Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"-in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself-and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape

Nonradioactive, ultrasensitive site-specific protein–protein photocrosslinking: interactions of α-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2

We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein–protein photocrosslinking, and we have applied the approach to the analysis of interactions of α-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA–TBP–DNA complex and in higher order transcription–initiation complexes, and we have mapped the crosslink to the ‘connector’ region of the TFIIA α/β subunit (TFIIAα/β). We further have found that TBP H2 can be crosslinked to NC2 in the NC2–TBP–DNA complex, and we have mapped the crosslink to the C-terminal ‘tail’ of the NC2 α-subunit (NC2α). Interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail were not observed in crystal structures of TFIIA–TBP–DNA and NC2–TBP–DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAα/β connector and the NC2α C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP–TFIIA interactions and TBP–NC2 interactions, and provide an explanation—steric exclusion—for competition between TFIIA and NC2

Design, construction and characterization of a set of insulated bacterial promoters

We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5′- or 3′-sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community

The 'values journey' of nursing and midwifery students selected using multiple mini interviews: evaluations from a longitudinal study

Values-based practice is deemed essential for healthcare provision world-wide. In England, values-based recruitment methods, such as multiple mini interviews (MMIs), are employed to ensure that healthcare students’ personal values align with the values of the National Health Service (NHS), which focus on compassion and patient-centeredness. However, values cannot be seen as static constructs. They can be positively and negatively influenced by learning and socialisation. We have conceptualised students’ perceptions of their values over the duration of their education programme as a ‘values journey’. The aim of this hermeneutic longitudinal focus group study was to explore the ‘values journey’ of student nurses and midwives, recruited through MMIs, across the three years of their education programme. The study commenced in 2016, with 42 nursing and midwifery students, originally recruited onto their programmes through multiple mini interviews. At the third and final point of data collection, 25 participants remained. Findings indicate that students’ confidence, courage and sense of accountability increased over the three years. However, their values were also shaped by time constraints, emotional experiences and racial discrimination. We argue that adequate psychological support is necessary as healthcare students embark on and progress through their values journey, and propose a framework for this

Mutational Analysis of EGFR and Related Signaling Pathway Genes in Lung Adenocarcinomas Identifies a Novel Somatic Kinase Domain Mutation in FGFR4

BACKGROUND: Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line. CONCLUSIONS/SIGNIFICANCE: This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Human plague: An old scourge that needs new answers

Yersinia pestis, the bacterial causative agent of plague, remains an important threat to human health. Plague is a rodent-borne disease that has historically shown an outstanding ability to colonize and persist across different species, habitats, and environments while provoking sporadic cases, outbreaks, and deadly global epidemics among humans. Between September and November 2017, an outbreak of urban pneumonic plague was declared in Madagascar, which refocused the attention of the scientific community on this ancient human scourge. Given recent trends and plague’s resilience to control in the wild, its high fatality rate in humans without early treatment, and its capacity to disrupt social and healthcare systems, human plague should be considered as a neglected threat. A workshop was held in Paris in July 2018 to review current knowledge about plague and to identify the scientific research priorities to eradicate plague as a human threat. It was concluded that an urgent commitment is needed to develop and fund a strong research agenda aiming to fill the current knowledge gaps structured around 4 main axes: (i) an improved understanding of the ecological interactions among the reservoir, vector, pathogen, and environment; (ii) human and societal responses; (iii) improved diagnostic tools and case management; and (iv) vaccine development. These axes should be cross-cutting, translational, and focused on delivering context-specific strategies. Results of this research should feed a global control and prevention strategy within a “One Health” approach

Maintaining the Environmental–Racial Order in Northern New Mexico

The environmental - racial order in northern New Mexico is maintained through a process of racial triangulation in which Anglos, Native Americans, and Hispanos are valued relative to one another along axes of environmental stewardship and victimization (Kim C J, 1999, "The racial triangulation of Asian Americans" Politics and Society 27 105 - 138). Both axes involve the juxtaposing of three long-standing images: (1) Spanish injustices to the Indians; (2) the inability of Mexicans to manage their land properly; and (3) Indians being preeminent environmental stewards. In contrast to Kim's formulation of racial triangulation, however, the axes also involve imagery that contradicts these images: the debauched, poverty-stricken Indian; and European culture as a despoiler of the environment. Also in contrast to Kim's formulation, racial triangulation can involve the creation of new identities. In the 1960s Hispano activists began claiming to be heirs to a hybrid culture that included elements of both Native American and Spanish cultures. While this claim to hybridity enabled the creation of new oppositional discourses, the reconciling of contradictory imagery by historicizing the discourses and by other means undermines the new Hispano oppositional discourses as well as Hispano identity itself Racial triangulation is thus a fluid and contested process in which identity formation and the interchange between predominant and oppositional discourses are constitutive of power relations. Contradictory imagery in the discourse facilitates the maintenance of the environmental - racial order, even as it enables subordinates to challenge their racialized positions and to effect change in the distribution of material wealth, rights, and privileges

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery