Heterogenous associations between Gender-Sexuality Alliances and LGBTQ adolescents' maladjustment across individual victimization level.

Gender-Sexuality Alliances (GSAs), which are student-initiated school clubs for LGBTQ youth and allies, can reduce victimization for lesbian, gay, bisexual, transgender, and queer (LGBTQ) youth. This preregistered study identified heterogeneous correlates of GSAs, based on data from an anonymous survey of LGBTQ adolescents aged 13-17 years living in the United States (N = 10,588). In line with the healthy context paradox (Pan et al. [Child Development, 92, 2021, and 1836]), the presence of a GSA exacerbated associations between LGBTQ-based victimization and depressive symptoms, lower self-esteem, and lower academic grades-particularly in transgender youth. Inclusive settings, such as GSAs, might prevent increasing disparities by including tailored strategies to monitor and support more vulnerable, victimized LGBTQ youth.

Associations of Relationship Experiences, Dating Violence, Sexual Harassment, and Assault With Alcohol Use Among Sexual and Gender Minority Adolescents.

Sexual and gender minority (SGM) adolescents report higher rates of dating violence victimization compared with their heterosexual and cisgender peers. Research on dating violence often neglects diversity in sexual and gender identities and is limited to experiences in relationships. Further, given that dating violence and alcohol use are comorbid, research on experiences of dating violence could provide insights into alcohol use disparities among SGM adolescents. We aimed to map patterns of relationship experiences, sexual and physical dating violence, and sexual and physical assault and explored differences in these experiences among SGM adolescents. Further, we examined how these patterns explained alcohol use. We used a U.S. non-probability national web-based survey administered to 13-17-year-old SGM adolescents (N = 12,534). Using latent class analyses, four patterns were identified: low relationship experience, dating violence and harassment and assault (72.0%), intermediate dating experiences, sexual harassment, and assault and low levels of dating violence (13.1%), high dating experiences, dating violence, and sexual assault (8.6%), and high dating experiences, dating violence, and sexual harassment and assault (6.3%). Compared to lesbian and gay adolescents, bisexual adolescents reported more experiences with dating, dating violence, and sexual assault, whereas heterosexual adolescents reported fewer experiences with dating, dating violence, and sexual harassment and assault. Compared to cisgender boys, cisgender girls, transgender boys, and non-binary/assigned male at birth adolescents were more likely to experience dating violence inside and outside of relationship contexts. Experiences of dating, dating violence, and sexual harassment and assault were associated with both drinking frequency and heavy episodic drinking. Together, the findings emphasize the relevance of relationship experiences when studying dating violence and how dating violence and sexual harassment and assault might explain disparities in alcohol use.

Scan-directed mini-incision focused parathyroidectomy: how accurate is accurate enough?

INTRODUCTION Mini-incision focused parathyroidectomy (MI-FP) is advocated as an alternative to bilateral neck exploration (BNE), owing to its reduced morbidity. The site and side of the affected gland is identified preoperatively using a combination of ultrasound and sestamibi scans. However, the acceptable degree of inter-scan concordance required to prompt MI-FP without compromising accuracy is undetermined. METHODS Accuracy of preoperative imaging was determined both individually and in combination for all parathyroidectomies (2007-2014). A grading system (excellent, good, poor) was devised to describe the interscan concordance, which was validated by the operative and histological findings. RESULTS Eighty-nine patients (17 male, 68 female) underwent parathyroidectomy (MI-FP 44, BNE 45). The accuracy of scans interpreted individually was 53% for ultrasound and 60% for sestamibi, with no difference according to surgical technique (P = 0.43, P = 1, respectively). The proportion of interscan concordance was: excellent - 35%, good - 40%, poor 25%. Combined accuracy was 100% for both excellent and good grades but only 13% for those graded poor. Similar rates of normocalcaemia were observed for MI-FP and BNE, while postoperative hypocalcaemia was five times higher in those undergoing BNE. CONCLUSIONS Reduction in the inter-scan concordance from excellent to good does not compromise accuracy. MI-FP could be successfully performed in up to 75% of patients - 25% higher than recommended in national guidelines. Focused parathyroidectomy does not compromise surgical and endocrinological outcomes but boasts a far superior complication rate.

The predictive power of airborne gamma ray survey data on the locations of domestic radon hazards in Norway: A strong case for utilizing airborne data in large-scale radon potential mapping.

It is estimated that exposure to radon in Norwegian dwellings is responsible for as many as 300 deaths a year due to lung cancer. To address this, the authorities in Norway have developed a national action plan that has the aim of reducing exposure to radon in Norway (Norwegian Ministries, 2010). The plan includes further investigation of the relationship between radon hazard and geological conditions, and development of map-based tools for assessing the large spatial variation in radon hazard levels across Norway. The main focus of the present contribution is to describe how we generate map predictions of radon potential (RP), a measure of radon hazard, from available airborne gamma ray spectrometry (AGRS) surveys in Norway, and what impact these map predictions can be expected to have on radon protection work including land-use planning and targeted surveying. We have compiled 11 contiguous AGRS surveys centred on the most populated part of Norway around Oslo to produce an equivalent uranium map measuring 180 km × 102 km that represents the relative concentrations of radon in the near surface of the ground with a spatial resolution in the 100 s of metres. We find that this map of radon in the ground offers a far more detailed and reliable picture of the distribution of radon in the sub-surface than can be deduced from the available digital geology maps. We tested the performances of digital geology and AGRS data as predictors of RP. We find that digital geology explains approximately 40% of the observed variance in ln RP nationally, while the AGRS data in the Oslo area split into 14 bands explains approximately 70% of the variance in the same parameter. We also notice that there are too few indoor data to characterise all geological settings in Norway which leaves areas in the geology-based RP map in the Oslo area, and elsewhere, unclassified. The AGRS RP map is derived from fewer classes, all characterised by more than 30 indoor measurements, and the corresponding RP map of the Oslo area has no unclassified parts. We used statistics of proportions to add 95% confidence limits to estimates of RP on our predictive maps, offering public health strategists an objective measure of uncertainty in the model. The geological and AGRS RP maps were further compared in terms of their performances in correctly classifying local areas known to be radon affected and less affected. Both maps were accurate in their predictions; however the AGRS map out-performed the geology map in its ability to offer confident predictions of RP for all of the local areas tested. We compared the AGRS RP map with the 2015 distribution of population in the Oslo area to determine the likely impact of radon contamination on the population. 11.4% of the population currently reside in the area classified as radon affected. 34% of ground floor living spaces in this affected area are expected to exceed the maximum limit of 200 Bq/m3, while 8.4% of similar spaces outside the affected area exceed this same limit, indicating that the map is very efficient at separating areas with quite different radon contamination profiles. The usefulness of the AGRS RP map in guiding new indoor radon surveys in the Oslo area was also examined. It is shown that indoor measuring programmes targeted on elevated RP areas could be as much as 6 times more efficient at identifying ground floor living spaces above the radon action level compared with surveys based on a random sampling strategy. Also, targeted measuring using the AGRS RP map as a guide makes it practical to search for the worst affected homes in the Oslo area: 10% of the incidences of very high radon contamination in ground floor living spaces (≥800 Bq/m3) are concentrated in just 1.2% of the populated part of the area.

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 µL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Disruption of pocket protein dream complexes by E7 proteins of different types of human papillomaviruses.

It has been shown that the E7 protein of the high-risk HPV-16 transforms cells in vitro and binds pRB, p107 and p130, so called pocket proteins associated in cells with DREAM proteins, while that of the low-risk HPV-6 does not transform cells and binds p130 but not pRB or p107. These facts may indicate that p130 is essential for the HPV life cycle. To gain further insight into the relationship between HPV E7 proteins and pocket protein-DREAM complexes, E7 proteins of HPVs of various risk categories were expressed via appropriate vectors in T98G cells and the levels of various pocket proteins either total or associated with DREAM were analyzed. The obtained results demonstrated that high-risk HPV-16, HPV-18 and HPV-33, low-risk HPV-1 and HPV-11, and cutaneous HPV-48 disrupted pocket protein-DREAM complexes in T98G cells to a similar extent.

A method for making directed changes to the Fusarium graminearum genome without leaving markers or other extraneous DNA.

A method is described which allows exact targeted changes to the Fusarium graminearum genome, including changes of as little as one particular base pair to gene-size insertions, replacements or modifications. The technique leaves no other DNA in the genome, such as marker genes, and can be used serially to effect multiple complex changes in any desired chromosomal locations. The method is based on our previous finding that after transformation, DNA with homology to F. graminearum DNA recombines itself into the genome in a predictable manner involving multiple tandem copies. We designed a cloning vehicle with a built-in hygromycin-resistance marker (hygB) which can be used to transform the fungus, and with cloning sites to carry DNA with any desired genomic modifications. To effect a desired genomic change the sequence changes of interest are incorporated between two adjacent borders homologous to F. graminearum DNA which will target them to the desired location. This modified DNA is attached within the cloning sites within the vehicle. Transformants are readily obtained in which tandem copies of the vehicle plus insert are inserted between the two genomic border sequence homologues. Progeny of a transformant are subsequently screened for those with a decreased resistance to the antibiotic, and then for those which have completely lost the marker and the entire vehicle, leaving only the desired sequence modifications between the two genomic border sequences which were targeted. This method is demonstrated by exactly replacing the trichodiene synthase (tri5) gene coding sequence (CDS) with that of a green fluorescence protein (gfp) gene with no other genomic changes. This derivative was then re-engineered to replace the gfp CDS with that of the wild type, exactly regenerating the original F. graminearum genome.

A Lin-9 complex is recruited by B-Myb to activate transcription of G2/M genes in undifferentiated embryonal carcinoma cells.

It has recently been discovered that cell-cycle gene transcription is regulated by a core complex named LINC that switches from a transcriptionally repressive complex in G(0)-G(1) with the p130 or p107 pocket proteins and E2F4 to a transcriptionally active complex in S-G(2) containing B-Myb. We have studied the function of LINC in F9 embryonal carcinoma cells, which are distinguished by a rapid cell cycle resulting from an extremely short G(1) phase. We show that suppressing expression of the LINC component, Lin-9, in F9 cells causes arrest in mitosis, and we have used this system to screen for transcriptional targets. In these cells, B-Myb was found in complexes with Lin-9 and several other LINC constituents, however, the pocket proteins did not associate with LINC unless F9 cells were differentiated. Lin-9 and B-Myb were both required for transcription of G(2)/M genes such as Cyclin B1 and Survivin. Moreover, B-Myb was demonstrated to recruit Lin-9 to the Survivin promoter through multiple Myb-binding sites. The demonstration that a B-Myb/LINC complex is vital for progression through mitosis in cells lacking a G(1)/S checkpoint has implications for both undifferentiated embryonal cells and for cancers in which pocket protein function is compromised.

A model for integration of DNA into the genome during transformation of Fusarium graminearum.

Transformants of Fusarium graminearum were derived using linearized DNA of plasmids designed to replace the trichodiene synthase gene, a cutinase gene or a xylanase gene with a hygromycin-resistance marker cassette by homologous recombination between 1-kbp segments of flanking DNA. Most transformants did not exhibit the DNA structure expected of integration by classical double recombination. Instead, they contained linearized plasmid joined end-to-end and variably incorporated into the genome. Transformant types included ectopic integrations and integrations at the target site with or without removal of the targeted gene. We have analyzed a large number of transformants using cloning, PCR and DNA sequencing to determine the structures of their integrated DNA, and describe a model to explain their derivations. The data indicate that 1-3 copies of input DNA are first joined end-to-end to produce either linear or circular structures, probably mediated by the non-homologous end-joining (NHEJ) system. The end-joins typically have 1-5 nucleotides in common and are near or within the original cleavage site of the plasmid. Ectopic integrations occur by attaching linear DNA to two ends of genomic DNA via the same joining mechanism. Integration at the target site is consistent with replication around circularized input DNA, beginning and ending within the flanking homologous DNA, resulting in the integration of multiple copies of the entire structure. This results in deletion or duplication of the target site, or leaves one copy at either end of the integrated multimer. Reiterated DNA in the more complex structures is unstable due to homologous recombination, such that conversion to simpler forms is detected.

Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells.

Previous studies have shown that the cell cycle-regulated B-myb promoter contains a conserved E2F binding site that is critical for repressing transcription in quiescent cells. To investigate its significance for permanent promoter silencing, we have inactivated this binding site in the mouse genome. Mice homozygous for the mutant B-mybmE2F allele were fully viable, however, B-myb transcription was derepressed during quiescence in mouse embryo fibroblasts (MEFs) derived from mutant animals. Moreover, it was found that mutation of the E2F site resulted in abnormal maintenance of B-myb expression in senescent MEFs and in differentiated brain tissue. These findings therefore reveal a direct and primary role for repressive E2F complexes in silencing gene expression in post-mitotic cells. Analysis of histone modifications at the promoter showed that histone H3 lysine 9 was constitutively acetylated throughout the cell cycle in homozygous mutant MEFs. This mouse system is the first description of an E2F site mutation in situ and will facilitate the study of E2F function in vivo.

Cell cycle regulation by the B-Myb transcription factor.

The expression of genes required for progression through the cell cycle is highly modulated through a regulatory axis containing the E2F transcription factor and retinoblastoma tumour suppressor protein families. One of the genes regulated through this mechanism encodes the B-Myb transcription factor, which has been shown to be critically required for early embryonal development in the mouse. Transcriptional activity of B-Myb is substantially enhanced in S phase through modification by cyclin A/cdk2, and the evidence points squarely to the major role being played by B-Myb during this phase of the cell cycle. We discuss in this review recent findings suggesting that B-Myb is a multifunctional protein that has, in addition to its transcriptional properties, the ability to interact directly with other regulators of the cell cycle.

In vitro acoustic characterisation of four intravenous ultrasonic contrast agents at 30 MHz.

The acoustic properties of four ultrasonic contrast agents (Optison, Definity, SonoVue and Sonazoid) were studied at 30 MHz using a Boston Scientific ClearView Ultra intravascular ultrasound (US) scanner modified to allow access to the unprocessed US data. A range of contrast agent concentrations were studied using either saline or glucose as the diluent of choice. Mean backscatter power was measured over regions-of-interest (ROI) at distances of 1, 1.5, 2, 3, 4 and 5 mm from the centre of the intravascular probe and normalised to the US data collected from a standard glass reflector. For all of the agents, the mean backscatter power at 30 MHz varied in a linear manner with concentration between 0.01 million microbubbles/mL and 1 million microbubbles/mL. Furthermore, for two of the agents, mean backscatter enhancement was detectable at concentrations as low as 2 microbubbles/sample volume.

Three-dimensional forward-viewing intravascular ultrasound imaging of human arteries in vitro.

The aim of this work was to investigate the suitability of a novel forward-viewing intravascular ultrasound (IVUS) technique for three-dimensional imaging of severely stenosed or totally occluded vessels, where the conventional side-viewing IVUS systems are of limited use. A stiff 3.8 mm diameter forward-viewing catheter was manufactured to scan a 72 degrees sector ahead of its tip. Conical volume data were acquired by rotating the catheter over 180 degrees by means of a motorised mechanical system. Operating at 30 MHz, the catheter was integrated with an IVUS scanner and a radiofrequency data acquisition system. Postmortem carotid and femoral arteries were scanned in vitro. Correlation of the reconstructed images with histology demonstrated the ability of this forward-viewing IVUS system to visualise healthy lumens, bifurcations, thickened atherosclerotic walls and, most importantly, severe and complete vessel occlusions. A rotating-sector forward-viewing IVUS system is suitable for anatomical assessment of severely diseased vessels in three dimensions.

Sinorhizobium meliloti cells require biotin and either cobalt or methionine for growth.

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.

Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator.

Expression of the B-Myb transcription factor is directed by an E2F-dependent transcriptional mechanism to late G1 and S phases of the cell cycle, where its transactivation properties are enhanced post-translationally by cyclin A/Cdk2-mediated phosphorylation. Other experiments have shown that removal of the B-Myb C-terminus constitutively activates both transactivation and DNA-binding activities, suggesting that autoregulation by this inhibitory domain is counteracted by phosphorylation. We report here on further experiments to examine this hypothesis. The importance of this modification was first emphasized by showing that co-transfected dominant-negative Cdk2 (Cdk2DN) substantially reduced B-Myb transactivation activity. We then attempted to map the autoregulatory domain by analysing a series of progressively deleted C-terminal B-Myb mutants. Removal of just 29 C-terminal aa increased transactivation appreciably, however, maximal activity required removal of 143 amino acids (as in B-Myb + 561). Enhanced B-Myb + 561 function correlated with the acquisition of DNA binding activity to a single Myb binding site (MBS) oligonucleotide as determined by bandshift assays, however, further assays showed that even wt B-Myb could bind a DNA fragment containing three MBS. Although transactivation by B-Myb was severely dependent on hyperphosphorylation, neither inhibiting this activity by co-transfecting Cdk2DN nor augmenting it with cyclin A resulted in significant effects on DNA-binding. We also found that B-Myb could synergize with the CBP coactivator and that this cooperativity was cyclin A/Cdk2-dependent. Despite this, the physical association between these proteins was not influenced by the B-Myb phosphorylation status. We discuss these findings in relation to the autoregulation of B-Myb by the C-terminal domain.

Arylsulphonyl hydroxamic acids: potent and selective matrix metalloproteinase inhibitors.

A series of novel matrix metalloproteinase inhibitors is described in which selectivity between MMP and 'sheddase' activity has been achieved and which demonstrate potent in vivo activity in models of arthritis and cancer.

Synthesis and structure-activity relationships of guanine analogues as phosphodiesterase 7 (PDE7) inhibitors.

The synthesis of a novel series of guanine analogues is reported. The compounds have been assessed in vitro and some analogues have been found to be inhibitors of phosphodiesterase type 7 (PDE7).

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Regulation of the cell cycle by B-Myb.

B-Myb is a cell-cycle-regulated member of the Myb transcription factor and, like c-Myb, has been implicated in regulation of hematopoietic cell proliferation and differentiation. In this study we have examined the mechanisms by which B-Myb regulates the cell cycle. We found that the ability of B-Myb both to promote Saos-2 cells into the S phase of the cell cycle and to overcome G1 arrest mediated by overexpression of the retinoblastoma-related p107 protein was correlated with the capacity of B-Myb to form an in vivo complex with p107, but was independent of its transactivation function. Further experiments using a B-Myb dominant-negative protein suggested that transcriptional activation of genes regulated through Myb DNA-binding sequences was required for cell proliferation. Our experiments suggest, therefore, that B-Myb influences cell cycle progression at two distinct levels: by inhibiting p107 and by inducing transcription of specific target genes.

Purification and characterization of a common soil component which inhibits the polymerase chain reaction.

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.