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Abstract Aging manifests as progressive deteriorations in homeostasis, requiring systems-level perspectives to investigate the gradual molecular dysregulation of underlying biological processes. Here, we report systemic changes in the molecular regulation of biological processes under multiple lifespan-extending interventions. Differential Rank Conservation (DIRAC) analyses of mouse liver proteomics and transcriptomics data show that mechanistically distinct lifespan-extending interventions (acarbose, 17α-estradiol, rapamycin, and calorie restriction) generally tighten the regulation of biological modules. These tightening patterns are similar across the interventions, particularly in processes such as fatty acid oxidation, immune response, and stress response. Differences in DIRAC patterns between proteins and transcripts highlight specific modules which may be tightened via augmented cap-independent translation. Moreover, the systemic shifts in fatty acid metabolism are supported through integrated analysis of liver transcriptomics data with a mouse genome-scale metabolic model. Our findings highlight the power of systems-level approaches for identifying and characterizing the biological processes involved in aging and longevity.

Abstract The SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2013 Statement provides evidence‐based recommendations for the minimum content of clinical trial protocols. The Cellular Molecular Pathology Initiative, hosted by the UK National Cancer Research Institute, developed an extension, SPIRIT‐Path, describing how to effectively incorporate pathology support into clinical trial protocols. The current study assessed the inclusion of SPIRIT‐Path items in protocols of active clinical trials. Publicly available clinical trial protocols were identified for assessment against the new guidelines using a single UK hospital as the ‘test site’. One hundred and ninety interventional clinical trials were identified as receiving support from the pathology department. However, only 38 had publicly available full trial protocols (20%) and following application of the inclusion/exclusion criteria, 19 were assessed against the SPIRIT‐Path guidelines. The reviewed clinical trial protocols showed some areas of compliance and highlighted other items that were inadequately described. The latter lacked information about the individuals responsible for the pathology content of the trial protocol, how pathology activities and roles were organised in the trial, where the laboratory work would be carried out, and the accreditation status of the laboratory. Only one trial had information specific to digital pathology, a technology certain to become more prevalent in the future. Adoption of the SPIRIT‐Path checklist will facilitate comprehensive trial protocols that address all the key cellular and molecular pathology aspects of interventional clinical trials. This study highlights once again the lack of public availability of trial protocols. Full trial protocols should be available for scrutiny by the scientific community and the public who participate in the studies, increasing the transparency of clinical trial activity and improving quality.

Genetic susceptibility loci for oropharyngeal cancer have been reported but these studies have not always examined human papillomavirus (HPV) status. Here, the authors perform genome-wide analysis taking into account HPV16 serology status and report two independent loci in the HLA region, suggesting the protective role of HLA variants against HPV infection.

Abstract Background Sodium valproate (VPA) has been associated with a reduced risk of head and neck cancer development. The potential protective mechanism of action is believed to be via inhibition of histone deacetylase and subsequent epigenetic reprogramming. SAVER is a phase IIb open-label, randomised control trial of VPA as a chemopreventive agent in patients with high-risk oral epithelial dysplasia (OED). The aim of the trial is to gather preliminary evidence of the clinical and biological effects of VPA upon OED and assess the feasibility and acceptability of such a trial, with a view to inform a future definitive phase III study. Methods One hundred and ten patients with high-risk OED will be recruited from up to 10 secondary care sites in the UK and randomised into either VPA or observation only for 4 months. Women of childbearing potential will be excluded due to the teratogenic properties of VPA. Tissue and blood samples will be collected prior to randomisation and on the last day of the intervention/observation-only period (end of 4 months). Clinical measurement and additional safety bloods will be taken at multiple time points during the trial. The primary outcome will be a composite, surrogate endpoint of change in lesion size, change in grade of dysplasia and change in LOH profile at 8 key microsatellite regions. Feasibility outcomes will include recruitment targets, compliance with the study protocol and adverse effects. A qualitative sub-study will explore patient experience and perception of the trial. Discussion The current management options for patients with high-risk OED are limited and mostly include surgical resection and clinical surveillance. However, there remains little evidence whether surgery can effectively lead to a notable reduction in the risk of oral cancer development. Similarly, surveillance is associated with concerns regarding delayed diagnosis of OED progressing to malignancy. The SAVER trial provides an opportunity to investigate the effects of a repurposed, inexpensive and well-tolerated medication as a potential chemopreventive strategy for patients with high-risk OED. The clinical and biological findings of SAVER will inform the appropriateness, design and feasibility of a definitive phase III trial. Trial registration The trial is registered with the European Clinical Trials Database ( Eudra-CT 2018-000197-30 ). ( http://www.isrctn.com/ISRCTN12448611 ). The trial was prospectively registered on 24/04/2018.

Longitudinal multi-omics measurements are highly valuable in studying heterogeneity in health and disease phenotypes. Here, the authors apply Pareto Task Inference to analyze the clinical lab tests of 3094 individuals and find three wellness states, and one aberrant health state defining this cohort.

Summary: The 1000 Genomes Project (TGP) is a foundational resource that serves the biomedical community as a standard reference cohort for human genetic variation. There are now seven public versions of these genomes. The TGP Consortium produced the first by mapping its final data release against human reference sequence GRCh37, then “lifted over” these genomes to the improved reference sequence (GRCh38) when it was released, and remapped the original data to GRCh38 with two similar pipelines. As best-practice quality validation, the pipelines that generated these versions were benchmarked against the Genome In A Bottle Consortium’s “platinum quality” genome (NA12878). The New York Genome Center recently released the results of independently resequencing the cohort at greater depth (30×), a phased version informed by the inclusion of related individuals, and independently remapped the original variant calls to GRCh38. We performed a cross-comparison evaluation of all seven versions using genome fingerprinting, which supports ultrafast genome comparison even across reference versions. We noted multiple issues, including discrepancies in cohort membership, disagreement on the overall level of variation, evidence of substandard pipeline performance on specific genomes and in specific regions of the genome, cryptic relationships between individuals, inconsistent phasing, and annotation distortions caused by the history of the reference genome itself. We therefore recommend global quality assessment by rapid genome comparisons, alongside benchmarking as part of best-practice quality assessment of large genome datasets. Our observations also help inform the decision of which version to use, to support analyses by individual researchers.

Abstract The SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2013 Statement was developed to provide guidance for inclusion of key methodological components in clinical trial protocols. However, these standards do not include guidance specific to pathology input in clinical trials. This systematic review aims to synthesise existing recommendations specific to pathology practice in clinical trials for implementation in trial protocol design. Articles were identified from database searches and deemed eligible for inclusion if they contained: (1) guidance and/or a checklist, which was (2) pathology‐related, with (3) content relevant to clinical trial protocols or could influence a clinical trial protocol design from a pathology perspective and (4) were published in 1996 or later. The quality of individual papers was assessed using the AGREE‐GRS (Appraisal of Guidelines for REsearch & Evaluation – Global Rating Scale) tool, and the confidence in cumulative evidence was evaluated using the GRADE‐CERQual (Grading of Recommendations Assessment, Development and Evaluation–Confidence in Evidence from Reviews of Qualitative research) approach. Extracted recommendations were synthesised using the best fit framework method, which includes thematic analysis followed by a meta‐aggregative approach to synthesis within the framework. Of the 10 184 records screened and 199 full‐text articles reviewed, only 40 guidance resources met the eligibility criteria for inclusion. Recommendations extracted from 22 guidance documents were generalisable enough for data synthesis. Seven recommendation statements were synthesised as follows: (1) multidisciplinary collaboration in trial design with early involvement of pathologists, particularly with respect to the use of biospecimens and associated biomarker/analytical assays and in the evaluation of pathology‐related parameters; (2) funding and training for personnel undertaking trial work; (3) selection of an accredited laboratory with suitable facilities to undertake scheduled work; (4) quality assurance of pathology‐related parameters; (5) transparent reporting of pathology‐related parameters; (6) policies regarding informatics and tracking biospecimens across trial sites; and (7) informed consent for specimen collection and retention for future research.

Abstract Introduction Passive immunotherapies targeting Aβ continue to be evaluated as Alzheimer’s disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aβ deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aβ immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors’ mRNA levels are altered in human AD and an APP mouse model. Methods rAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aβ1–16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aβ levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages. Results Priming TgCRND8 mice with Il10 increases Aβ loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aβ loads by itself and subsequent Aβ immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors. Conclusions The underlying immune activation state can markedly affect the efficacy of passive Aβ immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aβ deposition.

We present a rare challenging case of metastatic non-small cell lung cancer with Epstein-Barr virus positivity that was also diagnosed with pulmonary tuberculosis at the same time. Palliative chemotherapy gemcitabine and carboplatin was started after two weeks of anti-tuberculosis treatment with the hopes that this period would be sufficient to keep acid fast bacilli non-viable to minimise risk of tuberculosis re-activation due to chemotherapy induced immunosuppression. She completed four cycles of chemotherapy and six months of anti-tuberculosis treatment with good results and minimal side effects. Two years later, there was disease recurrence in cervical and mediastinal lymph nodes which was treated with local treatment i.e. surgery and palliative radiotherapy. It has been two years since last radiotherapy and overall more than five years since diagnosis with no active disease at present. Given the complexity and rarity of this case, significant multidisciplinary team involvement, including oncologists and radiation oncologists, pulmonologists with special interest in tuberculosis and pathologists was necessary throughout.

Background: Angiotensin-converting enzyme inhibitors (ACEi) and angiotensin-II receptor blockers (ARB), the most commonly prescribed antihypertensive medications, counter renin-angiotensin-aldosterone system (RAAS) activation via induction of angiotensin-converting enzyme 2 (ACE2) expression. Considering that ACE2 is the functional receptor for SARS-CoV-2 entry into host cells, the association of ACEi and ARB with COVID-19 outcomes needs thorough evaluation. Methods: We conducted retrospective analyses using both unmatched and propensity score (PS)-matched cohorts on electronic health records (EHRs) to assess the impact of RAAS inhibitors on the risk of receiving invasive mechanical ventilation (IMV) and 30-day mortality among hospitalized COVID-19 patients. Additionally, we investigated the immune cell gene expression profiles of hospitalized COVID-19 patients with prior use of antihypertensive treatments from an observational prospective cohort. Results: The retrospective analysis revealed that there was no increased risk associated with either ACEi or ARB use. In fact, the use of ACEi showed decreased risk for mortality. Survival analyses using PS-matched cohorts suggested no significant relationship between RAAS inhibitors with a hospital stay and in-hospital mortality compared to non-RAAS medications and patients not on antihypertensive medications. From the analysis of gene expression profiles, we observed a noticeable up-regulation in the expression of 1L1R2 (an anti-inflammatory receptor) and RETN (an immunosuppressive marker) genes in monocytes among prior users of ACE inhibitors. Conclusion: Overall, the findings do not support the discontinuation of ACEi or ARB treatment and suggest that ACEi may moderate the COVID-19 hyperinflammatory response.

Abstract Digital pathology and image analysis potentially provide greater accuracy, reproducibility and standardisation of pathology‐based trial entry criteria and endpoints, alongside extracting new insights from both existing and novel features. Image analysis has great potential to identify, extract and quantify features in greater detail in comparison to pathologist assessment, which may produce improved prediction models or perform tasks beyond manual capability. In this article, we provide an overview of the utility of such technologies in clinical trials and provide a discussion of the potential applications, current challenges, limitations and remaining unanswered questions that require addressing prior to routine adoption in such studies. We reiterate the value of central review of pathology in clinical trials, and discuss inherent logistical, cost and performance advantages of using a digital approach. The current and emerging regulatory landscape is outlined. The role of digital platforms and remote learning to improve the training and performance of clinical trial pathologists is discussed. The impact of image analysis on quantitative tissue morphometrics in key areas such as standardisation of immunohistochemical stain interpretation, assessment of tumour cellularity prior to molecular analytical applications and the assessment of novel histological features is described. The standardisation of digital image production, establishment of criteria for digital pathology use in pre‐clinical and clinical studies, establishment of performance criteria for image analysis algorithms and liaison with regulatory bodies to facilitate incorporation of image analysis applications into clinical practice are key issues to be addressed to improve digital pathology incorporation into clinical trials.

Abstract While pathologists have always played a pivotal role in clinical trials ensuring accurate diagnosis and staging, pathology data from prognostic and predictive tests are increasingly being used to enrol, stratify and randomise patients to experimental treatments. The use of pathological parameters as primary and secondary outcome measures, either as standalone classifiers or in combination with clinical data, is also becoming more common. Moreover, reporting of estimates of residual disease, termed ‘pathological complete response’, have been incorporated into neoadjuvant clinical trials. Pathologists have the expertise to deliver this essential information and they also understand the requirements and limitations of laboratory testing. Quality assurance of pathology‐derived data builds confidence around trial‐specific findings and is necessarily focused on the reproducibility of pathological data, including ‘estimates of uncertainty of measurement’, emphasising the importance of pathologist education, training, calibration and demonstration of satisfactory inter‐observer agreement. There are also opportunities to validate objective image analysis tools alongside conventional histological assessments. The ever‐expanding portfolio of clinical trials will demand more pathologist engagement to deliver the reliable evidence‐base required for new treatments. We provide guidance for quality assurance of pathology scoring and reporting in clinical trials.

Quality assured pathology services are integral to provision of optimal management for patients with head and neck cancer. Pathology services vary globally and are dependent on resources in terms of both laboratory provision and availability of a highly trained and accredited workforce. Ensuring a high-quality pathology service depends largely on close working and effective communication between the clinical team providing treatment and the pathologists providing laboratory input. Laboratory services should be quality assured by achieving external accreditation, most often by conforming to International Organization for Standardization (ISO) standards such as ISO15189 sometimes with ISO17025 or alternatively ISO17020. Quality of diagnostic reporting can be assured by the ISO but clinical teams should endeavor to work with pathologists who engage in continuing professional development, external quality assurance and audit. Research also contributes to diagnostic reporting quality. A number of initiatives in the UK such as the EPSRC/MRC funded Molecular Pathology Nodes and the National Cancer Research Institute Cellular-Molecular Pathology initiative (C-M Path), for example, have linked pathologists, industry and researchers. This has resulted in centers leading in digital innovation, artificial intelligence, translational research and clinical trials supported by pathologists. For rare tumors and contemporary molecular diagnostics, biopsy material can increasingly be shared with expert specialist pathologists working in specialist centers, particularly by using digital pathology platforms with potentially global reach. High quality services for the majority of diagnostic processes required for head and neck cancer management is best provided by local pathologists where communication with the treating team is more effective than with pathologists working in remote centers. Quality assurance is an increasingly important aspect of pathology, assuring not only effective turnaround times and accuracy for the diagnostic service but also high quality consistent reporting for clinical trials where even small pathology errors can potentially produce a significant bias and in the worst case negate the value of a completed trial. Better outcomes have been associated with centers engaged in clinical trials than in non-participating centers. Provision of a quality assured pathology service should extend to both the research and diagnostic services.

Abstract Background Bacterial genomes have characteristic compositional skews, which are differences in nucleotide frequency between the leading and lagging DNA strands across a segment of a genome. It is thought that these strand asymmetries arise as a result of mutational biases and selective constraints, particularly for energy efficiency. Analysis of compositional skews in a diverse set of bacteria provides a comparative context in which mutational and selective environmental constraints can be studied. These analyses typically require finished and well-annotated genomic sequences. Results We present three novel metrics for examining genome composition skews; all three metrics can be computed for unfinished or partially-annotated genomes. The first two metrics, (dot-skew and cross-skew) depend on sequence and gene annotation of a single genome, while the third metric (residual skew) highlights unusual genomes by subtracting a GC content-based model of a library of genome sequences. We applied these metrics to 7738 available bacterial genomes, including partial drafts, and identified outlier species. A phylogenetically diverse set of these outliers (i.e., Borrelia, Ehrlichia, Kinetoplastibacterium, and Phytoplasma) display similar skew patterns but share lifestyle characteristics, such as intracellularity and biosynthetic dependence on their hosts. Conclusions Our novel metrics appear to reflect the effects of biosynthetic constraints and adaptations to life within one or more hosts on genome composition. We provide results for each analyzed genome, software and interactive visualizations at http://db.systemsbiology.net/gestalt/ skew_metrics.

Abstract Background The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been rising in Western countries and this has been attributed to human papillomavirus (HPV) infection. p16 expression is a marker for HPV infection and p16 positive OPSCC is now recognized as a separate disease entity. There are only limited data available regarding HPV-related OPSCC in Asian countries and no data from Malaysia. Methods We identified 60 Malaysian patients with OPSCC over a 12-year period (2004–2015) from four different hospitals in two major cities, Kuala Lumpur and Penang. The detection of HPV was carried out using p16 immunohistochemistry and high risk HPV DNA in situ hybridisation. Results Overall, 15 (25%) tumours were p16 positive by immunohistochemistry, 10 of which were also positive for high risk HPV DNA by in situ hybridisation. By comparison, a matched cohort of UK patients had a p16 positive rate of 49%. However, between 2009 and 2015, where cases were available from all four hospitals, 13 of 37 (35%) cases were p16 positive. In our Malaysian cohort, 53% of patients were of Chinese ethnicity and 80% of the p16 positive cases were found in these patients; no Indian patients had p16 positive disease, despite representing 35% of the total cohort. Conclusion The proportion of OPSCCs associated with HPV in Malaysia appears to be lower than in European and American cohorts and could possibly be more prevalent amongst Malaysians of Chinese ethnicity. Further, our data suggests that the burden of HPV-related OPSCC could be increasing in Malaysia. Larger cross-sectional studies of Malaysian patients are required to determine the public health implications of these preliminary findings.

Data normalization is a crucial step in the gene expression analysis as it ensures the validity of its downstream analyses. Although many metrics have been designed to evaluate the existing normalization methods, different metrics or different datasets by the same metric yield inconsistent results, particularly for the single-cell RNA sequencing (scRNA-seq) data. The worst situations could be that one method evaluated as the best by one metric is evaluated as the poorest by another metric, or one method evaluated as the best using one dataset is evaluated as the poorest using another dataset. Here raises an open question: principles need to be established to guide the evaluation of normalization methods. In this study, we propose a principle that one normalization method evaluated as the best by one metric should also be evaluated as the best by another metric (the consistency of metrics) and one method evaluated as the best using scRNA-seq data should also be evaluated as the best using bulk RNA-seq data or microarray data (the consistency of datasets). Then, we designed a new metric named Area Under normalized CV threshold Curve (AUCVC) and applied it with another metric mSCC to evaluate 14 commonly used normalization methods using both scRNA-seq data and bulk RNA-seq data, satisfying the consistency of metrics and the consistency of datasets. Our findings paved the way to guide future studies in the normalization of gene expression data with its evaluation. The raw gene expression data, normalization methods, and evaluation metrics used in this study have been included in an R package named NormExpression. NormExpression provides a framework and a fast and simple way for researchers to select the best method for the normalization of their gene expression data based on the evaluation of different methods (particularly some data-driven methods or their own methods) in the principle of the consistency of metrics and the consistency of datasets.

We present an ultrafast method for comparing personal genomes. We transform the standard genome representation (lists of variants relative to a reference) into “genome fingerprints” via locality sensitive hashing. The resulting genome fingerprints can be meaningfully compared even when the input data were obtained using different sequencing technologies, processed using different pipelines, represented in different data formats and relative to different reference versions. Furthermore, genome fingerprints are robust to up to 30% missing data. Because of their reduced size, computation on the genome fingerprints is fast and requires little memory. For example, we could compute all-against-all pairwise comparisons among the 2504 genomes in the 1000 Genomes data set in 67 s at high quality (21 μs per comparison, on a single processor), and achieved a lower quality approximation in just 11 s. Efficient computation enables scaling up a variety of important genome analyses, including quantifying relatedness, recognizing duplicative sequenced genomes in a set, population reconstruction, and many others. The original genome representation cannot be reconstructed from its fingerprint, effectively decoupling genome comparison from genome interpretation; the method thus has significant implications for privacy-preserving genome analytics.

Background: Planned neck dissection (ND) after radical chemoradiotherapy (CRT) for locally advanced nodal metastases in patients with head and neck squamous cell carcinoma (HNSCC) remains controversial. Thirty per cent of ND specimens show histological evidence of tumour. Consequently, a significant proportion of clinicians still practise planned ND. Fludeoxyglucose positron emission tomography (PET)–computerised tomography (CT) scanning demonstrated high negative predictive values for persistent nodal disease, providing a possible alternative paradigm to ND. Evidence is sparse and drawn mainly from retrospective single-institution studies, illustrating the need for a prospective randomised controlled trial. Objectives: To determine the efficacy and cost-effectiveness of PET–CT-guided surveillance, compared with planned ND, in a multicentre, prospective, randomised setting. Design: A pragmatic randomised non-inferiority trial comparing PET–CT-guided watch-and-wait policy with the current planned ND policy in HNSCC patients with locally advanced nodal metastases and treated with radical CRT. Patients were randomised in a 1 : 1 ratio. Primary outcomes were overall survival (OS) and cost-effectiveness [incremental cost per incremental quality-adjusted life-year (QALY)]. Cost-effectiveness was assessed over the trial period using individual patient data, and over a lifetime horizon using a decision-analytic model. Secondary outcomes were recurrence in the neck, complication rates and quality of life. The recruitment of 560 patients was planned to detect non-inferior OS in the intervention arm with a 90% power and a type I error of 5%, with non-inferiority defined as having a hazard ratio (HR) of no higher than 1.50. An intention-to-treat analysis was performed by Cox’s proportional hazards model. Settings: Thirty-seven head and neck cancer-treating centres (43 NHS hospitals) throughout the UK. Participants: Patients with locally advanced nodal metastases of oropharynx, hypopharynx, larynx, oral or occult HNSCC receiving CRT and fit for ND were recruited. Intervention: Patients randomised to planned ND before or after CRT (control), or CRT followed by fludeoxyglucose PET–CT 10–12 weeks post CRT with ND only if PET–CT showed incomplete or equivocal response of nodal disease (intervention). Balanced by centre, planned ND timing, CRT schedule, disease site and the tumour, node, metastasis stage. Results: In total, 564 patients were recruited (ND arm, n = 282; and surveillance arm, n = 282; 17% N2a, 61% N2b, 18% N2c and 3% N3). Eighty-four per cent had oropharyngeal cancer. Seventy-five per cent of tested cases were p16 positive. The median time to follow-up was 36 months. The HR for OS was 0.92 [95% confidence interval (CI) 0.65 to 1.32], indicating non-inferiority. The upper limit of the non-inferiority HR margin of 1.50, which was informed by patient advisors to the project, lies at the 99.6 percentile of this estimate (p = 0.004). There were no differences in this result by p16 status. There were 54 NDs performed in the surveillance arm, with 22 surgical complications, and 221 NDs in the ND arm, with 85 complications. Quality-of-life scores were slightly better in the surveillance arm. Compared with planned ND, PET–CT surveillance produced an incremental net health benefit of 0.16 QALYs (95% CI 0.03 to 0.28 QALYs) over the trial period and 0.21 QALYs (95% CI –0.41 to 0.85 QALYs) over the modelled lifetime horizon. Limitations: Pragmatic randomised controlled trial with a 36-month median follow-up. Conclusions: PET–CT-guided active surveillance showed similar survival outcomes to ND but resulted in considerably fewer NDs, fewer complications and lower costs, supporting its use in routine practice. Future work: PET–CT surveillance is cost-effective in the short term, and long-term cost-effectiveness could be addressed in future work. Trial registration: Current Controlled Trials ISRCTN13735240. Funding: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 17. See the NIHR Journals Library website for further project information.

Karaginan adalah koloid hidrofilik yang diperlukan secara komersial dan materialnya terdapat dalam beberapa spesies rumput laut merah (Rhodophyta) termasuk didalamnya Kappaphycus alvarezii. Karaginan dapat digunakan sebagai bahan baku untuk industri farmasi, kosmetik, makanan, pembentuk gel, bahan pengikat, bahan pengemulsi dan bahan penstabil. Kualitas karaginan dipengaruhi oleh beberapa faktor, satu di antaranya adalah umur panen dari rumput laut, yang berkaitan dengan lokasi dan parameter lingkungan. Penelitian ini menggunakan Rancangan Acak Lengkap dengan satu faktor utama yaitu umur panen dengan 4 taraf, yaitu 40, 45, 50, dan 55 hari. Data hasil pengamatan diolah dengan analisis ragam dan dilanjutkan dengan uji beda Duncan. Karaginan terbaik yang dihasilkan dari penelitian ini adalah yang diekstrak dari rumput laut dengan umur panen 50 hari. Karakteristik fisiko-kimia yang dihasilkan dari karaginan terbaik yaitu kekuatan gel 330 g/cm2, viskositas 30,73 cP, titik jendal 33,20oC, titik leleh 43,50oC, derajat putih 38,36%, kadar air 10,86%, kadar abu 22,76%, kadar abu tidak larut asam 0,88%, dan kadar sulfat 27,43%.

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

Deep sequencing of transcriptomes has become an indispensable tool for biology, enabling expression levels for thousands of genes to be compared across multiple samples. Since transcript counts scale with sequencing depth, counts from different samples must be normalized to a common scale prior to comparison. We analyzed fifteen existing and novel algorithms for normalizing transcript counts, and evaluated the effectiveness of the resulting normalizations. For this purpose we defined two novel and mutually independent metrics: (1) the number of "uniform" genes (genes whose normalized expression levels have a sufficiently low coefficient of variation), and (2) low Spearman correlation between normalized expression profiles of gene pairs. We also define four novel algorithms, one of which explicitly maximizes the number of uniform genes, and compared the performance of all fifteen algorithms. The two most commonly used methods (scaling to a fixed total value, or equalizing the expression of certain 'housekeeping' genes) yielded particularly poor results, surpassed even by normalization based on randomly selected gene sets. Conversely, seven of the algorithms approached what appears to be optimal normalization. Three of these algorithms rely on the identification of "ubiquitous" genes: genes expressed in all the samples studied, but never at very high or very low levels. We demonstrate that these include a "core" of genes expressed in many tissues in a mutually consistent pattern, which is suitable for use as an internal normalization guide. The new methods yield robustly normalized expression values, which is a prerequisite for the identification of differentially expressed and tissue-specific genes as potential biomarkers.