Urinary Plasminogen as a Marker of Disease Progression in Human Glomerular Disease.

RATIONALE & OBJECTIVE: Glomerular disorders have a highly variable clinical course, and biomarkers that reflect the molecular mechanisms underlying their progression are needed. Based on our previous work identifying plasminogen as a direct cause of podocyte injury, we designed this study to test the association between urine plasmin(ogen) (ie, plasmin and its precursor plasminogen) and end-stage kidney disease (ESKD). STUDY DESIGN: Multicenter cohort study. SETTING & PARTICIPANTS: 1,010 patients enrolled in the CureGN Cohort with biopsy-proven glomerular disease (focal segmental glomerulosclerosis, membranous nephropathy, and immunoglobulin A nephropathy). PREDICTORS: The main predictor was urine plasmin(ogen) at baseline. Levels were measured by an electrochemiluminescent immunoassay developed de novo. Traditional clinical and analytical characteristics were used for adjustment. The ratio of urine plasmin(ogen)/expected plasmin(ogen) was evaluated as a predictor in a separate model. OUTCOME: Progression to ESKD. ANALYTICAL APPROACH: Cox regression was used to examine the association between urinary plasmin(ogen) and time to ESKD. Urinary markers were log2 transformed to approximate normal distribution and normalized to urinary creatinine (Log2uPlasminogen/cr, Log2 urinary protein/cr [UPCR]). Expected plasmin(ogen) was calculated by multiple linear regression. RESULTS: Adjusted Log2uPlasminogen/cr was significantly associated with ESKD (HR per doubling Log2 uPlasminogen/cr 1.31 [95% CI, 1.22-1.40], P<0.001). Comparison of the predictive performance of the models including Log2 uPlasminogen/cr, Log2 UPCR, or both markers showed the plasmin(ogen) model superiority. The ratio of measured/expected urine plasmin(ogen) was independently associated with ESKD: HR, 0.41 (95% CI, 0.22-0.77) if ratio<0.8 and HR 2.42 (95% CI, 1.54-3.78) if ratio>1.1 (compared with ratio between 0.8 and 1.1). LIMITATIONS: Single plasmin(ogen) determination does not allow for the study of changes over time. The use of a cohort of mostly white patients and the restriction to patients with 3 glomerular disorders limits the external validity of our analysis. CONCLUSIONS: Urinary plasmin(ogen) and the ratio of measured/expected plasmin(ogen) are independently associated with ESKD in a cohort of patients with glomerular disease. Taken together with our previous experimental findings, urinary plasmin(ogen) could be a useful biomarker in prognostic decision making and a target for the development of novel therapies in patients with proteinuria and glomerular disease. PLAIN-LANGUAGE SUMMARY: Glomerular diseases are an important cause of morbidity and mortality in patients of all ages. Knowing the individual risk of progression to dialysis or transplantation would help to plan the follow-up and treatment of these patients. Our work studies the usefulness of urinary plasminogen as a marker of progression in this context, since previous studies indicate that plasminogen may be involved in the mechanisms responsible for the progression of these disorders. Our work in a sample of 1,010 patients with glomerular disease demonstrates that urinary plasminogen (as well as the ratio of measured to expected plasminogen) is associated with the risk of progression to end-stage kidney disease. Urine plasminogen exhibited good performance and, if further validated, could enable risk stratification for timely interventions in patients with proteinuria and glomerular disease.

Plasminogenuria is associated with podocyte injury, edema, and kidney dysfunction in incident glomerular disease.

Urinary plasminogen/plasmin, or plasmin (ogen) uria, has been demonstrated in proteinuric patients and exposure of cultured podocytes to plasminogen results in injury via oxidative stress pathways. A causative role for plasmin (ogen) as a "second hit" in kidney disease progression has yet to have been demonstrated in vivo. Additionally, association between plasmin (ogen) uria and kidney function in glomerular diseases remains unclear. We performed comparative studies in a puromycin aminonucleoside (PAN) nephropathy rat model treated with amiloride, an inhibitor of plasminogen activation, and measured changes in plasmin (ogen) uria. In a glomerular disease biorepository cohort (n = 128), we measured time-of-biopsy albuminuria, proteinuria, and plasmin (ogen) uria for correlations with kidney outcomes. In cultured human podocytes, plasminogen treatment was associated with decreased focal adhesion marker expression with rescue by amiloride. Increased glomerular plasmin (ogen) was found in PAN rats and focal segmental glomerulosclerosis (FSGS) patients. PAN nephropathy was associated with increases in plasmin (ogen) uria and proteinuria. Amiloride was protective against PAN-induced glomerular injury, reducing CD36 scavenger receptor expression and oxidative stress. In patients, we found associations between plasmin (ogen) uria and edema status as well as eGFR. Our study demonstrates a role for plasmin (ogen)-induced podocyte injury in the PAN nephropathy model, with amiloride having podocyte-protective properties. In one of the largest glomerular disease cohorts to study plasminogen, we validated previous findings while suggesting a potentially novel relationship between plasmin (ogen) uria and estimated glomerular filtration rate (eGFR). Together, these findings suggest a role for plasmin (ogen) in mediating glomerular injury and as a viable targetable biomarker for podocyte-sparing treatments.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

The Hippo pathway regulator KIBRA promotes podocyte injury by inhibiting YAP signaling and disrupting actin cytoskeletal dynamics.

Kidney podocytes represent a key constituent of the glomerular filtration barrier. Identifying the molecular mechanisms of podocyte injury and survival is important for better understanding and management of kidney diseases. KIBRA (kidney brain protein), an upstream regulator of the Hippo signaling pathway encoded by the Wwc1 gene, shares the pro-injury properties of its putative binding partner dendrin and antagonizes the pro-survival signaling of the downstream Hippo pathway effector YAP (Yes-associated protein) in Drosophila and MCF10A cells. We recently identified YAP as an essential component of the glomerular filtration barrier that promotes podocyte survival by inhibiting dendrin pro-apoptotic function. Despite these recent advances, the signaling pathways that mediate podocyte injury remain poorly understood. Here we tested the hypothesis that, similar to its role in other model systems, KIBRA promotes podocyte injury. We found increased expression of KIBRA and phosphorylated YAP protein in glomeruli of patients with biopsy-proven focal segmental glomerulosclerosis (FSGS). KIBRA/WWc1 overexpression in murine podocytes promoted LATS kinase phosphorylation, leading to subsequent YAP Ser-127 phosphorylation, YAP cytoplasmic sequestration, and reduction in YAP target gene expression. Functionally, KIBRA overexpression induced significant morphological changes in podocytes, including disruption of the actin cytoskeletal architecture and reduction of focal adhesion size and number, all of which were rescued by subsequent YAP overexpression. Conversely, constitutive KIBRA knockout mice displayed reduced phosphorylated YAP and increased YAP expression at baseline. These mice were protected from acute podocyte foot process effacement following protamine sulfate perfusion. KIBRA knockdown podocytes were also protected against protamine-induced injury. These findings suggest an important role for KIBRA in the pathogenesis of podocyte injury and the progression of proteinuric kidney disease.

Podocyte-Specific Deletion of Yes-Associated Protein Causes FSGS and Progressive Renal Failure.

FSGS is the most common primary glomerular disease underlying ESRD in the United States and is increasing in incidence globally. FSGS results from podocyte injury, yet the mechanistic details of disease pathogenesis remain unclear. This has resulted in an unmet clinical need for cell-specific therapy in the treatment of FSGS and other proteinuric kidney diseases. We previously identified Yes-associated protein (YAP) as a prosurvival signaling molecule, the in vitro silencing of which increases podocyte susceptibility to apoptotic stimulus. YAP is a potent oncogene that is a prominent target for chemotherapeutic drug development. In this study, we tested the hypothesis that podocyte-specific deletion of Yap leads to proteinuric kidney disease through increased podocyte apoptosis. Yap was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Yap silencing in podocytes resulted in podocyte apoptosis, podocyte depletion, proteinuria, and an increase in serum creatinine. Histologically, features characteristic of FSGS, including mesangial sclerosis, podocyte foot process effacement, tubular atrophy, interstitial fibrosis, and casts, were observed. In human primary FSGS, we noted reduced glomerular expression of YAP. Taken together, these results suggest a role for YAP as a physiologic antagonist of podocyte apoptosis, the signaling of which is essential for maintaining the integrity of the glomerular filtration barrier. These data suggest potential nephrotoxicity with strategies directed toward inhibition of YAP function. Further studies should evaluate the role of YAP in proteinuric glomerular disease pathogenesis and its potential utility as a therapeutic target.

Hippo signaling in the kidney: the good and the bad.

The Hippo signaling pathway is an evolutionarily conserved kinase cascade, playing multiple roles in embryonic development that controls organ size, cell proliferation, and apoptosis. At the center of this network lie the Hippo kinase target and downstream pathway effector Yes-associated protein (YAP) and its paralog TAZ. In its phosphorylated form, cytoplasmic YAP is sequestered in an inactive state. When it is dephosphorylated, YAP, a potent oncogene, is activated and relocates to the nucleus to interact with a number of transcription factors and signaling regulators that promote cell growth, differentiation, and survival. The identification of YAP activation in human cancers has made it an attractive target for chemotherapeutic drug development. Little is known to date about the function of the Hippo pathway in the kidney, but that is rapidly changing. Recent studies have shed light on the role of Hippo-YAP signaling in glomerular and lower urinary tract embryonic development, maintenance of podocyte homeostasis, the integrity of the glomerular filtration barrier, regulation of renal tubular cyst growth, renal epithelial injury in diabetes, and renal fibrogenesis. This review summarizes the current knowledge of the Hippo-YAP signaling axis in the kidney under normal and disease conditions.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress.

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap-/- model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2-) that promotes endothelin-1 synthesis. Plg via O2- also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a "second hit" in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization.

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMß causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.

Dendrin ablation prolongs life span by delaying kidney failure.

Podocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin.

Yes-associated protein (YAP) promotes cell survival by inhibiting proapoptotic dendrin signaling.

Kidney podocytes are highly specialized terminally differentiated cells that form the final barrier to urinary protein loss. Podocytes are a target for injury by metabolic, autoimmune, hereditary, inflammatory, and other stressors. Persistence of podocyte injury leads to podocyte death and loss, which results in progressive kidney damage and ultimately kidney failure. Dendrin is a dual compartment protein with proapoptotic signaling properties. Nuclear relocation of dendrin in response to glomerular injury promotes podocyte apoptosis. Here we show that Yes-associated protein (YAP), a downstream target of Hippo kinases and an inhibitor of apoptosis, is expressed in the nucleus of podocytes. The WW domains of YAP mediate the interaction with the PPXY motifs of dendrin. This interaction is functionally relevant because YAP binding to dendrin reduces dendrin-dependent, staurosporine-induced apoptosis in co-transfected HEK293 cells. Moreover gene silencing of YAP in podocytes increases adriamycin-induced podocyte apoptosis. It also increases staurosporine-induced caspase-3/7 activity, which is rescued by dendrin depletion in YAP knockdown cells. Our findings elucidate YAP binding to dendrin as a prosurvival mechanism. The antiapoptotic signaling properties of YAP in podocytes could hold significance in the quest for targeted therapeutics aimed at preventing podocyte loss.

Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma.

Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional analysis revealed that NKX2.2 is an EWS/FLI-regulated gene that is necessary for oncogenic transformation in this tumor. Thus, we developed a highly validated transcriptional profile for the EWS/FLI fusion protein and identified a critical target gene in Ewing's sarcoma development.

KIBRA upregulation increases susceptibility to podocyte injury and glomerular disease progression.

Despite recent progress in the identification of mediators of podocyte injury, mechanisms underlying podocyte loss remain poorly understood, and cell-specific therapy is lacking. We previously reported that kidney and brain expressed protein (KIBRA), encoded by WWC1, promotes podocyte injury in vitro through activation of the Hippo signaling pathway. KIBRA expression is increased in the glomeruli of patients with focal segmental glomerulosclerosis, and KIBRA depletion in vivo is protective against acute podocyte injury. Here, we tested the consequences of transgenic podocyte-specific WWC1 expression in immortalized human podocytes and in mice, and we explored the association between glomerular WWC1 expression and glomerular disease progression. We found that KIBRA overexpression in immortalized human podocytes promoted cytoplasmic localization of Yes-associated protein (YAP), induced actin cytoskeletal reorganization, and altered focal adhesion expression and morphology. WWC1-transgenic (KIBRA-overexpressing) mice were more susceptible to acute and chronic glomerular injury, with evidence of YAP inhibition in vivo. Of clinical relevance, glomerular WWC1 expression negatively correlated with renal survival among patients with primary glomerular diseases. These findings highlight the importance of KIBRA/YAP signaling to the regulation of podocyte structural integrity and identify KIBRA-mediated injury as a potential target for podocyte-specific therapy in glomerular disease.

Podocyte Foot Process Effacement Precedes Albuminuria and Glomerular Hypertrophy in CD2-Associated Protein Deficient Mice.

Background: Podocyte foot process effacement is a key histologic finding in proteinuric kidney disease. We previously showed that 3-week old CD2AP-deficient mice have significant proteinuria, glomerular hypertrophy and mesangial expansion. The goal of this study is to use morphometry to establish the temporal sequence of podocyte foot process effacement, glomerular volume expansion and albuminuria in Cd2ap -/- mice by measuring these parameters at the 2-week time point. Methods: Wild-type mice age 14 ± 1 days with the Cd2ap gene (WT, N = 5) and mice deficient for Cd2ap (Cd2ap KO, N = 5) were generated. Kidneys were harvested and fixed in 2.5% glutaraldehyde and processed for examination by light and electron microscopy. An average of 415.2 (range 268-716) grid points were counted for all the glomeruli, and quantification of glomerular volume from each kidney. Urine was collected the day prior to sacrifice for urine albumin-to-creatinine ratio (ACR) measurements. Results: There was no difference in albuminuria [median (range) mg/g] between WT [212.2 (177.6-388.4) mg/g] vs. Cd2ap KO mice [203.3 (164.7-910.2) mg/g], P = 0.89; or glomerular volume 68,307[10,931] vs. 66,844[13,022] µm3, p = 0.92. The volume densities of glomerular components of the podocyte, capillary lumen and mesangium were not different for the two groups, P = 0.14, 0.14 and 0.17 respectively. However, foot process width was increased in Cd2ap KO 1128[286] vs. WT [374 ± 42] nm, P = 0.02. Conclusion: Here we show that while 2-week old WT and Cd2ap KO mice have similar levels of albuminuria, glomerular and mesangial volume, Cd2ap KO mice have more extensive podocyte foot process effacement. The data suggests that podocyte injury is the initiating event leading to mesangial expansion and albuminuria in this model.

Molecular Analysis of the Kidney From a Patient With COVID-19-Associated Collapsing Glomerulopathy.

Recent case reports suggest that coronavirus disease 2019 (COVID-19) is associated with collapsing glomerulopathy in African Americans with apolipoprotein L1 gene (APOL1) risk alleles; however, it is unclear whether disease pathogenesis is similar to HIV-associated nephropathy. RNA sequencing analysis of a kidney biopsy specimen from a patient with COVID-19-associated collapsing glomerulopathy and APOL1 risk alleles (G1/G1) revealed similar levels of APOL1 and angiotensin-converting enzyme 2 (ACE2) messenger RNA transcripts as compared with 12 control kidney samples downloaded from the GTEx (Genotype-Tissue Expression) Portal. Whole-genome sequencing of the COVID-19-associated collapsing glomerulopathy kidney sample identified 4 indel gene variants, 3 of which are of unknown significance with respect to chronic kidney disease and/or focal segmental glomerulosclerosis. Molecular profiling of the kidney demonstrated activation of COVID-19-associated cell injury pathways such as inflammation and coagulation. Evidence for direct severe acute respiratory syndrome coronavirus 2 infection of kidney cells was lacking, which is consistent with the findings of several recent studies. Interestingly, immunostaining of kidney biopsy sections revealed increased expression of phospho-STAT3 (signal transducer and activator of transcription 3) in both COVID-19-associated collapsing glomerulopathy and HIV-associated nephropathy as compared with control kidney tissue. Importantly, interleukin 6-induced activation of STAT3 may be a targetable mechanism driving COVID-19-associated acute kidney injury.

Corynebacterium accolens-associated pelvic osteomyelitis.

Corynebacterium accolens is a rare human pathogen. We encountered a case of C. accolens isolated from a thigh collection in a man with osteomyelitis of the adjacent pubic symphysis.

Campylobacter fetus-associated epidural abscess and bacteremia.

Campylobacter fetus has been isolated as an infrequent cause of abscesses. We report a case of Campylobacter fetus epidural abscess and bacteremia in a debilitated elderly man who had a fatal outcome.

Disruption of podocyte cytoskeletal biomechanics by dasatinib leads to nephrotoxicity.

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Effect of a Novel Photoelectrochemical Oxidation Air Purifier on Nasal and Ocular Allergy Symptoms.

BACKGROUND: Photoelectrochemical oxidation (PECO) is a new air purification technology developed to reduce circulating indoor allergens. PECO removes particles as small as 0.1 nm with the destruction of organic matter otherwise not trapped by a traditional filter and removes volatile organic compounds. OBJECTIVE: We hypothesized that with daily use, the device would reduce user nasal and ocular allergy total symptom scores (TSS) within 4 weeks. METHODS: The study was performed among 46 individuals with self-reported allergies using a portable PECO air purifier. Self-reported TSS were calculated at baseline and weekly for 4 weeks following initiation of continuous use of the system. TSS was the sum of total nasal symptom scores (TNSS) and total ocular symptom scores (TOSS) for the week. RESULTS: There was a statistically significant change in overall TSS from baseline to 4 weeks (10.1 at baseline and 4.35 postintervention) resulting in a mean difference of 5.75 (95% confidence interval [CI] 4.32-7.18; P < .0001). There was a statistically significant change in TNSS from baseline to 4 weeks (6.3 at baseline and 3.04 postintervention) resulting in a mean difference of 3.26 (95% CI 2.33-3.19; P < .0001). There was a statistically significant change in TOSS from baseline to 4 weeks (3.82 at baseline and 1.3 postintervention) resulting in a mean difference of 2.52 (95% CI 1.74-3.3; P < .0001). CONCLUSION: With the use of PECO air purification technology, TSS, TNSS, and TOSS decreased significantly. These improvements were consistent over the 4-week course of device use.

Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma.

BACKGROUND: The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression-based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. METHODS AND FINDINGS: A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. CONCLUSIONS: We demonstrate that a gene expression-based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.

Emergence of blaKPC carbapenemase genes in Australia.

blaKPC genes encoding resistance to carbapenems are increasingly widely reported and are now endemic in parts of several countries, but only one Klebsiella pneumoniae isolate carrying blaKPC-2 had previously been reported in Australia, in 2010. Here we characterised this isolate, six additional K. pneumoniae and one Escherichia coli carrying blaKPC and another K. pneumoniae lacking blaKPC, all isolated in Australia in 2012. Seven K. pneumoniae belonged to clonal complex (CC) 292, associated with blaKPC in several countries. Five with blaKPC-2 plus the isolate lacking a blaKPC gene were sequence type 258 (ST258) and the seventh was the closely related ST512 with blaKPC-3. The eighth K. pneumoniae isolate, novel ST1048, and the E. coli (ST131) also carried blaKPC-2. blaKPC genes were associated with the most common Tn4401a variant, which gives the highest levels of expression, in all isolates. The ST258 isolates appeared to share a similar set of plasmids, with IncFIIK, IncX3 and ColE-type plasmids identified in most isolates. All K. pneumoniae isolates had a characteristic insertion in the ompK35 gene resulting in a frameshift and early termination, but only the ST512 isolate had a GlyAsp insertion in loop 3 of OmpK36 that may contribute to increased resistance. The clinical epidemiology of blaKPC emergence in Australia thus appears to reflect the global dominance of K. pneumoniae CC292 (and perhaps E. coli ST131). Some, but not all, patients carrying these isolates had previously been hospitalised outside Australia, suggesting multiple discrete importation events of closely related strains, as well as undetected nosocomial spread.