CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome ...engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.
•Multiplex CRISPR/Cas9 procedure for 1–5 marker-free genome edits in S. cerevisiae.•Genome re-sequencing shows that the tool is likely to have no off-target effects.•Generation of all strain combinations using 5 targets without selected phenotype.•Identification of strains with increased mevalonate levels of >41-fold compared to reference.
Ascent to high altitude is accompanied by a reduction in partial pressure of inspired oxygen, which leads to interconnected adjustments within the neuromuscular system. This review describes the ...unique challenge that such an environment poses to neuromuscular fatigability (peripheral, central and supraspinal) for individuals who normally reside near to sea level (SL) (<1000 m; ie, lowlanders) and for native highlanders, who represent the manifestation of high altitude‐related heritable adaptations across millennia. Firstly, the effect of acute exposure to high altitude‐related hypoxia on neuromuscular fatigability will be examined. Under these conditions, both supraspinal and peripheral fatigability are increased compared with SL. The specific mechanisms contributing to impaired performance are dependent on the exercise paradigm and amount of muscle mass involved. Next, the effect of chronic exposure to high altitude (ie, acclimatization of ~7‐28 days) will be considered. With acclimatization, supraspinal fatigability is restored to SL values, regardless of the amount of muscle mass involved, whereas peripheral fatigability remains greater than SL except when exercise involves a small amount of muscle mass (eg, knee extensors). Indeed, when whole‐body exercise is involved, peripheral fatigability is not different to acute high‐altitude exposure, due to competing positive (haematological and muscle metabolic) and negative (respiratory‐mediated) effects of acclimatization on neuromuscular performance. In the final section, we consider evolutionary adaptations of native highlanders (primarily Himalayans of Tibet and Nepal) that may account for their superior performance at altitude and lesser degree of neuromuscular fatigability compared with acclimatized lowlanders, for both single‐joint and whole‐body exercise.
The objectives of this study were to examine long-term effects of feeding forage rape (Brassica napus L.) on methane yields (g methane per kg of feed dry matter intake), and to propose mechanisms ...that may be responsible for lower emissions from lambs fed forage rape compared to perennial ryegrass (Lolium perenne L.). The lambs were fed fresh winter forage rape or ryegrass as their sole diet for 15 weeks. Methane yields were measured using open circuit respiration chambers, and were 22-30% smaller from forage rape than from ryegrass (averages of 13.6 g versus 19.5 g after 7 weeks, and 17.8 g versus 22.9 g after 15 weeks). The difference therefore persisted consistently for at least 3 months. The smaller methane yields from forage rape were not related to nitrate or sulfate in the feed, which might act as alternative electron acceptors, or to the levels of the potential inhibitors glucosinolates and S-methyl L-cysteine sulfoxide. Ruminal microbial communities in forage rape-fed lambs were different from those in ryegrass-fed lambs, with greater proportions of potentially propionate-forming bacteria, and were consistent with less hydrogen and hence less methane being produced during fermentation. The molar proportions of ruminal acetate were smaller and those of propionate were greater in forage rape-fed lambs, consistent with the larger propionate-forming populations and less hydrogen production. Forage rape contained more readily fermentable carbohydrates and less structural carbohydrates than ryegrass, and was more rapidly degraded in the rumen, which might favour this fermentation profile. The ruminal pH was lower in forage rape-fed lambs, which might inhibit methanogenic activity, shifting the rumen fermentation to more propionate and less hydrogen and methane. The significance of these two mechanisms remains to be investigated. The results suggest that forage rape is a potential methane mitigation tool in pastoral-based sheep production systems.
The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs ...as biocides. Unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics). Many of these eNPs are composed of heavy metals or metal oxides such as silver, gold, zinc, titanium dioxide, and zinc oxide. It is thought that since metallic/metallic oxide NPs impact so many aspects of bacterial physiology that it will difficult for bacteria to evolve resistance to them. This study utilized laboratory experimental evolution to evolve silver nanoparticle (AgNP) resistance in the bacterium Escherichia coli (K-12 MG1655), a bacterium that does not harbor any known silver resistance elements. After 225 generations of exposure to the AgNP environment, the treatment populations demonstrated greater fitness vs. control strains as measured by optical density (OD) and colony forming units (CFU) in the presence of varying concentrations of 10 nm citrate-coated silver nanoparticles (AgNP) or silver nitrate (AgNO3). Genomic analysis shows that changes associated with AgNP resistance were already accumulating within the treatment populations by generation 100, and by generation 200 three mutations had swept to high frequency in the AgNP resistance stocks. This study indicates that despite previous claims to the contrary bacteria can easily evolve resistance to AgNPs, and this occurs by relatively simple genomic changes. These results indicate that care should be taken with regards to the use of eNPs as biocides as well as with regards to unintentional exposure of microbial communities to eNPs in waste products.
Despite ongoing efforts aimed to improve treatment engagement for people with substance-related disorders, evidence shows modest rates of utilization as well as client-perceived barriers to care. ...Patient-centered care (PCC) is one widely recognized approach that has been recommended as an evidence-based practice to improve the quality of substance use disorder treatment. PCC includes four core principles: a holistic and individualized focus to care, shared decision-making and enhanced therapeutic alliance.
This scoping review aimed to explore which PCC principles have been described and how they have defined and measured among people with substance-related disorders.
Following the iterative stages of the Arksey and O'Malley scoping review methodology, empirical (from Medline, Embase, PsycINFO, CINAHL and ISI Web of Science) and grey literature references were eligible if they focused on people accessing treatment for substance-related disorders and described PCC. Two reviewers independently screened the title/abstract and full-texts of references. Descriptive analyses and a directed content analysis were performed on extracted data.
One-hundred and forty-nine references met inclusion from the 2951 de-duplicated references screened. Therapeutic alliance was the most frequent principle of PCC described by references (72%); this was consistently defined by characteristics of empathy and non-judgment. Shared decision-making was identified in 36% of references and was primarily defined by client and provider strategies of negotiation in the treatment planning process. Individualized care was described by 30% of references and included individualized assessment and treatment delivery efforts. Holistic care was identified in 23% of references; it included an integrated delivery of substance use, health and psychosocial services via comprehensive care settings or coordination. Substance use and treatment engagement outcomes were most frequently described, regardless of PCC principle.
This review represents a necessary first step to explore how PCC has been defined and measured for people accessing substance use disorder treatment. The directed content analysis revealed population and context-specific evidence regarding the defining characteristics of PCC-principles that can be used to further support the implementation of PCC.
The chicken gastrointestinal tract has a diverse microbial community. There is increasing evidence for how this gut microbiome affects specific molecular pathways and the overall physiology, nervous ...system and behavior of the chicken host organism due to a growing number of studies investigating conditions such as host diet, antibiotics, probiotics, and germ-free and germ-reduced models. Systems-level investigations have revealed a network of microbiome-related interactions between the gut and state of health and behavior in chickens and other animals. While some microbial symbionts are crucial for maintaining stability and normal host physiology, there can also be dysbiosis, disruptions to nutrient flow, and other outcomes of dysregulation and disease. Likewise, alteration of the gut microbiome is found for chickens exhibiting differences in feather pecking (FP) behavior and this alteration is suspected to be responsible for behavioral change. In chickens and other organisms, serotonin is a chief neuromodulator that links gut microbes to the host brain as microbes modulate the serotonin secreted by the host’s own intestinal enterochromaffin cells which can stimulate the central nervous system via the vagus nerve. A substantial part of the serotonergic network is conserved across birds and mammals. Broader investigations of multiple species and subsequent cross-comparisons may help to explore general functionality of this ancient system and its increasingly apparent central role in the gut-brain axis of vertebrates. Dysfunctional behavioral phenotypes from the serotonergic system moreover occur in both birds and mammals with, for example, FP in chickens and depression in humans. Recent studies of the intestine as a major site of serotonin synthesis have been identifying routes by which gut microbial metabolites regulate the chicken serotonergic system. This review in particular highlights the influence of gut microbial metabolite short chain fatty acids (SCFAs) on the serotonergic system. The role of SCFAs in physiological and brain disorders may be considerable because of their ability to cross intestinal as well as the blood-brain barriers, leading to influences on the serotonergic system via binding to receptors and epigenetic modulations. Examinations of these mechanisms may translate into a more general understanding of serotonergic system development within chickens and other avians.
•Comprehensive non-targeted method applied to monitoring tea manufacturing LC–MS.•Detected significant changes in product composition after the ‘de-greening’ manufacturing step.•Detected for the ...first time, increases in jasmonic acid levels during tea fermentation.•Demonstrated the potential of non-targeted LC–MS to monitor the progress of complex manufacturing processes.
Oolong tea is a semi-fermented tea that is partially oxidised during the manufacturing process to create a product unique in composition. In this study, we investigated the potential of non-targeted LC–MS with two complementary chromatographic modes to provide a “comprehensive and unbiased” view of biochemical compositional changes occurring during oolong tea manufacturing in New Zealand. Tea leaf samples from throughout the manufacturing/fermentation process during three different harvest periods (spring, summer and autumn) were analysed by four different LC–MS streams. Principal component analysis revealed the de-greening stage of the manufacturing process was responsible for major changes in the biochemical profile, with the methodology detecting changes in a wide range of metabolites of differing polarities, such as flavonoids, nucleosides and primeverosides. Changes during the fermentation phase of the manufacturing process were less marked, however significant increases in levels of free amino acids, a hydroxyjasmonic acid and related metabolites were observed.
Higher education institutions are increasingly seeking technological solutions to not only enhance the learning environment but also support students. In this study, we explored the case of an early ...alert system (EAS) at a regional university engaged in both on-campus and online teaching. Using a total of 16,142 observations captured between 2011 and 2013, we examined the relationship between EAS and the student retention rate. The results indicate that when controlling for demographic, institution, student performance and workload variables, the EAS is able to identify students who have a significantly higher risk of discontinuing from their studies. This implies that early intervention strategies are effective in addressing student retention, and thus an EAS is able to provide actionable information to the student support team.
Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a ...sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence-function relationships of small-molecule transport systems in bacteria.
Injectable opioid agonist treatment (iOAT) was designed as a pragmatic and compassionate approach for people who have not benefitted from medication assisted treatment with oral opioids (e.g., ...methadone). While, a substantial body of clinical trial evidence has demonstrated the safety and effectiveness of iOAT, considerably less is known about the patient-centered aspects of this treatment and their role in self-reported treatment goals and outcomes. The aim of this study was to explore participants' experiences in iOAT as they broadly relate to the domains of patient-centered care. A secondary goal was to explore how these experiences affected participants' self-reported treatment outcomes.
A qualitative methodology, and constructivist grounded theory approach, was used to guide sampling, data collection and analysis. A total of 30 in-depth interviews were conducted with people receiving iOAT in North America's first clinic. Audio-recordings for each semi-structured interview were transcribed and read repeatedly. The strategy of constant comparison was used through iterative stages of line-by-line, focused and theoretical coding until theoretical saturation was achieved.
"Building healthcare provider relationships for patient-centered care in iOAT" was the emergent core concept. Healthcare provider relationships were established through two interrelated processes: 'Opening up' was attributed to the positive environment, and to feeling understood and supported by healthcare providers. 'Being a part of care' emerged as participants felt safe to ask for what was needed and had opportunities to collaborate in treatment decisions. These processes established a foundation in which participants experienced care that was responsive to their individual dose, health and psychosocial needs.
The core concept suggested that therapeutic relationships were fundamental to experiences of patient-centered care in iOAT. When relationships were respectful and understanding, participants received individualized and holistic care in iOAT. These findings offer a valuable example of how therapeutic relationships can be strengthened in other substance use treatment settings, particularly when responding to the diverse treatment needs of clients.
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