Prediction of MIGS outcomes on second eyes using first eyes' response.

To evaluate how well outcomes following cataract extraction and microinvasive glaucoma surgery in one eye predict outcomes in sequential second eye. Retrospective study of 78 patients who underwent cataract extraction and microinvasive glaucoma surgery in both eyes. Linear regressions using Pearson correlation coefficients were used to evaluate correlations in intraocular pressure and glaucoma medication change between eyes. Multivariable logistic regression models were used to evaluate the associations between first-eye variables and the likelihood of second-eye surgical success at 6 months. Surgical success was defined as meeting target intraocular pressure without additional medications compared to baseline or secondary surgical interventions. Baseline ocular characteristics were comparable between fellow eyes, with the majority having mild glaucoma. Intraocular pressure changes between fellow eyes at 6 months were modestly correlated between eyes (R = 0.48; P < .001). Changes in glaucoma medications were strongly correlated between eyes at all time points, and month 6 demonstrated the most significant correlation (R = 0.80; P < .001). First and second eye cohorts achieved 82% and 83% surgical success. Multivariate analysis for predictive factors of successful second eye surgery showed patients with successful first eye surgery at 6 months were significantly more likely to have successful second eye surgery (odds ratio, 20.67; P < .001). Reductions in intraocular pressure and glaucoma medications at 6 months following surgery in first eyes are correlated to second eye reductions. Successful surgical outcomes at 6 months following first eye surgeries are strongly associated with successful sequential second eye outcomes.

Short-Term Outcomes of Hydrus Microstent With and Without Additional Canaloplasty During Cataract Surgery.

PRCIS: Compared with phacoemulsification and microstent alone, we observed that phacoemulsification with combined microstent and canaloplasty resulted in a significantly greater reduction in glaucoma medications while maintaining similar rates of intraocular pressure reduction and low complications. PURPOSE: The purpose of this study was to compare the outcomes of phacoemulsification combined with Hydrus Microstent (Alcon Inc.) implantation alone or in combination with canaloplasty (OMNI Surgical System, Sight Sciences Inc.). MATERIALS AND METHODS: Retrospective study of mild-to-moderate primary open angle glaucoma patients who underwent phacoemulsification with microstent alone (42 eyes of 42 patients) or in combination with canaloplasty (canaloplasty-microstent, 32 eyes of 32 patients). The mean number of ocular hypotensive medications and intraocular pressure were assessed preoperatively and postoperatively at 1 week and at 1, 3, and 6 months. Complications and secondary surgical interventions were recorded. Outcomes measures included the percentage of unmedicated eyes and surgical success at 6 months. Surgical success was defined as reaching the target intraocular pressure without medications or secondary surgical interventions. RESULTS: Mean intraocular pressure at 6 months was 14.1±3.5 mm Hg (13% reduction) after microstent alone and 13.6±3.1 mm Hg (17% reduction) after canaloplasty-microstent. Mean medications at 6 months were 0.57±0.9 (67% reduction) after microstent alone and 0.16±0.4 (88% reduction) after canaloplasty-microstent ( P< 0.05). At 6 months, 64.3% of microstent alone and 87.3% of canaloplasty-microstent were off all medications ( P =0.02). Success probabilities at 6 months were 44.5% for microstent alone and 70.0% for canaloplasty-microstent ( P =0.04). No secondary surgical interventions occurred in either group. CONCLUSIONS: Microstent combined with canaloplasty resulted in a significantly higher rate of medication-free status compared with microstent alone through 6 months.

Distinguishing Glaucoma, Cataract, and Glaucoma Suspect Based on Visual Symptoms.

PRCIS: Cataract, glaucoma, and glaucoma suspect patients report differing visual symptoms. Asking patients about their visual symptoms may provide useful diagnostic information and inform decision-making in patients with comorbid conditions. PURPOSE: To compare visual symptoms in glaucoma, glaucoma suspect (controls), and cataract patients. METHODS: Glaucoma, cataract, and glaucoma suspect patients at Wilmer Eye Institute responded to a questionnaire rating the frequency and severity of 28 symptoms. Univariate and multivariable logistic regression determined the symptoms that best differentiate each disease pair. RESULTS: In all, 257 patients (mean age: 67.4 ± 13.4 y; 57.2% female; 41.2% employed), including 79 glaucoma, 84 cataract, and 94 glaucoma suspect patients, participated. Compared with glaucoma suspects, glaucoma patients were more likely to report poor peripheral vision (OR 11.29, 95% CI: 3.73-34.16), better vision in 1 eye (OR 5.48, 95% CI: 1.33-22.64), and light sensitivity (OR 4.85, 95% CI: 1.78-13.24), explaining 40% of the variance in diagnosis (ie, glaucoma vs. glaucoma suspect). Compared with controls, cataract patients were more likely to report light sensitivity (OR 3.33, 95% CI: 1.56-7.10) and worsening vision (OR 12.20, 95% CI: 5.33-27.89), explaining 26% of the variance in diagnosis (ie, cataract vs. glaucoma suspect). Compared with cataract patients, glaucoma patients were more likely to report poor peripheral vision (OR 7.24, 95% CI: 2.53-20.72) and missing patches (OR 4.91, 95% CI: 1.52-15.84), but less likely to report worsening vision (OR 0.08, 95% CI 0.03-0.22), explaining 33% of the variance in diagnosis (ie, glaucoma vs. cataract). CONCLUSIONS: Visual symptoms distinguish disease state to a moderate degree in glaucoma, cataract, and glaucoma suspect patients. Asking about visual symptoms may serve as a useful diagnostic adjunct and inform decision-making, for example, in glaucoma patients considering cataract surgery.

Associations among Visual, Auditory, and Olfactory Functions in Community-Based Older Adults: The Atherosclerosis Risk in Communities (ARIC) Study.

Purpose: Objective examination of relationships among visual, hearing, and olfactory function may yield mechanistic insights and inform our understanding of the burden of multiple-sensory impairments. Methods: This cross-sectional study capitalized on continuous measures of visual acuity (VA), contrast sensitivity, pure tone audiometry, Quick Speech-in-Noise (QuickSIN), and Sniffin' Sticks from a subset of ARIC participants at two community sites (EyeDOC Study, 2017-2019). Scales of all measures were aligned such that higher values indicated greater impairment. Intersensory bivariate associations were assessed graphically, and correlations assessed using Kendall's tau. Intersensory associations, independent of age, education, smoking, diabetes, and hypertension, were examined using linear regression. Analyses were stratified by community/race (Washington County/White vs Jackson/Black) and sex (men vs women) to explore community-sex heterogeneity. Results: We included 834 participants (mean age, 79 years); 39% were from Jackson and 63% females. We found weak intersensory correlations (tau generally ≤0.15). In the demographics-adjusted regression models, results were heterogeneous across communities and sex. Worse near VA, contrast sensitivity, and olfaction were associated with worse QuickSIN and worse near VA was associated with worse olfaction in some but not all community/race-sex groups (e.g., Jackson/Black women, 0.1 logMAR worse near VA was associated with 0.27 units increase in QuickSIN [95% confidence interval, 0.10-0.45]). Associations were modestly attenuated by adjustment for the shared risk factors of smoking, diabetes, and hypertension. Conclusions: Visual dysfunction showed little or no association with hearing or olfaction impairments, suggesting a modest role for shared risk factors. Translational Relevance: Visually impaired individuals have only a modestly higher risk of other sensory impairment.

Home Self-tonometry Trials Compared with Clinic Tonometry in Patients with Glaucoma.

PURPOSE: This study examined characteristics of intraocular pressure (IOP) as measured during home tonometry in comparison with in-clinic tonometry in patients with glaucoma. DESIGN: Retrospective cross-sectional study of glaucoma patients who completed 1 week of self-tonometry at a single academic center. PARTICIPANTS: Patients with glaucoma who completed home tonometry trials with the iCare HOME tonometer (iCare USA) for any reason. METHODS: Home IOP measurements were compared with in-clinic tonometry performed during the 5 visits preceding home tonometry. Maximum daily IOP was correlated to time of day. Generalized estimating equations were used to evaluate patient characteristics and clinic-derived variables that predicted differences between home and clinic IOP. MAIN OUTCOME MEASURES: IOP mean, maximum, minimum, range, standard deviation and coefficient of variation were compared between clinic and home tonometry. IOP mean daily maximum (MDM) and mean daily range were calculated to describe recurrent IOP spiking. RESULTS: A total of 107 eyes from 61 patients were analyzed. Mean age was 63.2 years (standard deviation [SD], 14.0 years) and 59.0% were women. Mean clinic and home IOPs were 14.5 mmHg (SD, 4.7 mmHg) and 13.6 mmHg (SD, 5.1 mmHg). Home tonometry identified significantly higher maximum IOP, lower minimum IOP, and greater IOP range than clinic tonometry (P < 0.001). Maximum daily IOP occurred outside of clinic hours (8 am-5 pm) on 50% of days assessed and occurred between 4:30 am and 8 am on 24% of days. Mean daily maximum IOP exceeded maximum clinic IOP in 44% of patients and exceeded target IOP by 3 mmHg, 5 mmHg, or 10 mmHg in 31%, 15%, and 6% of patients, respectively. Patient characteristics that predicted significant deviations between MDM and mean clinic IOP or target IOP in multivariate models included younger age, male gender, and absence of prior filtering surgery. CONCLUSIONS: Self-tonometry provides IOP data that supplements in-clinic tonometry and would not be detectable over daytime in-clinic diurnal curves. A subset of patients in whom home tonometry was ordered by their glaucoma clinician because of suspicion of occult IOP elevation demonstrated reproducible IOP elevation outside of the clinic setting. Such patients tended to be younger and male and not to have undergone previous filtering surgery.

Comparison of Patient-Reported Functional Recovery From Different Types of Ophthalmic Surgery.

PURPOSE: To characterize and compare patient-reported recovery of function after cataract or glaucoma surgery using a novel visual analog scale. DESIGN: Prospective observational cohort study. METHODS: Daily for 2 weeks and weekly thereafter, patients recovering from trabeculectomy, tube shunt implantation, or cataract extraction (CE) completed a diary-style questionnaire including visual analog scales (VASs; scored 0-100) grading pain and global function. Clinical examination data and medical histories were collected. Generalized estimating equation models evaluated associations between VAS function scores and pain or visual acuity (VA) and compared scores between surgery types. RESULTS: Among 51 participants followed for 12 weeks, tube shunt placement reduced postoperative day 1 (POD1) function by 47 of 100 points vs CE (P = .006), while trabeculectomy did not reduce POD1 function vs CE (P = .33). After CE, trabeculectomy, and tube shunt placement, average VAS function scores increased 13.94 per week for 2 weeks (P < .001), 4.18 per week for 4 weeks (P = .02), and 7.76 per week for 7 weeks (P < .001), respectively. After those timepoints, there was no further significant change. Beyond 2 weeks, pain levels plateaued, and VA returned to baseline across surgery types; function was inversely related to pain or VA only for the first 2 or 4 weeks, respectively. CONCLUSIONS: Patients recovering from cataract and glaucoma surgery report reduced function in the postoperative period. Tube shunt implantation causes greater morbidity than trabeculectomy, and both are associated with slower improvement than CE. Early postoperative function is associated with VA and pain, but neither fully explains reported impairment. A VAS for function may efficiently capture postoperative recovery.

Statistical Assessment of Depth Normalization for Small RNA Sequencing.

PURPOSE: Methods for depth normalization have been assessed primarily with simulated data or cell-line-mixture data. There is a pressing need for benchmark data enabling a more realistic and objective assessment, especially in the context of small RNA sequencing. METHODS: We collected a unique pair of microRNA sequencing data sets for the same set of tumor samples; one data set was collected with and the other without uniform handling and balanced design. The former provided a benchmark for evaluating evidence of differential expression and the latter served as a test bed for normalization. Next, we developed a data perturbation algorithm to simulate additional data set pairs. Last, we assembled a set of computational tools to visualize and quantify the assessment. RESULTS: We validated the quality of the benchmark data and showed the need for normalization of the test data. For illustration, we applied the data and tools to assess the performance of 9 existing normalization methods. Among them, trimmed mean of M-values was a better scaling method, whereas the median and the upper quartiles were consistently the worst performers; one variation of remove unwanted variation had the best chance of capturing true positives but at the cost of increased false positives. In general, these methods were, at best, moderately helpful when the level of differential expression was extensive and asymmetric. CONCLUSION: Our study (1) provides the much-needed benchmark data and computational tools for assessing depth normalization, (2) shows the dependence of normalization performance on the underlying pattern of differential expression, and (3) calls for continued research efforts to develop more effective normalization methods.

Comparison of changes in corneal endothelial cell density and central corneal thickness between conventional and femtosecond laser-assisted cataract surgery: a randomised, controlled clinical trial.

BACKGROUND/AIM: To identify changes in endothelial cell density (ECD) and central corneal thickness (CCT) in eyes undergoing femtosecond laser-assisted cataract surgery (FLACS) compared with conventional phacoemulsification surgery (CPS). METHODS: This is an intraindividual randomised, controlled clinical trial. One eye was randomised to receive FLACS, while the contralateral eye of the same patient received CPS. The femtosecond laser pretreatment included creating main and side-port corneal incisions, capsulotomy and lens fragmentation. Non-contact endothelial cell microscopy and pachymetry were performed preoperatively and at postoperative day 1, week 1, month 1 and month 3. RESULTS: A total of 134 paired eyes from 67 patients were included in the analysis. ECD was not significantly different between the two groups at either postoperative month 1 (2370±580 cells/mm2 and 2467±564 cells/mm2 in FLACS and CPS groups, respectively; p=0.18) or at postoperative month 3 (2374±527 cells/mm2 and 2433±526 cells/mm2 in FLACS and CPS groups, respectively; p=0.19). No significant difference was observed in the mean CCT values between the two groups over the follow-up period (p>0.05). CONCLUSION: Postoperative corneal ECD and CCT were comparable between FLACS and CPS during the 3 months' follow-up period.

What Is a Falls Risk Factor? Factors Associated with Falls per Time or per Step in Individuals with Glaucoma.

OBJECTIVES: To determine whether visual field (VF) damage or other factors confer a similar risk of falling when falls are ascertained as a rate over time (falls/year) as when ascertained over activity (falls/step). DESIGN: Prospective, observational cohort study. SETTING: Clinic-based recruitment with real-world monitoring of falls and physical activity. PARTICPANTS: Individuals with glaucoma or suspected glaucoma (N=230). MEASUREMENTS: Participants recorded falls using daily calendars, with injuries identified in a follow-up questionnaire. Annual 1-week accelerometer trials were used to estimate steps. VF results from both eyes were merged to determine integrated VF sensitivity, with lower sensitivity indicating greater VF damage. Other potential risk factors for falls (age, sex, race, comorbid illness, polypharmacy) were determined using questionnaires. RESULTS: The cumulative probability of falls was 45.2% at 12 months and 61.6% at 24 months; cumulative probability of injurious falls was 23.3% at 12 months and 40.0% at 24 months. Greater VF damage was associated with higher rates of falls/steps (incident rate ratio = 1.40/5 dB decrement in sensitivity; p = .004) but not with more falls/year (incident rate ratio = 1.25/5 dB decrement in sensitivity; p = .07). Several additional variables (older age, female sex, more comorbid disease) were also associated with a higher rate of falls/step (p < .02 for all) but not with falls/year (p > 0.10). Black participants had fewer falls/year than whites (p = .002) but did not differ in falls/step (p = .07). Similar results were obtained when injurious falls were analyzed. CONCLUSIONS: Risk factors associated with frequent falls when walking (falls/step) are not properly identified when analyzing falls as a rate over time (falls/year). Given the clinical importance of preventing falls while preserving physical activity, falls assessment integrated with activity measurement is recommended when determining whether a risk factor is associated with falls. J Am Geriatr Soc 67:87-92, 2019.

miR-193b-Regulated Signaling Networks Serve as Tumor Suppressors in Liposarcoma and Promote Adipogenesis in Adipose-Derived Stem Cells.

Well-differentiated and dedifferentiated liposarcomas (WDLS/DDLS) account for approximately 13% of all soft tissue sarcoma in adults and cause substantial morbidity or mortality in the majority of patients. In this study, we evaluated the functions of miRNA (miR-193b) in liposarcoma in vitro and in vivo Deep RNA sequencing on 93 WDLS, 145 DDLS, and 12 normal fat samples demonstrated that miR-193b was significantly underexpressed in DDLS compared with normal fat. Reintroduction of miR-193b induced apoptosis in liposarcoma cells and promoted adipogenesis in human adipose-derived stem cells (ASC). Integrative transcriptomic and proteomic analysis of miR-193b-target networks identified novel direct targets, including CRK-like proto-oncogene (CRKL) and focal adhesion kinase (FAK). miR-193b was found to regulate FAK-SRC-CRKL signaling through CRKL and FAK. miR-193b also stimulated reactive oxygen species signaling by targeting the antioxidant methionine sulfoxide reductase A to modulate liposarcoma cell survival and ASC differentiation state. Expression of miR-193b in liposarcoma cells was downregulated by promoter methylation, resulting at least in part from increased expression of the DNA methyltransferase DNMT1 in WDLS/DDLS. In vivo, miR-193b mimetics and FAK inhibitor (PF-562271) each inhibited liposarcoma xenograft growth. In summary, miR-193b not only functions as a tumor suppressor in liposarcoma but also promotes adipogenesis in ASC. Furthermore, this study reveals key tyrosine kinase and DNA methylation pathways in liposarcoma, some with immediate implications for therapeutic exploration. Cancer Res; 77(21); 5728-40. ©2017 AACR.

Deep MicroRNA sequencing reveals downregulation of miR-29a in neuroblastoma central nervous system metastasis.

Central nervous system (CNS) is an increasingly common site of isolated metastasis for patients with Stage 4 neuroblastoma. To explore the microRNA (miRNA) profile of this metastatic process, miRNA sequencing was performed to identify miRNA sequence families with differential expression between tumor pairs (pre-CNS primary and CNS metastasis) from 13 patients with Stage 4 neuroblastoma. Seven miRNA sequence families had distinct expression in CNS metastases when compared with their corresponding pre-CNS primaries. MiR-7 was upregulated (3.75-fold), and miR-21, miR-22, miR-29a, miR-143, miR-199a-1-3p, and miR-199a-1-5p were downregulated (3.5-6.1-fold), all confirmed by quantitative reverse transcription-PCR. MiR-29a, previously shown to be downregulated in a broad spectrum of solid tumors including neuroblastoma, had the most significant decrease in all 13 CNS metastases (P = 0.001). Its known onco-targets CDC6, CDK6, and DNMT3A, as well as B7-H3, an inhibitory ligand for T cells, and natural killer cells, were found to have higher differential expression in these 13 CNS metastases when compared with their paired primaries. Additionally, miR-29a expression in primary tumors was significantly lower among patients who eventually relapsed in the CNS. Irrespective of the amplification status of MYCN, which is known to be associated with metastasis, pre-CNS primaries, and CNS metastases had significantly lower miR-29a expression than non-CNS primary tumors. Among MYCN amplified cell lines, those from CNS relapse also had lower miR-29a expression than non-CNS relapse. These findings raised the hypothesis that miR-29a could be a biomarker for neuroblastoma CNS metastasis, and its downregulation may play a pivotal role in CNS progression.

Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets.

BACKGROUND: Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference. RESULTS: To generate a biochemically-validated set of miRNA-binding sites, we performed argonaute-2 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO2-PAR-CLIP) in MCF7 cells. We then defined putative miRNA-target interactions using a computational model, which ranked and selected additional TargetScan-predicted interactions based on features of our AGO2-PAR-CLIP binding-site data. We subselected modeled interactions according to the abundance of their constituent miRNA and mRNA transcripts in tumors, and we took advantage of the variability of miRNA expression within molecular subtypes to detect miRNA repression. Interestingly, our data suggest that miRNA families control subtype-specific pathways; for example, miR-17, miR-19a, miR-25, and miR-200b show high miRNA regulatory activity in the triple-negative, basal-like subtype, whereas miR-22 and miR-24 do so in the HER2 subtype. An independent dataset validated our findings for miR-17 and miR-25, and showed a correlation between the expression levels of miR-182 targets and overall patient survival. Pathway analysis associated miR-17, miR-19a, and miR-200b with leukocyte transendothelial migration. CONCLUSIONS: We combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.

Multicolor microRNA FISH effectively differentiates tumor types.

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations.

We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.

The miR-17-92 cluster and its target THBS1 are differentially expressed in angiosarcomas dependent on MYC amplification.

Angiosarcomas (ASs) represent a heterogeneous group of malignant vascular tumors that may occur spontaneously as primary tumors or secondarily after radiation therapy or in the context of chronic lymphedema. Most secondary ASs have been associated with MYC oncogene amplification, whereas the role of MYC abnormalities in primary AS is not well defined. Twenty-two primary and secondary ASs were analyzed by array-comparative genomic hybridization (aCGH) and by deep sequencing of small RNA libraries. By aCGH and subsequently confirmed by fluorescence in situ hybridization, MYC amplification was identified in three out of six primary tumors and in 8 out of 12 secondary AS. We have also found MAML1 as a new potential oncogene in MYC-amplified AS. Significant upregulation of the miR-17-92 cluster was observed in MYC-amplified AS compared to AS lacking MYC amplification and the control group (other vascular tumors, nonvascular sarcomas). Moreover, MYC-amplified ASs were associated with a significantly lower expression of thrombospondin-1 (THBS1) than AS without MYC amplification or controls. Altogether, our study implicates MYC amplification not only in the pathogenesis of secondary AS but also in a subset of primary AS. Thus, MYC amplification may play a crucial role in the angiogenic phenotype of AS through upregulation of the miR-17-92 cluster, which subsequently downregulates THBS1, a potent endogenous inhibitor of angiogenesis.

Small RNA sequencing and functional characterization reveals MicroRNA-143 tumor suppressor activity in liposarcoma.

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21 and miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143 and miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, topoisomerase 2A, protein regulator of cytokinesis 1 (PRC1), and polo-like kinase 1 (PLK1). The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G(2)-M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma.

MicroRNA sequence and expression analysis in breast tumors by deep sequencing.

MicroRNAs (miRNA) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 noninvasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed sequence-dependent miRNA target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most noninvasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the noninvasive carcinomas. In addition, patients that went on to develop metastasis showed increased expression of mir-423, and triple-negative breast carcinomas were most distinct from other tumor subtypes due to upregulation of the mir~17-92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested.

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.

[Immunocytochemical characteristics of submucosal uterine myomas].

BACKGROUND/AIM: Myomas of the uterus, the most common benign tumors, have been studied for decades from the aspects of different basic and clinical disciplines. Despite this fact, their pathogenesis is still poorly understood. The aim of this study was to determine immunocytochemical characteristics of smooth muscle cells and connective tissue components of submucosal myomas of the uterus. METHOD: During the course of this study, 25 samples of submucosal myomas of the uterus were analyzed, all of them obtained during the surgery, after abdominal histerctomy by Aldridge. The samples were fixed in 4% formalin and embedded in paraffin. Sections of 5 microm thickness were stained immunocytochemically using the DAKO LSAB+/HRP technique to identify alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, CD34, CD45, CD68 and PCNA (DAKO specification). RESULTS: Our results suggest that submucosal myomas of the uterus are build-up of smooth muscle cells which are immunoreactive to alpha-SMA and desmin, but also to a certain number of smooth muscle cells which are immunoreactive to alpha-SMA and vimentin. Some of vimentin-immunoreactive cells also show an immunoreactivity of PCNA. In the build-up of connective stroma CD34-immunoreactive fibroblasts and neovascular formations are also present. By examining the distribution of CD45 antigen, at all the analyzed samples we observed a weak reaction. CONCLUSION: Submucosal myomas of the uterus are made-up of smooth muscle cells of the highly differentiated contractile phenotype (alpha-SMA- and desmin-immunoreactivity), as well as smooth muscle cell of the synthetic phenotype which proliferate (alpha-SMA-, vimentin- and PCNA-immunoreactivity). In submucosal myoma of the uterus there is a significant presence of connective tissue as a result of synthetic activity of fibroblasts, which clearly differ in their immunocytochemical characteristics from smooth muscle cells of the synthetic phenotype.

Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.

Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5' end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5' triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5' triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5' triphosphate was required. RIG-I activation was impaired by a 3' overhang at the 5' triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5' triphosphate RNA in the panhandle region of their single-stranded genome.