Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

Protective Efficacy of Menthol Propylene Glycol Carbonate Compared to N, N-diethyl-Methylbenzamide Against Mosquito Bites in Northern Tanzania.

The reduction of malaria parasite transmission by preventing human-vector contact is critical in lowering disease transmission and its outcomes. This underscores the need for effective and long lasting arthropod/insect repellents. Despite the reduction in malaria transmission and outcomes in Tanzania, personal protection against mosquito bites is still not well investigated. This study sought to determine the efficacy of menthol propylene glycol carbonate (MR08), Ocimum suave as compared to the gold standard repellent N, N-diethyl-methylbenzamide (DEET), either as a single dose or in combination (blend), both in the laboratory and in the field against Anopheles gambiae s.l and Culex quinquefasciatus. In the laboratory evaluations, repellents were applied on one arm while the other arm of the same individual was treated with a base cream. Each arm was separately exposed in cages with unfed female mosquitoes. Repellents were evaluated either as a single dose or as a blend. Efficacy of each repellent was determined by the number of mosquitoes that landed and fed on treated arms as compared to the control or among them. In the field, evaluations were performed by human landing catches at hourly intervals from 18:00  hr to 01:00  hr. A total of 2,442 mosquitoes were collected during field evaluations, of which 2,376 (97.30%) were An. gambiae s.l while 66 (2.70%) were Cx. quinquefaciatus. MR08 and DEET had comparatively similar protective efficacy ranging from 92% to 100 for both single compound and blends. These findings indicate that MR08 has a similar protective efficacy as DEET for personal protection outside bed nets when used singly and in blends. Because of the personal protection provided by MR08, DEET and blends as topical applicants in laboratory and field situations, these findings suggest that, these repellents could be used efficiently in the community to complement existing tools. Overall, Cx. quinquefasciatus were significantly prevented from blood feeding compared to An. gambiae s.l. The incorporation of these topical repellents for protection against insect bites can be of additional value in the absence or presence of IRS and ITNs coverage. However, a combination of both the physical (bed nets) and the repellent should be used in an integrated manner for maximum protection, especially before going to bed. Additional research is needed to develop repellents with longer duration of protection

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

INTRODUCTION Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years

Safety climate and increased risk: The role of deadlines in design work

Although much research indicates positive safety climate is associated with reduced safety risk, we argue this association is not universal and may even be reversed in some contexts. Specifically, we argue that positive safety climate can be associated with increased safety risk when there is pressure to prioritize production over safety and where workers have some detachment from the consequences of their actions, such as found in engineering design work. We used two indicators of safety risk: use of heuristics at the individual level and design complexity at the design team level. Using experience sampling data (N = 165, 42 design teams, k = 5752 observations), we found design engineers’ perceptions of team positive safety climate were associated with less use of heuristics when engineers were not working to deadlines, but more use of heuristics when engineers were working to deadlines. Independent ratings were obtained of 31 teams’ designs of offshore oil and gas platforms (N = 121). For teams that worked infrequently to deadlines, positive team safety climate was associated with less design complexity. For teams that worked frequently to deadlines, positive team safety climate was associated with more design complexity

Worry is associated with robust reductions in heart rate variability: a transdiagnostic study of anxiety psychopathology

Background Individuals with anxiety disorders display reduced resting-state heart rate variability (HRV), although findings have been contradictory and the role of specific symptoms has been less clear. It is possible that HRV reductions may transcend diagnostic categories, consistent with dimensional-trait models of psychopathology. Here we investigated whether anxiety disorders or symptoms of anxiety, stress, worry and depression are more strongly associated with resting-state HRV. Methods Resting-state HRV was calculated in participants with clinical anxiety (n = 25) and healthy controls (n = 58). Symptom severity measures of worry, anxiety, stress, and depression were also collected from participants, regardless of diagnosis. Results Participants who fulfilled DSM-IV criteria for an anxiety disorder displayed diminished HRV, a difference at trend level significance (p = .1, Hedges’ g = -.37, BF10 = .84). High worriers (Total n = 41; n = 22 diagnosed with an anxiety disorder and n = 19 not meeting criteria for any psychopathology) displayed a robust reduction in resting state HRV relative to low worriers (p = .001, Hedges’ g = -.75, BF10 = 28.16). Conclusions The specific symptom of worry – not the diagnosis of an anxiety disorder – was associated with the most robust reductions in HRV, indicating that HRV may provide a transdiagnostic biomarker of worry. These results enhance understanding of the relationship between the cardiac autonomic nervous system and anxiety psychopathology, providing support for dimensional-trait models consistent with the Research Domain Criteria framework

Clinical assessment of DSM-IV anxiety disorders in fragile X syndrome: prevalence and characterization

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID). Anxiety and social withdrawal are considered core features of the FXS phenotype, yet there is limited diagnostic evidence of the prevalence of formal anxiety disorders in FXS. This study assessed the prevalence of anxiety disorders in a sample of 58 males and 39 females with FXS (ages 5.0–33.3 years). Participants’ parents completed the Anxiety Disorders Interview Schedule (ADIS-IV), a clinical interview based on DSM-IV criteria, and the Anxiety Depression and Mood Scale (ADAMS), a psychiatric disorders screening instrument normed in ID. We conducted cognitive (IQ) and autism (AUT) assessments and surveyed medication use. Despite a high rate of psychopharmacological treatment, 86.2% of males and 76.9% of females met criteria for an anxiety disorder, with social phobia and specific phobia the most commonly diagnosed. Proband status, gender, and IQ were not significantly related to any anxiety disorders, however significantly higher rates of a few anxiety disorders were found in older age and AUT groups. Significant correlations between ADIS diagnoses and ADAMS scores provided cross-validation of instruments, indicating that the ADIS is suitable for use in FXS. A greater percentage of our sample met criteria for most anxiety disorders than has been reported in other ID groups or the general population. The rate of anxiety compared to general ID suggests that the FMR1 full mutation confers an especially high risk for these disorders, regardless of factors commonly associated with FXS clinical involvement. A thorough clinical assessment and treatment of anxiety should be included in the FXS standard of care

Evolutionary analysis of mitochondrially encoded proteins of toad-headed lizards, Phrynocephalus, along an altitudinal gradient.

BACKGROUND: Animals living at high altitude must adapt to environments with hypoxia and low temperatures, but relatively little is known about underlying genetic changes. Toad-headed lizards of the genus Phrynocephalus cover a broad altitudinal gradient of over 4000 m and are useful models for studies of such adaptive responses. In one of the first studies to have considered selection on mitochondrial protein-coding regions in an ectothermic group distributed over such a wide range of environments, we analysed nineteen complete mitochondrial genomes from all Chinese Phrynocephalus (including eight genomes sequenced for the first time). Initial analyses used site and branch-site model (program: PAML) approaches to examine nonsynonymous: synonymous substitution rates across the mtDNA tree. RESULTS: Ten positively selected sites were discovered, nine of which corresponded to subunits ND2, ND3, ND4, ND5, and ND6 within the respiratory chain enzyme mitochondrial Complex I (NADH Coenzyme Q oxidoreductase). Four of these sites showed evidence of general long-term selection across the group while the remainder showed evidence of episodic selection across different branches of the tree. Some of these branches corresponded to increases in altitude and/or latitude. Analyses of physicochemical changes in protein structures revealed that residue changes at sites that were under selection corresponded to major functional differences. Analyses of coevolution point to coevolution of selected sites within the ND4 subunit, with key sites associated with proton translocation across the mitochondrial membrane. CONCLUSIONS: Our results identify mitochondrial Complex I as a target for environment-mediated selection in this group of lizards, a complex that frequently appears to be under selection in other organisms. This makes these lizards good candidates for more detailed future studies of molecular evolution