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The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an \"off cassette\" between the promoter and the coding region of the desired product. The \"off cassette\" is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.

The global prevalence of mental health disorders is at a crisis point, particularly in the wake of COVID-19, prompting calls for the development of digital interdisciplinary mental health promotion interventions (MHPIs) for nonclinical cohorts. However, the influence of gender and age on the outcomes of and adherence to MHPIs is not well understood. The aim of this study was to determine the influence of gender and age on the outcomes of and adherence to a 10-week digital interdisciplinary MHPI that integrates strategies from positive psychology and lifestyle medicine and utilizes persuasive systems design (PSD) principles in a nonclinical setting. This study involved 488 participants who completed the digital interdisciplinary MHPI. Participants completed a pre and postintervention questionnaire that used: (1) the \"mental health\" and \"vitality\" subscales from the Short Form 36 (SF-36) Health Survey; (2) the Depression, Anxiety and Stress Scale (DASS-21); and (3) Satisfaction With Life Scale (SWL). Adherence to the digital interdisciplinary MHPI was measured by the number of educational videos the participants viewed and the extent to which they engaged in experiential challenge activities offered as part of the program. On average, the participants (N=488; mean age 47.1 years, SD 14.1; 77.5% women) demonstrated statistically significant improvements in all mental health and well-being outcome measures, and a significant gender and age interaction was observed. Women tended to experience greater improvements than men in the mental health and well-being measures, and older men experienced greater improvements than younger men in the mental health and vitality subscales. Multiple analysis of variance results of the adherence measures indicated a significant difference for age but not gender. No statistically significant interaction between gender and age was observed for adherence measures. Digital interdisciplinary MHPIs that utilize PSD principles can improve the mental health and well-being of nonclinical cohorts, regardless of gender or age. Hence, there may be a benefit in utilizing PSD principles to develop universal MHPIs such as that employed in this study, which can be used across gender and age groups. Future research should examine which PSD principles optimize universal digital interdisciplinary MHPIs. Australian New Zealand Clinical Trials Registry ACTRN12619000993190; http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=377889 and Australian New Zealand Clinical Trials Registry ACTRN12619001009101; http://www.anzctr.org.au/ACTRN12619001009101.aspx.

Background There is an urgent need for efficacious interventions to combat the global mental health crisis, and mental health promotion and primary prevention approaches are paramount. The aim of this study is to examine whether an online interdisciplinary intervention that incorporates evidence-based strategies from the disciplines of Lifestyle Medicine and Positive Psychology improves measures of mental health and emotional wellness. Methods A randomized controlled trial with a wait-list control (N = 425, aged 46.97 ± 14.5, 69.9% females) was conducted in Australia and New Zealand. The intervention group participated in a 10-week online interdisciplinary intervention. Primary outcome measures of mental health and emotional wellness were taken at baseline (Week 1), post-intervention (Week 12), and 12 weeks post-intervention (Week 24). The wait-list control completed the same assessments. Results General Linear Modelling analyses indicated that the intervention group experienced significantly greater improvements than the wait-list control group over time in all outcome measures: mental health (F(319) = 7.326, p  = 0.007) and vitality (F(319) = 9.445, p  = 0.002) subscales of the Short Form Survey (SF-36); depression (F(319) = 7.841, p  = 0.005), anxiety (F(319) = 4.440,  p  = 0.36) and stress (F(319) = 12.494, p  < 0.001) scales of the Depression, Anxiety and Stress Scale (DASS-21); and life satisfaction (F(319) = 8.731, p  = 0.003) as measured by the Satisfaction With Life Scale. Within the intervention group, significant improvements were observed from Week 1 to 12 in all outcome measures: mental health (10%, t(167) = − 6.423), p  < 0.001, dz = 0.50), vitality (22%, t(167) = − 7.043, p  < 0.001, dz = 0.54), depression (− 41%, t(167) = 6.189, p  < 0.001, dz = 0.48), anxiety (− 38%, t(167) = 5.030, p  < 0.001, dz = 0.39), stress (− 31%, t(167) = 6.702, p  < 0.001, dz = 0.52) and life satisfaction (8%, t(167) = − 6.199, p  < 0.001, dz = 0.48). Improvements in the outcome measures remained significant in the intervention group at 12 weeks post-intervention. Conclusion The online interdisciplinary intervention improved measures of mental health and emotional wellness suggesting that such interventions may be useful for mental health promotion and prevention. Trial registration The Australian New Zealand Clinical Trials Registry. ACTRN12619000993190. Registered on 12 July 2019 (Retrospectively registered). The ANZCTRN is part of the WHO Primary Registries.

Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo . This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the Fok I restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli , partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV−, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine. •This HNSCC xenograft model includes all clinical subtypes by location and HPV status.•Mutation profile is representative of seminal sequencing reports (Including NOTCH).•Morphology, gene expression and drug sensitivity are stable over generations.•Cases with PI3KCA alterations were sensitive to the novel PI3K inhibitor PX-866.•PI3K inhibition was effective in two cases harboring NOTCH1 mutations.

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, ≈20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.

Rocky reef habitat is common in many estuaries, yet its role as a habitat for fishes is poorly understood. There is also limited understanding of how access of coastal species into estuaries and habitat quality can affect the distribution of rocky reef fishes within estuaries. This study used baited remote underwater video stations to determine spatial patterns in fish assemblages associated with rocky reef habitat throughout a barrier estuary with a permanently open but restricted inlet. Estuarine rocky reefs provided habitat for a diverse assemblage of fishes, many of which were large juveniles and subadults. In the absence of a pronounced salinity or temperature gradient, a clear transition in fish assemblages occurred from coastal waters, through the inlet channel, to the central estuary, and into the inner estuary. The inlet channel, notably its narrowness and length, limits tidal input into this estuary, which acts as a significant impediment to the dispersal of many coastal fishes, and insufficient habitat excludes many coastal rocky reef species from the inner estuary. This study highlights the need to recognise estuarine rocky reefs as providing habitat for diverse fish assemblages and the role inlets play in restricting access of coastal species.

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG·CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.

The foraging behaviours and dietary compositions of three co-occurring labrids ( Ophthalmolepis lineolatus , Notolabrus gymnogenis and Pictilabrus laticlavius ), which are conspicuous on rocky reefs in temperate south-eastern Australia, were investigated between 2003 and 2005. SCUBA observations at two locations showed that the feeding intensity, and hence the associated effects of these fishes on rocky reef invertebrate prey, was temporally consistent. Relative differences in the contributions of ingested prey and use of different feeding microhabitats demonstrated that the feeding ecology differed significantly among the three species. Thus, O. lineolatus fed on proportionately higher volumes of polychaetes, polyplacophorans, marginellid gastropods (especially Austroginella sp.), bivalves and echinoids, which were sighted opportunistically in a wide selection of microhabitats, but particularly in sand/rubble. Ambush hunting was used regularly by smaller N. gymnogenis and all sizes of P. laticlavius to forage on amphipods, small decapods and small gastropods at algal bases or fronds and Diopatra dentata tubes. Amphipods were similarly important in the diet of smaller O. lineolatus . Larger N. gymnogenis foraged opportunistically over an increased reef area and made greater use of microhabitats that offered minimal prey refuge (e.g. sand/rubble, bare rock/steel) from which common prey, in particular decapods, were obtained. The significant intra- and inter-specific differences in dietary compositions, allied with differences in the use of feeding microhabitats, would facilitate co-occurrence of these three conspicuous species and contribute to maintaining high richness of labrid species in reef systems. Echinoids were regularly consumed by each species but they made a moderate contribution to the diet of only O. lineolatus , which suggests that only one of the three labrids is likely to play a significant role in regulation of echinoid densities in these rocky reef habitats. However, the broad diets and diverse forging strategies employed by these labrid species imply that they have a system-wide influence on invertebrate prey on rocky reefs.

Digital mental health promotion interventions (MHPIs) present a scalable opportunity to attenuate the risk of mental health distress among nonclinical cohorts. However, adherence is frequently suboptimal, and little is known about participants' perspectives concerning facilitators and barriers to adherence in community-based settings. This study aimed to examine participants' perceptions of facilitators and barriers to adherence in a web- and mobile app-based MHPI for a nonclinical cohort. This qualitative study used inductive, reflexive thematic analysis to explore free-text responses in a postintervention evaluation of a 10-week digital MHPI. The intervention was administered using a web and mobile app from September to December 2018. Participants (N=320) were Australian and New Zealand members of a faith-based organization who self-selected into the study, owned a mobile phone with messaging capability, had an email address and internet access, were fluent in English, provided informed consent, and gave permission for their data to be used for research. The postintervention questionnaire elicited participants' perceptions of facilitators and barriers to adherence during the intervention period. Key factors that facilitated adherence were engaging content, time availability and management, ease of accessibility, easy or enjoyable practical challenges, high perceived value, and personal motivation to complete the intervention. The primary perceived barrier to adherence was the participants' lack of time. Other barriers included completing and recording practical activities, length of video content, technical difficulties, and a combination of personal factors. Time scarcity was the foremost issue for the nonclinical cohort engaged in this digital MHPI. Program developers should streamline digital interventions to minimize the time investment for participants. This may include condensed content, optimization of intuitive web and app design, simplified recording of activities, and greater participant autonomy in choosing optional features. Nonetheless, participants identified a multiplicity of other interindividual factors that facilitated or inhibited adherence.