MinION-in-ARMS: Nanopore Sequencing to Expedite Barcoding of Specimen-Rich Macrofaunal Samples From Autonomous Reef Monitoring Structures

Autonomous Reef Monitoring Structure (ARMS) are standardized devices for sampling biodiversity in complex marine benthic habitats such as coral reefs. When coupled with DNA sequencing, these devices greatly expand our ability to document marine biodiversity. Unfortunately, the existing workflow for processing macrofaunal samples (\u3e2-mm) in the ARMS pipeline—which involves Sanger sequencing—is expensive, laborious, and thus prohibitive for ARMS researchers. Here, we propose a faster, more cost-effective alternative by demonstrating a successful application of the MinION-based barcoding approach on the \u3e2 mm-size fraction of ARMS samples. All data were available within 3.5–4 h, and sequencing costs relatively low at approximately US$3 per MinION barcode. We sequenced the 313-bp fragment of the cytochrome c oxidase subunit I (COI) for 725 samples on both MinION and Illumina platforms, and retrieved 507–584 overlapping barcodes. MinION barcodes were highly accurate (∼99.9%) when compared with Illumina reference barcodes. Molecular operational taxonomic units inferred between MinION and Illumina barcodes were consistently stable, and match ratios demonstrated highly congruent clustering patterns (≥0.96). Our method would make ARMS more accessible to researchers, and greatly expedite the processing of macrofaunal samples; it can also be easily applied to other small-to-moderate DNA barcoding projects (\u3c10,000 specimens) for rapid species identification and discovery

Seq’ and ARMS shall find: DNA (meta)barcoding of Autonomous Reef Monitoring Structures across the tree of life uncovers hidden cryptobiome of tropical urban coral reefs

Coral reefs are among the richest marine ecosystems on Earth, but there remains much diversity hidden within cavities of complex reef structures awaiting discovery. While the abundance of corals and other macroinvertebrates are known to influence the diversity of other reef-associated organisms, much remains unknown on the drivers of cryptobenthic diversity. A combination of standardized sampling with 12 units of the Autonomous Reef Monitoring Structure (ARMS) and high-throughput sequencing was utilized to uncover reef cryptobiome diversity across the equatorial reefs in Singapore. DNA barcoding and metabarcoding of mitochondrial cytochrome c oxidase subunit I, nuclear 18S and bacterial 16S rRNA genes revealed the taxonomic composition of the reef cryptobiome, comprising 15,356 microbial ASVs from over 50 bacterial phyla, and 971 MOTUs across 15 metazoan and 19 non-metazoan eukaryote phyla. Environmental factors across different sites were tested for relationships with ARMS diversity. Differences among reefs in diversity patterns of metazoans and other eukaryotes, but not microbial communities, were associated with biotic (coral cover) and abiotic (distance, temperature and sediment) environmental variables. In particular, ARMS deployed at reefs with higher coral cover had greater metazoan diversity and encrusting plate cover, with larger-sized non-coral invertebrates influencing spatial patterns among sites. Our study showed that DNA barcoding and metabarcoding of ARMS constitute a valuable tool for quantifying cryptobenthic diversity patterns and can provide critical information for the effective management of coral reef ecosystems

Seq’ and ARMS shall find: DNA (meta)barcoding of Autonomous Reef Monitoring Structures across the tree of life uncovers hidden cryptobiome of tropical urban coral reefs

Coral reefs are among the richest marine ecosystems on Earth, but there remains much diversity hidden within cavities of complex reef structures awaiting discovery. While the abundance of corals and other macroinvertebrates are known to influence the diversity of other reef-associated organisms, much remains unknown on the drivers of cryptobenthic diversity. A combination of standardized sampling with 12 units of the Autonomous Reef Monitoring Structure (ARMS) and high-throughput sequencing was utilized to uncover reef cryptobiome diversity across the equatorial reefs in Singapore. DNA barcoding and metabarcoding of mitochondrial cytochrome c oxidase subunit I, nuclear 18S and bacterial 16S rRNA genes revealed the taxonomic composition of the reef cryptobiome, comprising 15,356 microbial ASVs from over 50 bacterial phyla, and 971 MOTUs across 15 metazoan and 19 non-metazoan eukaryote phyla. Environmental factors across different sites were tested for relationships with ARMS diversity. Differences among reefs in diversity patterns of metazoans and other eukaryotes, but not microbial communities, were associated with biotic (coral cover) and abiotic (distance, temperature and sediment) environmental variables. In particular, ARMS deployed at reefs with higher coral cover had greater metazoan diversity and encrusting plate cover, with larger-sized non-coral invertebrates influencing spatial patterns among sites. Our study showed that DNA barcoding and metabarcoding of ARMS constitute a valuable tool for quantifying cryptobenthic diversity patterns and can provide critical information for the effective management of coral reef ecosystems

Effectiveness of a technology-based injury prevention program for enhancing mothers’ knowledge of child safety: protocol for a randomized controlled trial

Background: Provision of anticipatory guidance for parents is recommended as an effective strategy to prevent injuries among young children. Technology-based anticipatory guidance has been suggested to reinforce the effectiveness of injury prevention and improve parents’ knowledge of child safety. Objective: This study aims to examine the effectiveness of a technology-based injury prevention program with parental anticipatory guidance for enhancing mothers’ knowledge of child safety. Methods: In this randomized controlled trial, 308 mothers will be recruited from the antenatal clinics and postnatal wards of two major public hospitals in Hong Kong. Participating mothers will be randomly assigned into intervention and control groups. Mothers in the intervention group will be given free access to a technology-based injury prevention program with anticipatory guidance, whereas mothers in the control group will be given a relevant booklet on parenting. The injury prevention program, available as a website or on a mobile app, includes behavioral components based on the Theory of Planned Behavior. The primary outcome measure will be the change in the mother’s knowledge of child safety. The secondary outcome measures will be age-appropriate domestic safety knowledge, attitudes, intentions, perceived behavioral control, and self-reported behavior related to home safety practice. We will also determine dose-response relationships between the outcome measures and the website and mobile app usage. Results: Enrolment of participants will begin in October 2016. Results are expected by June 2018. Conclusions: Parents will be able to easily access the domestic injury prevention website to find information regarding child injury prevention. It is anticipated that the technology-based intervention will help parents improve their knowledge of child safety and raise their awareness about the consequences of domestic injuries and the importance of prevention. Trial Registration: Clinicaltrials.gov Clinicaltrials.gov NCT02835768; http://clinicaltrials.gov/ct2/show/NCT02835768 (Archived by WebCite at http://www.webcitation/6lbXYM6b9)

Milk Consumption Across Life Periods in Relation to Lower Risk of Nasopharyngeal Carcinoma: A Multicentre Case-Control Study

Background: The much higher incidence of nasopharyngeal carcinoma (NPC) in men suggests sex hormones as a risk factor, and dairy products contain measurable amounts of steroid hormones. Milk consumption has greatly increased in endemic regions of NPC. We investigated the association between NPC and milk consumption across life periods in Hong Kong.Methods: A multicentre case-control study included 815 histologically confirmed NPC incident cases and 1,502 controls who were frequency-matched on age and sex at five major hospitals in Hong Kong in 2014–2017. Odds ratios (ORs) of NPC (cases vs. controls) for milk consumption at different life periods were estimated by unconditional logistic regression, adjusting for sex, age, socioeconomic status score, smoking and alcohol drinking status, exposure to occupational hazards, family history of cancer, IgA against Epstein-Barr virus viral capsid antigen, and total energy intake.Results: Compared with abstainers, lower risks of NPC were consistently observed in regular users (consuming ≥5 glasses of milk [fresh and powdered combined] per month) across four life periods of age 6–12 (adjusted OR 0.74, 95% CI 0.54–0.86), 13–18 (0.68, 0.55–0.84), 19–30 (0.68, 0.55–0.84), and 10 years before recruitment (0.72, 0.59–0.87). Long-term average milk consumption of ≤2.5, >2.5, and ≤12.5, >12.5 glasses per month yielded adjusted OR (95% CI) of 1.00 (0.80–1.26), 0.98 (0.81–1.18), 0.95 (0.76–1.18), and 0.55 (0.43–0.70), respectively (all P-values for trend < 0.05).Conclusion: Consumption of milk across life periods was associated with lower risks of NPC. If confirmed to be causal, this has important implications for dairy product consumption and prevention of NPC

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

DNA BARCODING AND METABARCODING FOR ESTIMATING SINGAPORE’S CORAL REEF BIODIVERSITY

Ph.DDOCTOR OF PHILOSOPHY (FOS

Complete mitochondrial genome of the feather star Cenometra bella (Hartlaub, 1890) (Crinoidea: Colobometridae)

Mitochondrial DNA Part B76950-95