Ectopic expression of T in the paraxial mesoderm disrupts somite maturation in the mouse.

T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.

The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production.

The secreted Ly-6/uPAR related protein-1 (SLURP1) is an anti-angiogenic and anti-inflammatory peptide highly expressed by the mucosal epithelial cells. SLURP1 is abundantly expressed by the corneal epithelial cells and is significantly downregulated when these cells are transformed and adapted for culture in vitro. Here we studied the effect of overexpressing SLURP1 in Human Corneal Limbal Epithelial (HCLE) cells cultured in vitro. The expression of DSP1, DSG1, TJP1 and E-Cadherin was significantly upregulated in two different SLURP1-overexpressing HCLE cell (HCLE-SLURP1) clones. HCLE-SLURP1 cells also displayed a significant decrease in tumor necrosis factor-α (TNF-α)-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1-fold, (ii) IL-1ß from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the control, to 503 and 352 pg/ml in HCLE-SLURP1 cells. Consistently, cytosolic IκB expression was elevated in HCLE-SLURP1 cells with a concurrent suppression of TNF-α-activated nuclear translocation of NF-κB. Collectively, these results elucidate the beneficial effects of SLURP1 in stabilizing the HCLE intercellular junctions and suppressing the TNF-α-induced upregulation of inflammatory cytokines by suppressing NF-κB nuclear translocation.

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

Clinical Correlates of Benefit From Radium-223 Therapy in Metastatic Castration Resistant Prostate Cancer.

BACKGROUND: We sought to identify potential clinical variables associated with outcomes after radium-223 therapy in routine practice. METHODS: Consecutive non-trial mCRPC patients who received ≥1 dose of radium dichloride-223 at four academic and one community urology-specific cancer centers from May 2013 to June 2014 were retrospectively identified. Association of baseline and on-therapy clinical variables with number of radium doses received and clinical outcomes including overall survival were analyzed using chi-square statistics, cox proportional hazards, and Kaplan-Meier methods. Bone Scan Index (BSI) was derived from available bone scans using EXINI software. RESULTS: One hundred and forty-five patients were included. Radium-223 was administered for six cycles in 74 patients (51%). One-year survival in this heavily pre-treated population was 64% (95%CI: 54-73%). In univariate and multivariate analysis, survival was highly associated with receiving all six doses of Radium-223. Receipt of six doses was associated with ECOG PS of 0-1, lower baseline PSA & pain level, no prior abiraterone/enzalutamide, <5 BSI value, and normal alkaline phosphatase. In patients who reported baseline pain (n = 72), pain declined in 51% after one dose and increased in 7%. PSA declined ≥50% in 16% (18/110). Alkaline phosphatase declined ≥25% in 48% (33/69) and ≥50% in 16/69 patients. BSI declined in 17 (68%) of the 25 patients who had bone scan available at treatment follow-up. Grade ≥3 neutropenia, anemia, and thrombocytopenia occurred in 4% (n = 114), 4% (n = 125), and 5% (n = 123), respectively. CONCLUSIONS: Patients earlier in their disease course with <5 BSI, low pain score, and good ECOG performance status are optimal candidates for radium-223. Radium-223 therapy is well tolerated with most patients reporting declines in pain scores and BSI. Prostate 77:479-488, 2017. © 2016 Wiley Periodicals, Inc.

Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments.

In vitro selection experiments in biochemistry allow for the discovery of novel molecules capable of specific desired biochemical functions. However, this is not the only benefit we can obtain from such selection experiments. Since selection from a random library yields an unprecedented, and sometimes comprehensive, view of how a particular biochemical function is distributed across sequence space, selection experiments also provide data for creating and analyzing molecular fitness landscapes, which directly map function (phenotypes) to sequence information (genotypes). Given the importance of understanding the relationship between sequence and functional activity, reliable methods to build and analyze fitness landscapes are needed. Here, we present some statistical methods to extract this information from pools of RNA molecules. We also provide new computational tools to construct and study molecular fitness landscapes.

Statistical innovations in the medical device world sparked by the FDA.

The world of medical devices while highly diverse is extremely innovative, and this facilitates the adoption of innovative statistical techniques. Statisticians in the Center for Devices and Radiological Health (CDRH) at the Food and Drug Administration (FDA) have provided leadership in implementing statistical innovations. The innovations discussed include: the incorporation of Bayesian methods in clinical trials, adaptive designs, the use and development of propensity score methodology in the design and analysis of non-randomized observational studies, the use of tipping-point analysis for missing data, techniques for diagnostic test evaluation, bridging studies for companion diagnostic tests, quantitative benefit-risk decisions, and patient preference studies.

Utilizing national and international registries to enhance pre-market medical device regulatory evaluation.

Regulatory decisions are made based on the assessment of risk and benefit of medical devices at the time of pre-market approval and subsequently, when post-market risk-benefit balance needs reevaluation. Such assessments depend on scientific evidence obtained from pre-market studies, post-approval studies, post-market surveillance studies, patient perspective information, as well as other real world data such as national and international registries. Such registries provide real world evidence and are playing a more and more important role in enhancing the safety and effectiveness evaluation of medical devices. While these registries provide large quantities of data reflecting real world practice and can potentially reduce the cost of clinical trials, challenges arise concerning (1) data quality adequate for regulatory decision-making, (2) bias introduced at every stage and aspect of study, (3) scientific validity of study designs, and (4) reliability and interpretability of study results. This article will discuss related statistical and regulatory challenges and opportunities with examples encountered in medical device regulatory reviews.

Comprehensive experimental fitness landscape and evolutionary network for small RNA.

The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Purpose: Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Methods: Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Results: Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10 days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Conclusions: Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

The paradox of dual roles in the RNA world: resolving the conflict between stable folding and templating ability.

The hypothesized dual roles of RNA as both information carrier and biocatalyst during the earliest stages of life require a combination of features: good templating ability (for replication) and stable folding (for ribozymes). However, this poses the following paradox: well-folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes. Here, we describe a strategy to overcome this dilemma through G:U wobble pairing in RNA. Unlike Watson-Crick base pairs, wobble pairs contribute highly to the energetic stability of the folded structure of their sequence, but only slightly, if at all, to the stability of the folded reverse complement. Sequences in the RNA World might thereby combine stable folding of the ribozyme with an unstructured, reverse-complementary genome, resulting in a "division of labor" between the strands. We demonstrate this strategy using computational simulations of RNA folding and an experimental model of early replication, nonenzymatic template-directed RNA primer extension. Additional study is needed to solve other problems associated with a complete replication cycle, including separation of strands after copying. Interestingly, viroid RNA sequences, which have been suggested to be relics of an RNA World (Diener, Proc Natl Acad Sci USA 86:9370-9374, 1989), also show significant asymmetry in folding energy between the infectious (+) and template (-) strands due to G:U pairing, suggesting that this strategy may even be used by replicators in the present day.

Site specific probability of passive acoustic detection of humpback whale calls from single fixed hydrophones.

Passive acoustic monitoring of marine mammal calls is an increasingly important method for assessing population numbers, distribution, and behavior. A common mistake in the analysis of marine mammal acoustic data is formulating conclusions about these animals without first understanding how environmental properties such as bathymetry, sediment properties, water column sound speed, and ocean acoustic noise influence the detection and character of vocalizations in the acoustic data. The approach in this paper is to use Monte Carlo simulations with a full wave field acoustic propagation model to characterize the site specific probability of detection of six types of humpback whale calls at three passive acoustic monitoring locations off the California coast. Results show that the probability of detection can vary by factors greater than ten when comparing detections across locations, or comparing detections at the same location over time, due to environmental effects. Effects of uncertainties in the inputs to the propagation model are also quantified, and the model accuracy is assessed by comparing calling statistics amassed from 24,690 humpback units recorded in the month of October 2008. Under certain conditions, the probability of detection can be estimated with uncertainties sufficiently small to allow for accurate density estimates.

Gastric pneumatosis with associated eosinophilic gastritis in four black and white ruffed lemurs (Varecia variegata variegata).

Pneumatosis cystoides intestinalis (PCI) with associated eosinophilic inflammation was documented in the gastric tissues of four black and white ruffed lemurs (Varecia variegata variegata). Pneumatosis cystoides intestinalis is an uncommon disease described in humans and characterized by multilocular gas-filled cystic spaces located within the wall of the gastrointestinal tract. These cystic spaces can occur in any location along the gastrointestinal tract as well as within the associated connective and lymphatic tissues. The exact cause of this disease is unknown. The four black and white ruffed lemurs described in this case series were captive born and had been housed in zoological institutions at two separate locations. Three of the four cases were female lemurs, and two of the affected lemurs were directly related. The individual disease presentations spanned a 5-yr time period. Two lemurs presented dead with no premonitory signs, whereas the other two lemurs presented with clinical signs of gastrointestinal disease and nonspecific signs of weakness. Gastric pneumatosis, diagnosed either grossly or histopathologically in each of these four lemurs, is described as a subset of PCI in which cystic spaces are localized to the stomach wall. Significant eosinophilic inflammatory infiltrate was identified on histopathology of gastric tissues and found to be associated with the cystic lesions in each lemur. No classic etiology, such as a fungal infection or a parasitic infection, was identified as the cause of the eosinophilic gastritis. This case series demonstrates that gastric pneumatosis with associated eosinophilic gastritis may be a significant gastrointestinal disease in black and white ruffed lemurs.

Bayesian Statistics for Medical Devices: Progress Since 2010.

The use of Bayesian statistics to support regulatory evaluation of medical devices began in the late 1990s. We review the literature, focusing on recent developments of Bayesian methods, including hierarchical modeling of studies and subgroups, borrowing strength from prior data, effective sample size, Bayesian adaptive designs, pediatric extrapolation, benefit-risk decision analysis, use of real-world evidence, and diagnostic device evaluation. We illustrate how these developments were utilized in recent medical device evaluations. In Supplementary Material, we provide a list of medical devices for which Bayesian statistics were used to support approval by the US Food and Drug Administration (FDA), including those since 2010, the year the FDA published their guidance on Bayesian statistics for medical devices. We conclude with a discussion of current and future challenges and opportunities for Bayesian statistics, including artificial intelligence/machine learning (AI/ML) Bayesian modeling, uncertainty quantification, Bayesian approaches using propensity scores, and computational challenges for high dimensional data and models.

Secreted Ly-6/uPAR-related protein-1 (SLURP1) is a pro-differentiation factor that stalls G1-S transition during corneal epithelial cell cycle progression.

PURPOSE: Previously we demonstrated that the secreted Ly-6/uPAR related protein-1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an anti-inflammatory and anti-angiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal phenotype for the first time. METHODS: We compared the 10-week-old wild type (WT) and Slurp1X-/- mouse corneal (i) histology by hematoxylin-eosin and periodic acid-Schiff's reagent staining, (ii) cell proliferation by immunostaining for Ki67, (iii) cell adhesion molecules by immunostaining for desmosomal and tight junction proteins, (iv) barrier function by fluorescein staining and (v) wound-healing by epithelial debridement. Effect of SLURP1 on cell cycle was quantified in human corneal limbal epithelial (HCLE) cells engineered to express SLURP1 (HCLE-SLURP1). RESULTS: WT and Slurp1X-/- corneal histology was largely comparable, other than a few loosely attached superficial cells in Slurp1X-/- corneas. Compared with the WT, Slurp1X-/- corneas displayed (i) increase in Ki67+ cells, (ii) altered expression and/or localization of tight junction proteins Tjp1 and Pard3, and desmosomal Dsp, (iii) increased superficial fragility and (iv) slower CE wound healing. HCLE-SLURP1 cells displayed (i) decrease in Ki67+ cells, (ii) increased cell number doubling time, (iii) stalling in G1-S phase transition during cell cycle, and (iv) downregulation of cyclins CCNE and CCND1/D2, cyclin-dependent kinases CDK4 and CDK6, and upregulation of CDK inhibitor p15/CDKN2B. CONCLUSIONS: Collectively, these results elucidate that Slurp1X-/- CE cell homeostasis is altered and suggest that SLURP1 is a pro-differentiation factor that stalls G1-S transition during cell cycle progression by downregulating cyclins and upregulating p15/CDKN2B.

Comparing a Bayesian Approach (BEST) with the Two One-Sided t-Tests (TOSTs) for Bioequivalence Studies.

The two one-sided t-tests (TOST) procedure has been used to evaluate average bioequivalence (BE). As a regulatory standard, it is crucial that TOST distinguish BE from not-BE (NBE) when BE data are not lognormal. TOST was compared with a Bayesian procedure (BEST by Kruschke) in simulated datasets of test/reference ratios (T/R) which were BE and NBE, wherein (1) log(T/R) or T-R were normally distributed, (2) sample sizes ranged 10-50, and (3) extreme log(T/R) or T-R values were randomly included in datasets. The 90% "credible interval" (CrI) from BEST is a Bayesian alternative of the 90% confidence interval (CI) of TOST and it can be derived from a posterior distribution that is more reflective of the observed mean log(T/R) distribution that often deviates from normality. In the absence of extreme T/R values, both methods agreed BE when observed T/R were lognormal. BEST more accurately concluded BE or NBE, while requiring fewer subjects, when observed log(T/R) or T-R were normal in the presence of extreme values. Overall, TOST and BEST perform comparably on lognormal T/R, while BEST is more accurate, requiring fewer subjects when datasets are normal for T-R or contain extreme values. Of note, the normally distributed datasets only rarely contain extreme values. Our results imply that when BEST and TOST yield different BE assessment results from bioequivalent products, TOST may disadvantage applicants when T/R are not lognormal and/or include extreme T/R values. Application of BEST can address the situation when T/R are not lognormal or include extreme data values. Application of BEST to BE data can be considered a useful alternative to TOST for evaluation of BE and for efficient development of BE formulations.

Bayesian statistics in medical devices: innovation sparked by the FDA.

Bayesian statistical methodology has been used for more than 10 years in medical device premarket submissions to the U.S. Food and Drug Administration (FDA). A complete list of the publicly available information associated with these FDA applications is presented. In addition to the increasing number of Bayesian methodological papers in the statistical journals, a number of successful Bayesian clinical trials in the biomedical journals have been recently reported. Some challenges that require more methodological development are discussed. The promise of using Bayesian methods for incorporation of prior information as well as for conducting adaptive trials is great.

Missing data in the regulation of medical devices.

Handling missing data is an important consideration in the analysis of data from all kinds of medical device studies. Missing data in medical device studies can arise for all the reasons one might expect in pharmaceutical clinical trials. In addition, they occur by design, in nonrandomized device studies, and in evaluations of diagnostic tests. For dichotomous endpoints, a tipping point analysis can be used to examine nonparametrically the sensitivity of conclusions to missing data. In general, sensitivity analysis is an important tool to study deviations from simple assumptions about missing data, such as the data being missing at random. Approaches to missing data in Bayesian trials are discussed, including sensitivity analysis. Many types of missing data that can occur with diagnostic test evaluations are surveyed. Careful planning and conduct are recommended to minimize missing data. Although difficult, the prespecification of all missing data analysis strategies is encouraged before any data are collected.

Severe zinc responsive dermatosis in a litter of Pharaoh Hounds.

A litter of 3-month-old Pharaoh Hound puppies presented to the referring veterinarian with severe generalized erythematous-crusted papules with pruritus, accompanied by exfoliation and erythema of footpads, inappetence, lethargy, and retarded growth. Three of 5 puppies (2 male and 1 female) were affected. Representative areas were biopsied from 1 affected male puppy and were routinely processed. Histologically, there was marked epidermal hyperplasia with a disorganized appearance of the epidermis and massive parakeratotic hyperkeratosis, compatible with zinc-responsive dermatosis. Low serum zinc concentrations were documented, and the affected animals partially responded to intravenous zinc supplementation but did not respond to oral supplementation. One male puppy died as a result of unrelated causes and was necropsied. The remaining 4 puppies were followed over 2 years. Growth was stunted, and enamel hypoplasia of permanent dentition developed compared with unaffected littermates. Intravenous zinc supplementation at 3-4 week intervals was required to prevent further skin lesion development. One dog died at 3 years of age of renal failure.

Two to five year results of primary ACL reconstruction using doubled tibialis anterior allograft.

The aim of the study was to report the 2-5 year results of primary ACL reconstruction with doubled tibialis anterior allograft. Seventy-three patients who underwent primary ACL reconstruction with doubled tibialis anterior allografts with minimum 2 year follow-up were included in the study. Sixty-four patients were available for follow-up. The median age was 27 years (16-55). There were 33 men and 31 women. The median follow-up was 44.5 months (24-55 months). There were two complications, 1 DVT with subsequent PE, and 1 hardware problem. Four patients had failure of their graft, and six patients required repeat arthroscopy. The median Lysholm score was 88 (range 70-95), and the median Tegner activity score was 6.5 (range 3-10). The median IDKC was 92 (range 73-100). According to the IDKC score, 60% of patients were rated as excellent, 27% as good, and 13% as fair. A total of 25 were able to attend the clinical assessment. On KT-1000, 15 (60%) patients had less than 3 mm side-side difference. Eighteen patients (72%) had no pivot shift. ACL reconstruction with allograft tibialis anterior tendon provided good functional results with a low-failure rate at 2-5 years. There was a statistically significant difference in outcome between men and women, with men performing better on the Lysholm and the IDKC scales.