for an excess of rare deleterious genetic variation using whole-exome sequence data from 262 case subjects with pulmonary fibrosis and 4,141 control subjects drawn from among a set of individuals of European ancestry. Comparing genetic variation across 18,668 protein-coding genes, we found a study-wide significant (P < 4.5 × 10-7) case enrichment of qualifying variants in TERT, RTEL1, and PARN. A model qualifying ultrarare, deleterious, nonsynonymous variants implicated TERT and RTEL1, and a model specifically qualifying loss-of-function variants implicated RTEL1 and PARN. A subanalysis of 186 case subjects with sporadic IPF confirmed TERT, RTEL1, and PARN as study-wide significant contributors to sporadic IPF. Collectively, 11.3% of case subjects with sporadic IPF carried a qualifying variant in one of these three genes compared with the 0.3% carrier rate observed among control subjects (odds ratio, 47.7; 95% confidence interval, 21.5-111.6; P = 5.5 × 10-22). CONCLUSIONS: We identified TERT, RTEL1, and PARN-three telomere-related genes previously implicated in familial pulmonary fibrosis-as significant contributors to sporadic IPF. These results support the idea that telomere dysfunction is involved in IPF pathogenesis.

Sequence, biogenesis, and function of diverse small RNA classes bound to the Piwi family proteins of Tetrahymena thermophila.

PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23- to 24-nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether, Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length, while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs.

Evidence for the involvement of gamma delta T cells in the immune response in Rasmussen encephalitis.

BACKGROUND: Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) and brain-infiltrating lymphocytes (BILs) were isolated from 20 RE surgery specimens by standard methods, and CD3(+) T cell populations were analyzed by flow cytometry. Gamma delta T cell receptor spectratyping was carried out by nested PCR of reversed transcribed RNA extracted from RE brain tissue, followed by high resolution capillary electrophoresis. A MiSeq DNA sequencing platform was used to sequence the third complementarity determining region (CDR3) of δ1 chains. RESULTS: CD3(+) BILs from all of the RE brain specimens comprised both αß and γδ T cells. The median αß:γδ ratio was 1.9 (range 0.58-5.2) compared with a median ratio of 7.7 (range 2.7-40.8) in peripheral blood from the same patients. The αß T cells isolated from brain tissue were predominantly CD8(+), and the majority of γδ T cells were CD4(-) CD8(-). Staining for the early activation marker CD69 showed that a fraction of the αß and γδ T cells in the BILs were activated (median 42%; range 13-91%, and median 47%; range 14-99%, respectively). Spectratyping T cell receptor (TCR) Vδ1-3 chains from 14 of the RE brain tissue specimens indicated that the γδ T cell repertoire was relatively restricted. Sequencing δ1 chain PCR fragments revealed that the same prevalent CDR3 sequences were found in all of the brain specimens. These CDR3 sequences were also detected in brain tissue from 15 focal cortical dysplasia (FCD) cases. CONCLUSION: Neuroinflammation in RE involves both activated αß and γδ T cells. The presence of γδ T cells with identical TCR δ1 chain CDR3 sequences in all of the brain specimens examined suggests that a non-major histocompatibility complex (MHC)-restricted immune response to the same antigen(s) is involved in the etiology of RE. The presence of the same δ1 clones in CD brain implies the involvement of a common inflammatory pathway in both diseases.

Minotaur is critical for primary piRNA biogenesis.

Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur.

Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila.

In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. Here, we identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. We show that Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism that is absent in other fly tissues. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub- and Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi-Aub complexes and piRNAs engaged in the amplification cycle. We propose that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing.

An endogenous small interfering RNA pathway in Drosophila.

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.

CyberKnife for inoperable renal tumors: Canadian pioneering experience.

INTRODUCTION: Stereotactic ablative body radiotherapy (SABR) is currently under study regarding its clinical application in management of patients with kidney tumors. CyberKnife can accurately deliver ablative tumor radiation doses while preserving kidney function. We report Canada's first use of CyberKnife SABR system in treating primary kidney tumors. MATERIALS AND METHODS: Between January 2011 and February 2012, we treated three patients with renal tumors using CyberKnife SABR. Two patients had tumors in solitary kidney. The third patient had a recurrent tumor after two previous radiofrequency ablation treatments. Platinum seed fiducials were used for real time tumor tracking. Magnetic resonance imaging registration was used for tumor delineation in all cases. The patients were followed with regular renal scans and renal function tests. RESULTS: The mean age was 79 years. Mean tumor size was 21.3 cm3. A dose of 39 Gy in 3 fractions was delivered. The post treatment follow up times were 15 months, 13 months and 12 months. Local control was obtained in all three patients. No acute or chronic toxicity was reported. Kidney functions remained unaffected after treatment. CONCLUSION: CyberKnife is technically feasible for treatment of medically inoperable renal tumors or tumors in a solitary kidney.

Efficacy of extended microthrombolysis in an elderly lady with purpura fulminans.

Purpura fulminans is a thrombotic emergency affecting small vessels of skin and of internal organs that may rapidly progress into necrotizing fasciitis, critical limb ischaemia and multiorgan failure; it often develops during an infection or as a postinfective 'autoimmune' disorder. Although supportive care and hydration are important, anticoagulation ought to be started to prevent further occlusions alongside blood products according to need. Herein, we describe the case of an elderly woman who received extended intravenous low-dose recombinant tissue plasminogen activator at the onset of purpura fulminans that salvaged her skin and prevented the development of multiorgan failure.