16S rRNA sequencing reveals likely beneficial core microbes within faecal samples of the EU protected slug Geomalacus maculosus

The EU-protected slug Geomalacus maculosus Allman occurs only in the West of Ireland and in northern Spain and Portugal. We explored the microbial community found within the faeces of Irish specimens with a view to determining whether a core microbiome existed among geographically isolated slugs which could give insight into the adaptations of G. maculosus to the available food resources within its habitat. Faecal samples of 30 wild specimens were collected throughout its Irish range and the V3 region of the bacterial 16S rRNA gene was sequenced using Illumina MiSeq. To investigate the influence of diet on the microbial composition, faecal samples were taken and sequenced from six laboratory reared slugs which were raised on two different foods. We found a widely diverse microbiome dominated by Enterobacteriales with three core OTUs shared between all specimens. While the reared specimens appeared clearly separated by diet in NMDS plots, no significant difference between the slugs fed on the two different diets was found. Our results indicate that while the majority of the faecal microbiome of G. maculosus is probably dependent on the microhabitat of the individual slugs, parts of it are likely selected for by the host

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water

Background: Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW. Results: A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteriaStenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescenswhich are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively. Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia andBurkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines. Conclusions: The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards

Community and functional shifts in ammonia oxidizers across terrestrial and marine (soil/sediment) boundaries in two coastal Bay ecosystems

Terrestrial–marine boundaries are significant sites of biogeochemical activity with delineated gradients from land to sea. While niche differentiation of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) driven by pH and nitrogen is well known, the patterns and environmental drivers of AOA and AOB community structure and activity across soil‐sediment boundaries have not yet been determined. In this study, nitrification potential rate, community composition and transcriptional activity of AOA and AOB in soil, soil/sediment interface and sediments of two coastal Bays were characterized using a combination of field investigations and microcosm incubations. At DNA level, amoA gene abundances of AOA were significantly greater than AOB in soil, while in sediments AOB were significantly more abundant than AOA, but at the soil/sediment interface there were equal numbers of AOA and AOB amoA genes. Microcosm incubations provided further evidence, through qPCR and DGGE‐sequencing analysis of amoA transcripts, that AOA were active in soil, AOB in sediment and both AOA and AOB were active at the soil/sediment interface. The AOA and AOB community composition shifted across the coastal soil‐interface‐sediment gradient with salinity and pH identified as major environmental drivers

Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples

Reliability and reproducibility of transcriptomics‐based studies are dependent on RNA integrity. In microbial ecology, microfluidics‐based techniques, such as the Ribosomal Integrity Number (RIN), targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present an integrity index, the Ratio Amplicon, Ramp, adapted from human clinical studies, to directly monitor mRNA integrity from complex environmental samples. We show, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of 16S rRNA, amoA and glnA transcripts. We successfully sequenced transcripts for all three targets even from highly‐degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcript profiles. Since both RT‐Q‐PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study

Retail Salesperson’s Influence on India’s Lonely Consumers

Many consumers in India experience loneliness and social isolation. Some may resort to shopping for a social experience, attempting to mitigate or remedy their situation. Looking to this key emerging market, we investigate how emotional loneliness, social loneliness, and social isolation influence enjoyment of social interaction with an in-store salesperson (ESIS). We also consider whether adaptive selling and predisposition to comply with salesperson input (PCSI) influence consumers’ trust in salesperson, purchase intention, and retail patronage. We utilize partial least squares structural equation modeling with a sample of 303 Indian consumers. Additionally, we provide two importance-performance mapping analyses, which offer additional insights for retail managers trying to prioritize attention to constructs driving improvement of ESIS and PCSI. In this study, the degree of consumer loneliness and social isolation were proposed to influence Indian consumers’ enjoyment of social interaction with an in-store salesperson (ESIS), à la shopping as a social experience (Hu & Jasper, 2006; Jayasankaraprasad & Kathyayani, 2014; Rajamma et al., 2010). As posited and supported, two varieties of loneliness—emotional (EL) and social (SE)—along with social isolation (SI) were shown to be positively associated with Indian consumers’ predisposition to comply with salesperson input (PCSI). Moreover, adaptive selling, which exhibited a positive relationship with PCSI, was propounded to demonstrate a salutary impact on three outcome variables: consumer trust in salesperson (TRUST), consumer purchase intention (PI), and consumer retail patronage (PATRON). Key values for variance explained (R2), relationship strength and direction (β), effect size (f2), and predictive relevance (Q2) as well as our two importance-performance map analyses underscore the quality of the model presented. The findings suggest that consumers’ perceptions of loneliness and isolation can lead them to seek out social experiences that specifically involve visiting stores and interacting with salespeople. Additionally, given Indian consumers’ realization of a store’s function and a salesperson’s primary purpose, seemingly consumers anticipate their willingness to be influenced by product recommendations and other inputs from salespersons. If, over time, social relationships develop between consumers and salespersons, discrete transactions can evolve or graduate into more regular interactions that enhance trust, purchase intention, and retail patronage. At present, there is relatively little research that deals with lonely and/or socially isolated consumers in the world’s emerging markets. For countries such as India, most scholarship on loneliness relates almost exclusively to elderly persons or youth (Bhatia et al., 2007; Bowker & Raja, 2011; Tiwari, 2013; Upmanyu, Sehgal, & Upmanyu, 1994), providing very few insights into the scale or impact of loneliness for adult consumers and the middle class. This study is step towards bridging the research gaps in this area

Conceptualizing a mobile-assisted learning environment featuring funds of knowledge for English learners’ narrative writing development

The purpose of this exploratory sequential mixed-methods study is to investigate a group of middle-school aged Latinx English learners (ELs) in a rural town in the Midwestern United States and to facilitate their narrative writing development via a mobile-assisted language learning (MALL) environment from a funds of knowledge perspective. In particular, we first explored the existing funds of knowledge sources drawing from the ELs’ lived experience and cultural practice through a multimethodological approach over a span of three months. We conceptualized the explored funds of knowledge sources into ELs’ narrative writing practice through the integration of mobile-based writing tools (MBWTs). Second, we employed a multiple pre-and post- non-experimental design for the ELs to complete two non- funds of knowledge and three funds of knowledge-featured narrative writing activities over ten weeks using Google Docs as an MBWT. Results showed a statistically significant positive learning effect of funds of knowledge as an intervention for developing the ELs’ literacy skills in narrative writing within a collaborative MALL environment

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included

Seasonal variation in denitrification and dissimilatory nitrate reduction to ammonia process rates and corresponding key functional genes along an estuarine nitrate gradient

This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) processes and abundances of corresponding genes and transcripts at three sites—the estuary-head, mid-estuary and the estuary mouth (EM) along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site. Abundance of gene markers for nitrate-reduction (nitrate reductase narG and napA), denitrification (nitrite reductase nirS) and DNRA (DNRA nitrite reductase nrfA) declined along the estuary with significant relationships between denitrification and nirS abundance, and DNRA and nrfA abundance. Spatially, rates of denitrification, DNRA and corresponding functional gene abundances decreased along the estuary. However, temporal correlations between rate processes and functional gene and transcript abundances were not observed

Doctoral Students’ Perceived Barriers that Slow the Progress toward Completing a Doctoral Dissertation: A Mixed Analysis

The non-completion of doctoral degrees has been a concern due to its economic, social, and personal consequences. In the current study, the researchers investigated perceived barriers of select doctoral students in completing their doctoral degrees by utilizing a fully mixed sequential mixed research design. The quantitative and qualitative data were concurrently collected using identical samples (n = 205) via a Reading Interest Survey questionnaire. A sequential mixed analysis revealed 6 emergent themes: external obligations (36%), challenges to doctoral-level researchers (34%), practical/logistical constraints (23%), emotional concerns (15%), program structure (9%), and support for completion (8%). Also, 3 meta-themes were identified (i.e., dissociation, external/internal barriers, and institutional/personal barriers), which aided in explaining the relationships among the 6 primary themes. Implications of the findings are discussed