Poor Template Research Articles

Fabrication of AAO-Based Four-Stack Thin Film Solid Oxide Fuel Cells for the Low-Temperature Operation Using Sputtering Method

Anodic Aluminium Oxide (AAO) based four thin-film solid oxide fuel cell (TF-SOFC) stack was fabricated. The fuel cell stack was tested and analyzed at the low-temperature window. Since the low-temperature operation has many advantages, such as lowering the system cost and widening the material selection, there has been much effort to reduce conductive resistance, mainly at the electrolyte, which is the most problematic issue in low-temperature operation. One of the solutions is TF-SOFC deposited on the various substrates or supporting materials. AAO, a non-conductive and nanoporous substrate, has been widely used in fabricating TF-SOFC as a substrate because of its uniformly arranged pores and highly developed manufacturing process.However, AAO is a poor template for stacking TF-SOFCs because it has lower mechanical strength than metal, has no electrical conductivity, and has a different coefficient of thermal expansion from metal. In this study, therefore, planarly stacked TF-SOFC were fabricated to expand its limits of use as a template for the stacked fuel cell. Nickel-based anode, yttria-stabilized zirconia (YSZ) electrolyte with functional layers, and platinum cathode and current collector were sequentially deposited using sputtering and atomic layer deposition methods. All thin-film components of four cells were deposited on the AAO at the same time, making good connections between each electrode. By effectively setting the deposition process, unnecessary pores of AAO were blocked, and current collecting layers were stably formed in between the structure of the cells with a step difference.Then, the stacked TF-SOFC were electrochemically analyzed, including the current density-voltage-power density curve (i-V-P curve) and electrochemical impedance spectroscopy (EIS) being compared to a single cell. The microstructure of the fuel cell and its electrically connected structures were observed by scanning electron microscopy (SEM) and focused ion beam (FIB) cross-sectional SEM analyses. The cell’s overall voltage was comparable to four times that of a single cell, and the power density was obtained. We revealed the correlation between the electrochemical performance and the microstructure of individual cells and the whole stack. The full stack achieved electrochemical and mechanical stability by forming a complete fuel cell structure and enhanced the applicability of the planar cell stack on the AAO substrate.

A multi-criteria decision-making approach to help resource-exhausted areas choose suitable transformation templates—The example of Wansheng in Chongqing, China

• Differences in conditions lead choose suitable transformation templates difficult. • Interaction between problems are critical in select suitable transformation templates. • ANP and DEMATEL can complement each other to make evaluation more accurate. • Administrative-led models are suitable for solving current problems. • Market-led models are suitable for further transformation development. China and other major economies of the world have invested considerable resources to assist resource-exhausted areas in their development. But few regions have improved the stagnant state of development. The literature suggests that the lack of assistance provided to decision-makers when choosing suitable transformation templates often results in the selection of poor templates that are not suitable for a given region. This is an important factor that contributes to the difficulties among resource-exhausted areas to achieve transformation. This paper describes an application of hybrid Multi Criteria Decision Making (MCDM) technique for the selection of optimum template for the resource-exhausted region. The proposed model, Fuzzy Analytical Network Process (Fuzzy-ANP) is integrated with Fuzzy Decision-Making Trial and Evaluation Laboratory (Fuzzy-DEMATEL) to evaluate the research object and candidate templates. Evaluation is based on problems (criteria) including the developing new industries, improving governance capabilities, enhancing the ability to attract production, reducing unemployment, restoring an ecological environment and transformation the original resource-mining industry. Fuzzy-ANP and Fuzzy-DEMATEL are used to determine the priority of these criteria for the research region and for candidate template regions from two viewpoints: the importance of each issue, and the ability of each issue to influence other issues. This paper selects Wangsheng as research region and Ruhr, Chikuho, Lorraine and Houston as templates. The obtain preference order of the templates for Fuzzy-ANP and Fuzzy-DEMATEL are Houston > Lorraine > Chikuho > Ruhr and Ruhr > Chikuho > Houston > Lorraine respectively. Combining two results, the order of the templates is Chkuho > Lorranie > Houston > Ruhr. Chikuho is selected as template for Wansheng transformation.

Discovery of Novel Activators of Large-Conductance Calcium-Activated Potassium Channels for the Treatment of Cerebellar Ataxia.

Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative spinocerebellar ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and are therefore poor templates for future drug development. We implemented an automated patch clamp platform for high-throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channel activators that were then retested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that exhibited a more potent half-maximal activation of BK current (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on small-conductance calcium-activated potassium (SK)1-3 channels but interestingly was a potent blocker of the T-type calcium channel, Cav3.1 (IC50 = 1.05 μM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. SIGNIFICANCE STATEMENT: Motor impairment associated with altered Purkinje cell spiking due to dysregulation of large-conductance calcium-activated potassium (BK) channel expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug development for the treatment of these disabling disorders.

Detection of chlorpyrifos based on molecular imprinting with a conducting polythiophene copolymer loaded on multi-walled carbon nanotubes

Molecular imprinting technique (MIT) with electrochemical sensing provides an attractive tool for the fabrication of sensors. Incorporation of conducting copolymer and surface imprinting strategies in the sensing device improves the conducting properties and poor template accessibility, slow binding kinetics at the same time. Here, this technique was employed with conducting polymers with multi-walled carbon nanotubes (MWCNT) to build an electrochemical sensor for detecting Chlorpyrifos (CPF) in vegetable sample solutions. In this work, we aimed at synthesizing a copolymer of 3-thiophene acetic acid and 3,4-ethylene dioxythiophene on the surface of MWCNT. We further constructed a polymer drop-casted glassy carbon electrode sensor for ultrasensitive detection CPF. Under optimal conditions, the sensor exhibited a very low limit of detection (LOD) of 4.0 × 10-12 M for CPF. Due to the excellent repeatability and reusability of the materials, this study and findings have potential applications in the monitoring of pesticides from vegetable samples.

A quality improvement project to increase palliative care team involvement in pediatric oncology patients.

Pediatric palliative care (PPC) for oncology patients improves quality of life and the likelihood of goal-concordant care. However, barriers to involvement exist. We aimed to increase days between PPC consult and death for patients with refractory cancer from a baseline median of 13.5 days to ≥30 days between March 2019 and March 2020. Outcome measure was days from PPC consult to death; process measure was days from diagnosis to PPC consult. The project team surveyed oncologists to identify barriers. Plan-do-study-act cycles included establishing target diagnoses, offering education, standardizing documentation, and sending reminders. The 24-month baseline period included 30 patients who died and 25 newly diagnosed patients. The yearlong intervention period included six patients who died and 16 newly diagnosed patients. Interventions improved outcome and process measures. Targeted patients receiving PPC ≥30 days prior to death increased from 43% to 100%; median days from consult to death increased from 13.5 to 159.5. Targeted patients receiving PPC within 30 days of diagnosis increased from 28% to 63%; median days from diagnosis to consult decreased from 221.5 to 14. Of those without PPC consult≤30 days after diagnosis, 17% had template documentation of the rationale. Interventions utilized met the global aim, outcome, and process measures. Use of QI methodology empowered providers to involve PPC. Poor template use was a barrier to identifying further drivers. Future directions for this project relate to expanding the target list, creating long-term sustainability, formalizing standards, and surveying patients and families.

DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

Characterization of FFPE-induced bacterial DNA damage and development of a repair method.

Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterized. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterize this FFPE-induced bacterial DNA damage and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised – an optimized decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.

Abstract 3520: Detection of low-frequency variants in highly degraded DNA and RNA samples

Abstract Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and formalin-fixed paraffin-embedded (FFPE) DNA. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity and variant detection power. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by “duplex sequencing”, where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. Here, we present the IDT library preparation kit optimized for low-input and degraded samples. Our novel library construction chemistry relies on an engineered DNA ligase and proprietary duplexed sequencing adapters that prevent chimeras, suppress dimer-formation (negating the need for size-selection), and enhance variant calling sensitivity. We adapted the workflow for both DNA and RNA applications and demonstrated efficacy using diverse sample types. To assess sensitivity, we created libraries with varied inputs using mixtures of Genome in a Bottle gDNA (NA12878 and NA24385) and performed hybrid capture using a 52 kb custom panel targeting: single nucleotide variants (SNVs), copy number variants (CNVs), and gene fusions. When compared to commercially available methods, our approach yielded a 1.5- to 4-fold increase in library complexity with improved sensitivity to 0.25% variants using 1-25 ng of cfDNA, and 0.5% using 25-250 ng FFPE DNA. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. RNA libraries were constructed from FFPE NGS reference standards to evaluate the detection of ALK, RET, ROS, NTRK1, and NTRK3 fusions and sequenced to an average target depth of 10,000X. Our method provides superior sensitivity and specificity for detection of low-frequency variants, even with highly degraded DNA. Citation Format: Ariel Royall, Ushati Das Chakravarty, Katharine Dilger, Manqing Hong, Kevin Lai, Kristina Giorda, Keith Bryan, Yu Wang, Lynette Lewis, Scott Rose, Yu Zheng. Detection of low-frequency variants in highly degraded DNA and RNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3520.

DESTINI: A deep-learning approach to contact-driven protein structure prediction

The amino acid sequence of a protein encodes the blueprint of its native structure. To predict the corresponding structural fold from the protein’s sequence is one of most challenging problems in computational biology. In this work, we introduce DESTINI (deep structural inference for proteins), a novel computational approach that combines a deep-learning algorithm for protein residue/residue contact prediction with template-based structural modelling. For the first time, the significantly improved predictive ability is demonstrated in the large-scale tertiary structure prediction of over 1,200 single-domain proteins. DESTINI successfully predicts the tertiary structure of four times the number of “hard” targets (those with poor quality templates) that were previously intractable, viz, a “glass-ceiling” for previous template-based approaches, and also improves model quality for “easy” targets (those with good quality templates). The significantly better performance by DESTINI is largely due to the incorporation of better contact prediction into template modelling. To understand why deep-learning accomplishes more accurate contact prediction, systematic clustering reveals that deep-learning predicts coherent, native-like contact patterns compared to co-evolutionary analysis. Taken together, this work presents a promising strategy towards solving the protein structure prediction problem.

Pressure Injury Documentation Practices in the Department of Veteran Affairs: A Quality Improvement Project.

Evidence suggests that inaccurate and incomplete pressure injury (PI) documentation threatens the validity of treatment and undermines policy and quality improvement. This quality improvement project sought to identify barriers and facilitators when conducting and documenting the daily comprehensive skin assessment in 31 Department of Veterans Affairs (VA) facilities. Evaluators in this 1-year, cross-sectional quality improvement project, using a qualitative approach, interviewed nurses of medical-surgical and critical care units. Participants (N = 62) from 12 high reassessment units (HRUs) and 13 low reassessment units (LRUs) were interviewed using telephone focus groups. Staff from HRUs reported 9 activities that ensured consistency in clinical practices, in validating data, and in correcting inaccuracies. The LRU staff tended to report performing only 2 of the 9 activities. The main barriers to accurate documentation were lack of knowledge, poor templates, and staffing issues such as understaffing and turnover, and main facilitators were an internal data validation process and a documentation template to local practices. Findings from this project led to increased VA leadership engagement, development of 3 innovative, award-winning VA mobile PI prevention and management applications, updated policies and directives on PI prevention, and upgrading of the national VA HAPI workgroup to an advisory committee and improved collaboration between the PI advisory committee and nursing informatics.

Perspectives on Self-Compassion From Adult Female Survivors of Sexual Abuse and the Counselors Who Work With Them.

While compassion-focused therapy (CFT) holds significant promise as an intervention for survivors of sexual abuse, a history of abuse can uniquely impact an individual's capacity to cultivate compassion and may generate a fear of compassion. Understanding the specific perspectives of sexual abuse survivors may inform the application of CFT-based interventions with this client group. Two separate focus groups were established for this purpose, one with adult female survivors of sexual abuse (n = 7) and another with sexual abuse counselors (n = 7). Transcripts were analyzed according to a consensual qualitative research design. Analysis of the survivor focus group identified two core domains, Barriers to Compassion, including poor relational templates, negative perception of self, low coping self-efficacy, and fears, resistance, and misperceptions regarding self-compassion, and Factors Supporting Compassion, including support from others, compassion for others, high coping self-efficacy, motivation and hope for change, and timing and readiness for change. Analysis of the counselor focus group revealed three domains, Therapeutic Factors to Support Compassion, including counselor authenticity and modeling, gradual introduction with consideration to individual needs, acknowledgment of suffering and offering an alternative perspective; Factors Affecting Client Readiness and Capacity, including shame, self-blame, and negative sense of self, response from others, and difficulty in changing self-critical habits; and Anticipated Outcomes, including providing a hope and recovery focus, offering an alternative perspective and coping strategy, and restoring trust. Findings are discussed in relation to clinical implications and relevance to a CFT model of intervention.

Abstract 430: Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method

Abstract Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and FFPE DNA. Furthermore, clinical samples are often available in limited quantity, making the detection of pathologic variants particularly challenging. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by “duplex sequencing”, where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. We present a novel library construction chemistry that eliminates adapter dimers and utilizes duplexed UMIs to increase sensitivity. The method employs a two-step ligation procedure that does not require size-selection or adjustment of AMPure bead ratios during cleanup. We achieve maximal library conversion using a unique, mutant DNA ligase and proprietary sequencing adapters that increase ligation efficiency and suppress chimera formation. We demonstrate performance using three sample types: sheared genomic DNA, cfDNA, and FFPE DNA, across a wide input range (1-1000 ng). To mimic performance with degraded DNA, we created libraries from mixtures of NA12878 and NA24385 cell line DNA sheared to 150 bp with inputs of 1-25 ng and mutant allele fractions (MAFs) down to 0.25%. Libraries were enriched using a 75 kb panel of xGen Lockdown Probes, followed by ultra-deep sequencing and variant calling. When compared to commercially available methods, our approach yielded a 50-100% increase in library complexity with significantly improved sensitivity to <1% variants. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. To highlight clinical utility, we extended our study to cfDNA samples with inputs of 5-25 ng and MAFs down to 1%, and FFPE DNA samples with inputs of 25-100 ng and MAFs down to 1%. Citation Format: Ushati Das Chakravarty, Zac Zwirko, Yu Zheng, Madelyn Light, Kevin Lai, Keith Bryan, Scott Rose, Yun Bao, Mirna Jarosz, Caifu Chen. Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 430.

C60 layer growth on intact and Tl-modified Si(1 1 1)5 [formula omitted] 2-Au surfaces

The Si(1 1 1)5 × 2-Au surface comprises a quasi-one-dimensional surface reconstruction consisting of Au stripes mediated by honeycomb Si chains. In the present study, using STM observations we explored adsorption of thallium (Tl) onto Si(1 1 1)5 × 2-Au surface as well as growth of C60 layers on Tl-modified and intact Si(1 1 1)5 × 2-Au surfaces. Analysis of the results was based on the recently proposed KK model of Si(1 1 1)5 × 2-Au surface [Kwon and Kang, PRL 113 (2014) 086101]. It was found that intact Si(1 1 1)5 × 2-Au surface produces a poor template effect on the overgrown C60 layers which have a close-packed-like structure without distinct ordering. In contrast, the C60 layers grown on the Tl-adsorbed Si(1 1 1)5 × 2-Au surface where Tl atoms occupy various adsorption sites on the honeycomb Si chains have a well-ordered quasi-one-dimensional structure consisting of straight C60 chains. Ordering of the C60 molecules is a result of an intricate interplay between adsorbed C60 molecules with each other and with a substrate surface. Separation between neighboring C60 chains is dictated by the periodicity of the underlying Si(1 1 1)5 × 2-Au template, while the C60-C60 spacing within the chains of ∼12 Å that is ∼3.2a0 reflects the peculiarity of the C60-C60 intermolecular interaction on the Tl-adsorbed Si(1 1 1)5 × 2-Au surface. Structural information on (Au, Tl)/Si(1 1 1) and C60/(Au, Tl)/Si(1 1 1) systems gained in the present study can be useful for the prospective works on elucidating the ways to control electronic properties of the Si(1 1 1)5 × 2-Au surface using atomic and molecular adsorbates.

Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics

The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.

The inadequate bladder template: Its effect on outcomes in classic bladder exstrophy

Newborns with classic bladder exstrophy (CBE) may present with a bladder template that is inadequate for closure in the neonatal period (figure). In these cases, a delayed primary closure (DPC) is conducted to permit growth of the bladder template. This study reports the surgical and long-term urinary continence outcomes of poor template CBE patients undergoing DPC and compares them to patients who underwent DPC for reasons unrelated to bladder quality (i.e., prematurity, comorbidities, or a late referral). An institutionally approved, prospectively maintained database of 1330 exstrophy-epispadias complex patients was reviewed for CBE patients who underwent DPC at the authors' institution. A bladder template was considered inadequate for neonatal closure if found to be inelastic, <3cm in diameter, and/or covered in hamartomatous polyps. In total, 63 patients (53 male and 10 female) undergoing DPC were identified. Of these, 36 had poor bladder templates (group 1). The remaining 27 patients (group 2) had adequate templates and their bladder closure was delayed for reasons unrelated to bladder quality. At the time of DPC, those in group 1 were relatively than those in group 2 (median of 229 vs. 128 days, p=0.094). All 36 group 1 patients and 26 (96%) group 2 patients underwent pelvic osteotomy during DPC (p=0.429). All patients in this study had a successful primary closure. There was little difference in longitudinal bladder capacities between group 1 and group 2 (p=0.518). Also, there was minimal difference in the median number of continence procedures between groups, with both groups having 1 (IQR 1-1) continence procedure (p=0.880). Eight patients in group 1, and three patients in group 2 underwent a bladder neck transection with urinary diversion. Of the 13 and 16 patients who have undergone a continence procedure in group 1 and 2, respectively, 11 (84.6%) and 13 (81.3%) are continent of urine. The age of first continence procedure was different between groups 1 and 2 at 8.0 years (5.8-9.9 years) and 4.8 (3.5-6.0 years), respectively p=0.009. The majority of patients in group 1 established continence at a relatively later age when compared to those in group 2, at 11.4 (8.0-14.8) years and 7.9 (2.6-13.2) years of age respectively p=0.087. In the authors' view, neonatal bladder closure is ideal for CBE patients as it minimizes potential damage to exposed bladder mucosa. However, prior studies indicate that the rate of bladder growth for patients undergoing a delayed primary closure does not differ from patients with a neonatal closure. Results from this study show continued evidence that patients with poor templates who undergo delayed closure have excellent primary closure outcomes, which is critical for further management. Furthermore, this study shows that an inadequate bladder does not affect DPC outcomes or the continence outcomes in DPC patients. However, the inadequate template does affect the type of continence procedure available to a DPC patient, the age of first continence procedure, and the age of continence. DPC of the exstrophic bladder has a high rate of success when pelvic osteotomy is utilized as an adjunct. Patients having a DPC for reasons of an inadequate bladder template have comparable rates of bladder growth when compared to DPC of an adequate bladder template. The inadequate bladder template affects the type of continence procedure, with the majority of patients requiring urinary diversion for continence. Patients with an inadequate bladder template have a later age of first continence procedure and a relatively later age of continence, because of an inherently smaller bladder template at birth. The inadequate bladder template patients require a longer period of surveillance to access bladder growth and capacity in preparation of a continence procedure. Furthermore, as the majority of inadequate bladder template patients require a catheterizable channel for continence, the age of continence is also likely influenced by the patient's preparation as they transition from volitional voiding to catheterization.

Cystectomy in the Pediatric Exstrophy Population: Indications and Outcomes

To explore the long-term outcomes and indications for cystectomy in patients with bladder exstrophy. Although rare, cystectomy is the final surgical alternative to bladder repair among these patients with a poor quality bladder template. A prospectively maintained database of 1298 patients with exstrophy-epispadias complex was reviewed for patients who underwent cystectomy between 1970 and 2015 at the authors' institution. Demographic data, indication for cystectomy, surgical history, postoperative outcomes, and continence status were collected. Eighteen (6 male; 12 female) patients with exstrophy (15 classic bladder exstrophy; 2 bladder exstrophy variants; 1 cloacal exstrophy) underwent cystectomy at a median age of 3.8 years. Six patients (33.3%) underwent primary cystectomy without attempted bladder closure. Eight patients (44.4%) had a history of failed primary closure with loss of capacity or inadequate growth after closure. Four patients (22.2%) had successful primary closure but underwent cystectomy secondary to poor bladder compliance with declining renal function or poor bladder growth or quality. Urinary diversion included 6 cutaneous ureterostomies, 4 bowel conduits (1 ileal; 3 colon), 6 continent urinary diversions with ileosigmoid reservoir, and 1 ureterosigmoidostomy. Of 8 patients who underwent a continence procedure, all were dry at a median of 25.3 months after cystectomy. Cystectomy was most commonly indicated in intrinsically diseased bladder templates that remained too small despite permitting time for interval growth. These bladders often were of poor quality and compliance and did not reduce into the pelvis on examination. It was, however, possible to achieve urinary continence in these patients with cystectomy and urinary diversion.

High-Resolution Gene Expression Profiling of RNA Synthesis, Processing, and Decay by Metabolic Labeling of Newly Transcribed RNA Using 4-Thiouridine.

Cellular RNA levels are orchestrated by highly regulated processes involving RNA synthesis (transcription), processing (e.g., splicing, polyadenylation, transport), and degradation. Profiling these changes provides valuable information on the regulation of gene expression. Total cellular RNA is a poor template for revealing short-term changes in gene expression, alterations in RNA decay rates, and the kinetics of RNA processing as well as the differentiation thereof. Here, we describe the metabolic labeling and purification of newly transcribed RNA with 4-thiouridine, by which these limitations are overcome.

Improving optical performance of GaN nanowires grown by selective area growth homoepitaxy: Influence of substrate and nanowire dimensions

Series of GaN nanowires (NW) with controlled diameters (160–500 nm) and heights (420–1100 nm) were homoepitaxially grown on three different templates: GaN/Si(111), GaN/AlN/Si(111), and GaN/sapphire(0001). Transmission electron microscopy reveals a strong influence of the NW diameter on dislocation filtering effect, whereas photoluminescence measurements further relate this effect to the GaN NWs near-bandgap emission efficiency. Although the templates' quality has some effects on the GaN NWs optical and structural properties, the NW diameter reduction drives the dislocation filtering effect to the point where a poor GaN template quality becomes negligible. Thus, by a proper optimization of the homoepitaxial GaN NWs growth, the propagation of dislocations into the NWs can be greatly prevented, leading to an exceptional crystal quality and a total dominance of the near-bandgap emission over sub-bandgap, defect-related lines, such as basal stacking faults and so called unknown exciton (UX) emission. In addition, a correlation between the presence of polarity inversion domain boundaries and the UX emission lines around 3.45 eV is established.

Assessment of template-based modeling of protein structure in CASP11.

We present the assessment of predictions submitted in the template-based modeling (TBM) category of CASP11 (Critical Assessment of Protein Structure Prediction). Model quality was judged on the basis of global and local measures of accuracy on all atoms including side chains. The top groups on 39 human-server targets based on model 1 predictions were LEER, Zhang, LEE, MULTICOM, and Zhang-Server. The top groups on 81 targets by server groups based on model 1 predictions were Zhang-Server, nns, BAKER-ROSETTASERVER, QUARK, and myprotein-me. In CASP11, the best models for most targets were equal to or better than the best template available in the Protein Data Bank, even for targets with poor templates. The overall performance in CASP11 is similar to the performance of predictors in CASP10 with slightly better performance on the hardest targets. For most targets, assessment measures exhibited bimodal probability density distributions. Multi-dimensional scaling of an RMSD matrix for each target typically revealed a single cluster with models similar to the target structure, with a mode in the GDT-TS density between 40 and 90, and a wide distribution of models highly divergent from each other and from the experimental structure, with density mode at a GDT-TS value of ∼20. The models in this peak in the density were either compact models with entirely the wrong fold, or highly non-compact models. The results argue for a density-driven approach in future CASP TBM assessments that accounts for the bimodal nature of these distributions instead of Z scores, which assume a unimodal, Gaussian distribution. Proteins 2016; 84(Suppl 1):200-220. © 2016 Wiley Periodicals, Inc.

A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples

A multiplex PCR-based method was developed to overcome the limitations of microscopic examination as a means of identifying individual infective larvae from the wide range of strongylid parasite species commonly encountered in sheep in mixed sheep-cattle grazing situations in New Zealand. The strategy employed targets unique species-specific sequence markers in the second internal transcribed spacer (ITS-2) region of ribosomal DNA of the nematodes and utilises individual larval lysates as reaction templates. The basic assay involves two sets of reactions designed to target the ten strongylid species most often encountered in ovine faecal cultures under New Zealand conditions (viz. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticei, Cooperia oncophora, Nematodirus spathiger, Chabertia ovina, and Oesophagostomum venulosum). Five species-specific primers, together with a pair of “generic” (conserved) primers, are used in each of the reactions. Two products are generally amplified, one by the generic primer pair regardless of species (providing a positive PCR control) and the other (whose size is indicative of the species present) by the appropriate species-specific primer in combination with one or other of the generic primers. If necessary, any larvae not identified by these reactions can subsequently be tested using primers designed specifically to detect those species less frequently encountered in ovine faecal cultures (viz. Ostertagia ostertagi, Ostertagia leptospicularis, Cooperia punctata, Nematodirus filicollis, and Bunostomum trigonocephalum). Results of assays undertaken on >5500 nematode larvae cultured from lambs on 16 different farms distributed throughout New Zealand indicated that positive identifications were initially obtained for 92.8% of them, while a further 4.4% of reactions gave a generic but no visible specific product and 2.8% gave no discernible PCR products (indicative of insufficient or poor quality DNA template). Of the reactions which yielded only generic products, 91% gave positive identifications in an assay re-run, resulting in a failure rate of just ∼0.4% for reactions containing amplifiable template. Although the method was developed primarily to provide a reliable way to identify individual strongylid larvae for downstream molecular applications, it potentially has a variety of other research and practical applications which are not readily achievable at present using other methods.