High-throughput identification of RNA nuclear enrichment sequences.

In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever-growing catalogs require high-throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA-based functionality. We applied MPRNA to identify RNA-based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine-rich motif. These nuclear enrichment sequences are highly conserved and over-represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single-molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities.

A gene expression atlas of embryonic neurogenesis in Drosophila reveals complex spatiotemporal regulation of lncRNAs.

Cell type specification during early nervous system development in Drosophila melanogaster requires precise regulation of gene expression in time and space. Resolving the programs driving neurogenesis has been a major challenge owing to the complexity and rapidity with which distinct cell populations arise. To resolve the cell type-specific gene expression dynamics in early nervous system development, we have sequenced the transcriptomes of purified neurogenic cell types across consecutive time points covering crucial events in neurogenesis. The resulting gene expression atlas comprises a detailed resource of global transcriptome dynamics that permits systematic analysis of how cells in the nervous system acquire distinct fates. We resolve known gene expression dynamics and uncover novel expression signatures for hundreds of genes among diverse neurogenic cell types, most of which remain unstudied. We also identified a set of conserved long noncoding RNAs (lncRNAs) that are regulated in a tissue-specific manner and exhibit spatiotemporal expression during neurogenesis with exquisite specificity. lncRNA expression is highly dynamic and demarcates specific subpopulations within neurogenic cell types. Our spatiotemporal transcriptome atlas provides a comprehensive resource for investigating the function of coding genes and noncoding RNAs during crucial stages of early neurogenesis.

A Machine Learning Approach Enables Quantitative Measurement of Liver Histology and Disease Monitoring in NASH.

BACKGROUND AND AIMS: Manual histological assessment is currently the accepted standard for diagnosing and monitoring disease progression in NASH, but is limited by variability in interpretation and insensitivity to change. Thus, there is a critical need for improved tools to assess liver pathology in order to risk stratify NASH patients and monitor treatment response. APPROACH AND RESULTS: Here, we describe a machine learning (ML)-based approach to liver histology assessment, which accurately characterizes disease severity and heterogeneity, and sensitively quantifies treatment response in NASH. We use samples from three randomized controlled trials to build and then validate deep convolutional neural networks to measure key histological features in NASH, including steatosis, inflammation, hepatocellular ballooning, and fibrosis. The ML-based predictions showed strong correlations with expert pathologists and were prognostic of progression to cirrhosis and liver-related clinical events. We developed a heterogeneity-sensitive metric of fibrosis response, the Deep Learning Treatment Assessment Liver Fibrosis score, which measured antifibrotic treatment effects that went undetected by manual pathological staging and was concordant with histological disease progression. CONCLUSIONS: Our ML method has shown reproducibility and sensitivity and was prognostic for disease progression, demonstrating the power of ML to advance our understanding of disease heterogeneity in NASH, risk stratify affected patients, and facilitate the development of therapies.

Assessing the possibilities of designing a unified multistep continuous flow synthesis platform.

The multistep flow synthesis of complex molecules has gained momentum over the last few years. A wide range of reaction types and conditions have been integrated seamlessly on a single platform including in-line separation as well as monitoring. Beyond merely getting considered as 'flow version' of conventional 'one-pot synthesis', multistep flow synthesis has become the next generation tool for creating libraries of new molecules. Here we give a more 'engineering' look at the possibility of developing a 'unified multistep flow synthesis platform'. A detailed analysis of various scenarios is presented considering 4 different classes of drugs already reported in the literature. The possible complexities that an automated and controlled platform needs to handle are also discussed in detail. Three different design approaches are proposed: (i) one molecule at a time, (ii) many molecules at a time and (iii) cybernetic approach. Each approach would lead to the effortless integration of different synthesis stages and also at different synthesis scales. While one may expect such a platform to operate like a 'driverless car' or a 'robo chemist' or a 'transformer', in reality, such an envisaged system would be much more complex than these examples.

Automating multistep flow synthesis: approach and challenges in integrating chemistry, machines and logic.

The implementation of automation in the multistep flow synthesis is essential for transforming laboratory-scale chemistry into a reliable industrial process. In this review, we briefly introduce the role of automation based on its application in synthesis viz. auto sampling and inline monitoring, optimization and process control. Subsequently, we have critically reviewed a few multistep flow synthesis and suggested a possible control strategy to be implemented so that it helps to reliably transfer the laboratory-scale synthesis strategy to a pilot scale at its optimum conditions. Due to the vast literature in multistep synthesis, we have classified the literature and have identified the case studies based on few criteria viz. type of reaction, heating methods, processes involving in-line separation units, telescopic synthesis, processes involving in-line quenching and process with the smallest time scale of operation. This classification will cover the broader range in the multistep synthesis literature.

Current challenges in bioequivalence, quality, and novel assessment technologies for topical products.

This paper summarises the proceedings of a recent workshop which brought together pharmaceutical scientists and dermatologists from academia, industry and regulatory agencies to discuss current regulatory issues and industry practices for establishing therapeutic bioequivalence (BE) of dermatologic topical products. The methods currently available for assessment of BE were reviewed as well as alternatives and the advantages and disadvantages of each method were considered. Guidance on quality and performance of topical products was reviewed and a framework to categorise existing and alternative methods for evaluation of BE was discussed. The outcome of the workshop emphasized both a need for greater attention to quality, possibly, via a Quality-By-Design (QBD) approach and a need to develop a "whole toolkit" approach towards the problem of determination of rate and extent in the assessment of topical bioavailability. The discussion on the BE and clinical equivalence of topical products revealed considerable concerns about the variability present in the current methodologies utilized by the industry and regulatory agencies. It was proposed that academicians, researchers, the pharmaceutical industry and regulators work together to evaluate and validate alternative methods that are based on both the underlying science and are adapted to the drug product itself instead of single "universal" method.

Feasibility and Advantages of Continuous Synthesis of Bioinspired Silica Using CO2 as an Acidifying Agent.

In this work, we present a method for the continuous synthesis of bioinspired porous silica (BIS) particles using carbon dioxide (CO2) as an acidifying agent. Typical BIS synthesis uses strong mineral acids (e.g., HCl) to initiate the hydrolysis and subsequent condensation reactions. The use of strong acids leads to challenges in controlling the reaction pH. The synthesis approach proposed in this work offers for the first time CO2 as an attractive alternative for the synthesis of BIS and demonstrates the continuous process. The developed method leverages the mild acidic and the self-buffering nature of the CO2 combined with additional options for controlling mass transfer rates to facilitate enhanced control of pH, which is crucial for controlling the properties of synthesized BIS. Proof of concept experiments conducted in continuous mode demonstrated a yield of over 70% and a surface area exceeding 500 m2/g. These results indicate the successful synthesis of BIS using CO2 with properties in the desired range. The enhanced pH control offered by this CO2-based process will facilitate the implementation of a sustainable and robust continuous process for BIS synthesis.

Novel Machine Learning-Based Method for Estimation of the Surface Area of Porous Silica Particles.

This work reports a novel and quick method to estimate the surface area of porous materials. Conventionally, surface area measurement requires the BET method/N2 adsorption experiment which is time-consuming. In this work, we developed a method based on machine learning (ML) and the adsorption of a conductive dye on porous materials. The rate and quantity of dye adsorption, which is characterized by dynamic measurement of conductivity, provide an indirect measure of surface area and zeta potential. An ML-based soft sensor is developed to relate the measured conductivity profiles with surface area and zeta potential. A phenomenological model on dye adsorption is also developed, validated, and used to augment experimental data for training the soft sensor. The developed method was tested for porous silica particles with a range of surface areas (250-1100 m2/g) and zeta potential (-17 mV: -29 mV). The developed soft sensor was able to estimate the surface area and zeta potential quite well. The developed approach and method reduce overall measurement time for surface area from several hours to a few minutes. The method can potentially be implemented in continuous plants producing porous materials like silica.

Human radiolabeled mass balance studies supporting the FDA approval of new drugs.

Human radiolabeled mass balance studies are an important component of the clinical pharmacology programs supporting the development of new investigational drugs. These studies allow for understanding of the absorption, distribution, metabolism, and excretion of the parent drug and metabolite(s) in the human body. Understanding the drug's disposition as well as metabolite profiling and abundance via mass balance studies can help inform the overall drug development program. A survey of the US Food and Drug Administration (FDA)-approved new drug applications (NDAs) indicated that about 66% of the drugs had relied on findings from the mass balance studies to help understand the pharmacokinetic characteristics of the drug and to inform the overall drug development program. When such studies were not available in the original NDA, adequate justifications were routinely provided. Of the 104 mass balance studies included in this survey, most of the studies were conducted in healthy volunteers (90%) who were mostly men (>86%). The studies had at least six evaluable participants (66%) and were performed using the final route(s) of administration (98%). Eighty-five percent of the studies utilized a dose within the pharmacokinetic linearity range with 54% of the studies using a dose the same as the approved dose. Nearly all studies were performed as a single-dose (97%) study using a fit-for-purpose radiolabeled formulation. In this analysis, we summarized the current practices for conducting mass balance studies and highlighted the importance of conducting appropriately designed human radiolabeled mass balance studies and the challenges associated with inadequately designed or untimely studies.

Pharmacokinetics of amitriptyline in rabbit skin and plasma following iontophoretic administrations.

BACKGROUND: Amitriptyline (AMT) is a tricyclic antidepressant with demonstrated local analgesic effects in human skin. AIM: We investigated the feasibility and mechanisms of iontophoretic delivery of AMT to rabbit dermis and plasma. METHOD: Two microdialysis probes were inserted into the upper dorsal shaved skin of tranquilized rabbits. After 1 hour, an iontophoresis cartridge was placed on top of one probe. The cartridge consisted of a stainless steel electrode covered with a pad that was filled with a 4.3 % AMT glycerin/water (50:50). Iontophoresis was performed at 100, 200, or 300 microA/cm(2) constant-current density for 60 minutes. Dialysate samples were collected every 8 minutes for 3 hours and analyzed for AMT via a validated high-performance liquid chromatographic assay. Retrodialysis was performed at the other site. Blood samples were collected serially for 4 hours. RESULTS: In vivo retrodialysis recovery was 89 +/- 2%. AMT skin exposure increased non-proportionally with current density: AUCs were 19 +/- 7, 119 +/- 56, and 615 +/- 302 mg/L/min for the 100, 200, and 300 muA/cm(2), respectively, and C(max) were 107 +/- 15, 1070 +/- 537, and 5870 +/- 1289 microg/L. In vivo plasma concentrations were always below LLOQ (0.1 microg/mL). CONCLUSION: AMT can be administered by iontophoresis and produces significant skin concentrations and negligible plasma levels.

Quantification and prediction of skin pharmacokinetics of amoxicillin and cefuroxime.

The purpose of this project was to develop and validate a pharmacokinetic model and to quantify the rate and extent of distribution between plasma and skin of two beta-lactam antibiotics, amoxicillin (AMX) and cefuroxime (CFX), which are frequently administered systemically to treat skin and skin structure infections. Dosing regimens are usually based on plasma concentration, however, concentrations at the target site are better correlated with the effect. For each antibiotic, three different i.v. bolus doses were administered to three female rabbits according to a randomized cross-over design and plasma samples were collected serially. Skin concentrations were obtained by continuous microdialysis. Skin and unbound plasma concentrations were fitted simultaneously using a semi-physiological model and the transfer constants plasma/skin (K(in)) and skin/plasma (K(out)) were estimated. K(in) and K(out) were then used to predict skin concentrations from the plasma levels obtained from an oral administration of AMX or from an i.v. bolus of CFX. The predicted skin profiles were similar to those measured by microdialysis during the actual experiments. In conclusion, this study shows that it is possible to generate a reasonable prediction of skin pharmacokinetics from any plasma level once a careful characterization of the transfer process between plasma and skin has been made.

A practical guide to methods controlling false discoveries in computational biology.

BACKGROUND: In high-throughput studies, hundreds to millions of hypotheses are typically tested. Statistical methods that control the false discovery rate (FDR) have emerged as popular and powerful tools for error rate control. While classic FDR methods use only p values as input, more modern FDR methods have been shown to increase power by incorporating complementary information as informative covariates to prioritize, weight, and group hypotheses. However, there is currently no consensus on how the modern methods compare to one another. We investigate the accuracy, applicability, and ease of use of two classic and six modern FDR-controlling methods by performing a systematic benchmark comparison using simulation studies as well as six case studies in computational biology. RESULTS: Methods that incorporate informative covariates are modestly more powerful than classic approaches, and do not underperform classic approaches, even when the covariate is completely uninformative. The majority of methods are successful at controlling the FDR, with the exception of two modern methods under certain settings. Furthermore, we find that the improvement of the modern FDR methods over the classic methods increases with the informativeness of the covariate, total number of hypothesis tests, and proportion of truly non-null hypotheses. CONCLUSIONS: Modern FDR methods that use an informative covariate provide advantages over classic FDR-controlling procedures, with the relative gain dependent on the application and informativeness of available covariates. We present our findings as a practical guide and provide recommendations to aid researchers in their choice of methods to correct for false discoveries.

Function and evolution of local repeats in the Firre locus.

More than half the human and mouse genomes are comprised of repetitive sequences, such as transposable elements (TEs), which have been implicated in many biological processes. In contrast, much less is known about other repeats, such as local repeats that occur in multiple instances within a given locus in the genome but not elsewhere. Here, we systematically characterize local repeats in the genomic locus of the Firre long noncoding RNA (lncRNA). We find a conserved function for the RRD repeat as a ribonucleic nuclear retention signal that is sufficient to retain an otherwise cytoplasmic mRNA in the nucleus. We also identified a repeat, termed R0, that can function as a DNA enhancer element within the intronic sequences of Firre. Collectively, our data suggest that local repeats can have diverse functionalities and molecular modalities in the Firre locus and perhaps more globally in other lncRNAs.

Navigating sticky areas in transdermal product development.

The benefits of transdermal delivery over the oral route to combat such issues of low bioavailability and limited controlled release opportunities are well known and have been previously discussed by many in the field (Prausnitz et al. (2004) [1]; Hadgraft and Lane (2006) [2]). However, significant challenges faced by developers as a product moves from the purely theoretical to commercial production have hampered full capitalization of the dosage forms vast benefits. While different technical aspects of transdermal system development have been discussed at various industry meetings and scientific workshops, uncertainties have persisted regarding the pharmaceutical industry's conventionally accepted approach for the development and manufacturing of transdermal systems. This review provides an overview of the challenges frequently faced and the industry's best practices for assuring the quality and performance of transdermal delivery systems and topical patches (collectively, TDS). The topics discussed are broadly divided into the evaluation of product quality and the evaluation of product performance; with the overall goal of the discussion to improve, advance and accelerate commercial development in the area of this complex controlled release dosage form.

Maximal Usage Trial: An Overview of the Design of Systemic Bioavailability Trial for Topical Dermatological Products.

Dermatologic diseases can present in varying forms and severity, ranging from the individual lesion and up to almost total skin involvement. Pharmacokinetic assessment of topical drug products has previously been plagued by bioanalytical assay limitations and the lack of a standardized study design. Since the mid-1990's the US Food and Drug Administration has developed and implemented a pharmacokinetic maximal usage trial (MUsT) design to help address these issues. The MUsT design takes into account the following elements: the enrollment of patients rather than normal volunteers, the frequency of dosing, duration of dosing, use of highest proposed strength, total involved surface area to be treated at one time, amount applied per square centimeter, application method and site preparation, product formulation, and use of a sensitive bioanalytical method that has been properly validated. This paper provides a perspective of pre-MUsT study designs and a discussion of the individual elements that make up a MUsT.

Pharmacokinetics of methotrexate in rabbit skin and plasma after iv-bolus and iontophoretic administrations.

The pharmacokinetics of methotrexate (MTX) in rabbit's skin and plasma after iv-bolus and iontophoretic delivery at different current densities was studied. Linear microdialysis probes were introduced into the upper dorsal shaved skin of tranquilized rabbits. Commercially available patches were used to deliver MTX for 1 h at different current densities (100, 200, and 300 microA/cm2) on different occasions. Iv-boluses (10 mg/kg) of MTX were also administered. Retrodialysis was performed at the end of the experiments to estimate probe recovery. Plasma and microdialysis samples were analyzed using a validated HPLC assay. Following iv-bolus, MTX showed a bi-exponential decay both in plasma and in skin. Cmax in skin occurred with a delay of 22 min compared with plasma. No quantifiable concentration of MTX was detected in the skin on passive drug delivery. Systemic exposure to MTX (AUC) and Cmax increased linearly with current density. Nevertheless, exposure to MTX in the skin did not increase linearly with current density, whereas Cmax did. In conclusion, iontophoresis remarkably improved the dermal delivery of MTX over passive diffusion. However, total exposure did not increase with current density in the skin, suggesting that for local applications lower current densities may achieve the same effects with minimal systemic exposure.

Bioanalytical method validation: concepts, expectations and challenges in small molecule and macromolecule--a report of PITTCON 2013 symposium.

The concepts, importance, and implications of bioanalytical method validation has been discussed and debated for a long time. The recent high profile issues related to bioanalytical method validation at both Cetero Houston and former MDS Canada has brought this topic back in the limelight. Hence, a symposium on bioanalytical method validation with the aim of revisiting the building blocks as well as discussing the challenges and implications on the bioanalysis of both small molecules and macromolecules was featured at the PITTCON 2013 Conference and Expo. This symposium was cosponsored by the American Chemical Society (ACS)-Division of Analytical Chemistry and Analysis and Pharmaceutical Quality (APQ) Section of the American Association of Pharmaceutical Scientists (AAPS) and featured leading speakers from the Food & Drug Administration (FDA), academia, and industry. In this symposium, the speakers shared several unique examples, and this session also provided a platform to discuss the need for continuous vigilance of the bioanalytical methods during drug discovery and development. The purpose of this article is to provide a concise report on the materials that were presented.

Skin microdialysis-based estimation of systemic bioavailability fraction.

Systemic bioavailability is usually determined from plasma data. However, when plasma is difficult to access, as in young children, alternative methods would be particularly beneficial. The present study investigates the possibility of calculating systemic bioavailability fraction (F) from skin concentrations measured by two microdialysis (MD) sampling methods: continuous microdialysis and intermittent microdialysis. When the drug concentration in skin is a linear and time-invariant function of plasma concentration, the area under the drug concentration curve in skin is directly proportional to the drug absorbed systemically. To verify this theory, we compared the F estimated from MD concentrations in the skin with that obtained from the plasma data in the same experiment. Two model drugs were selected for the study: amoxicillin and ketoprofen. Drugs were administered to rabbits as intravenous infusion or oral suspension according to a randomized crossover design. F estimated by either MD method was not significantly different from that obtained from the plasma for both drugs tested. However, the skin data exhibited a larger variability. These results confirm that skin MD could be an alternative way to obtain data for the calculation of systemic fraction of drug absorbed.

In vivo quantification of acyclovir exposure in the dermis following iontophoresis of semisolid formulations.

The objective was to quantify acyclovir (ACV) exposure in the dermis following iontophoresis of nontraditional (pH-11 gels) and traditional (neutral cream) topical formulations of ACV. Given that the application time of a formulation on the skin can be significantly reduced with iontophoresis, the use of formulations optimized for iontophoretic delivery was explored to maximize the delivery of ACV to the site of action for the treatment of Herpes-labialis. Microdialysis probes were inserted into the shaved skin of tranquilized rabbits. Iontophoresis was performed at a constant current density of 200 microA/cm(2) for 60 min using a single-use drug cartridge filled with the following formulations: neutral cream, soluble fraction of the same cream (anodal and cathodal-current), or two formulations of pH-11 gels, one without and one with stabilizers and preservatives (cathodal-current). Results showed that only the ACV in the water phase of the cream is available for transport. All of the pH-11 gels exhibited a statistically significant (p < 0.001) increase in ACV dermis exposure compared to the neutral cream formulations without any additional sign of skin irritation. In conclusion, iontophoresis of ACV pH-11 gels provides higher ACV concentrations in the dermis than iontophoresis of neutral cream formulations, which could result in a better clinical outcome.