Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria

Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2.

The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast's autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known "point" CEN elements, and their biological activity is retained within ~900-1300 bp DNA segments. CEN1 and CEN2 have features of both "point" and "regional" centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast's enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches

Women's considerations and experiences for breast cancer screening and surveillance during the COVID-19 pandemic in the United States: A focus group study

The COVID-19 pandemic resulted in numerous changes in delivery of healthcare services, including breast cancer screening and surveillance. Although facilities have implemented a number of strategies to provide services, women's thoughts and experiences related to breast cancer screening and surveillance during a pandemic are not well known. This focus group study with women across seven states recruited through the Breast Cancer Surveillance Consortium aims to remedy this gap in information. Thirty women ranging in age from 31 to 69 participated in five virtual focus groups, eight of whom had prior breast cancer. The first three focus groups covered a range of topics related to screening and surveillance during the pandemic while the last two groups covered experiences and then a review of sample communications to women about screening and surveillance during the pandemic to obtain reactions and recommendations. More than half of the women had screening or surveillance during the pandemic. Coding and analyses resulted in nine themes in three topic areas: decision factors, screening experiences, and preferred communications. Themes included weighing the risks of COVID-19 versus cancer; feelings that screening and surveillance were mostly safe but barriers may be heightened; feeling safe when undergoing screening but receiving a range of pandemic-specific communications from none to a lot; and wanting communications that are personalized, clear and concise. Based on these findings, providers and facilities should assure women of pandemic safety measures, review methods and content of communications, and assess for barriers to screening that may be amplified during the pandemic, including anxiety and access

Breast Density Knowledge in a Screening Mammography Population Exposed to Density Notification

ObjectiveWomen are increasingly informed about their breast density due to state density reporting laws. However, accuracy of personal breast density knowledge remains unclear. We compared self-reported with clinically assessed breast density and assessed knowledge of density implications and feelings about future screening.MethodsFrom December 2017 to January 2020, we surveyed women aged 40 to 74 years without prior breast cancer, with a normal screening mammogram in the prior year, and ≥1 recorded breast density measures in four Breast Cancer Surveillance Consortium registries with density reporting laws. We measured agreement between self-reported and BI-RADS breast density categorized as "ever-dense" if heterogeneously or extremely dense within the past 5 years or "never-dense" otherwise, knowledge of dense breast implications, and feelings about future screening.ResultsSurvey participation was 28% (1,528 of 5,408), and 59% (896 of 1,528) of participants had ever-dense breasts. Concordance between self-report versus clinical density was 76% (677 of 896) among women with ever-dense breasts and 14% (89 of 632) among women with never-dense breasts, and 34% (217 of 632) with never-dense breasts reported being told they had dense breasts. Desire for supplemental screening was more frequent among those who reported having dense breasts 29% (256 of 893) or asked to imagine having dense breasts 30% (152 of 513) versus those reporting nondense breasts 15% (15 of 102) (P = .003, P = .002, respectively). Women with never-dense breasts had 6.3-fold higher odds (95% confidence interval:3.39-11.80) of accurate knowledge in states reporting density to all compared to states reporting only to women with dense breasts.DiscussionStandardized communications of breast density results to all women may increase density knowledge and are needed to support informed screening decisions

The analysis of <i>CEN1</i> and <i>CEN2</i> properties.

<p>(a) The <i>CEN1</i> locus has 65.6% AT content and carries the ser-tRNA gene; it carries part of a gene homologous to the <i>RPT2</i> gene, the <i>TVP15</i> gene, and the <i>COPI</i>-coated vesicle-protein encoding gene. The <i>CEN2</i> locus has 62.4% AT content and it carries the gene coding for the MATE efflux family protein. (b) The AT-peaks occur at the intergenic region in <i>CEN1</i> and within a 3’-region of <i>CEN2</i>. This subfigure was drawn using the DNA/RNA GC Content Calculator (<a href="http://www.endmemo.com/bio/gc.php" target="_blank">http://www.endmemo.com/bio/gc.php</a>). (c) The transformation efficiency of three plasmid types. (1) The plasmid with <i>CEN1</i>. (2) Two plasmids with <i>CEN2</i>, specifically the Y881 strain possessing the transposon and the Y879 strain lacking the transposon. (3) The deletion plasmids of <i>CEN2</i>, specifically <i>CEN2-1</i>, <i>CEN2-2</i>, <i>CEN2-3</i>, <i>CEN2-4</i>, <i>CEN2-5</i>; the plasmid <i>CEN2-2</i> has a transposon. The P892/BI plasmid, which carried the <i>URA3</i> gene and which had been linearized by <i>Bam</i>HI (BI), was used as a control. (d) The schematic representation of the <i>CEN1</i>, <i>CEN2</i> and <i>CEN2</i> deletion-plasmids. The sequences that promote autonomous replication and stable maintenance are marked with a plus sign (+); the remaining sequences are marked with a minus sign (-). (e) The MITE-like transposon found within the <i>CEN2</i> fragment (from 1925 to 2120 bp), named <i>CEN2-2</i>, was used as a query in a search through the <i>D</i>. <i>bruxellensis</i> genome (Y879 strain). 10 sequences (and their surroundings) were randomly sampled from the 51 present in the genome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161741#pone.0161741.s012" target="_blank">S2 Table</a>) and aligned with <i>CEN2-2</i> (reference sequence) to present the “structural” parts of the transposon: inverted and direct repeats. The bases identical to the reference sequence are shown in grey. Inverted repeats are 7 bp long and they are at the beginning and end of the transposon; marked with red arrows only on the reference sequence, inverted repeats are present in all sequences in the genome. Direct repeats are marked with blue arrows; they are 5 bp long but have different sequences, and they immediately flank the transposon (in the genome of Y879 strain).</p

Decision quality and regret with treatment decisions in women with breast cancer: Pre-operative breast MRI and breast density

Purpose: We evaluated self-report of decision quality and regret with breast cancer surgical treatment by pre-operative breast MRI use in women recently diagnosed with breast cancer. Methods: We conducted a survey with 957 women aged 18 + with stage 0-III breast cancer identified in the Breast Cancer Surveillance Consortium. Participants self-reported receipt of pre-operative breast MRI. Primary outcomes were process measures in the Breast Cancer Surgery Decision Quality Instrument (BCS-DQI) (continuous outcome) and Decision Regret Scale (dichotomized outcome as any/none). Generalized estimating equations with linear and logit link were used to estimate adjusted associations between breast MRI and primary outcomes. All analyses were also stratified by breast density. Results: Survey participation rate was 27.9% (957/3430). Study population was primarily \u3e 60 years, White, college educated, and diagnosed with early-stage breast cancer. Pre-operative breast MRI was reported in 46% of women. A higher proportion of women who were younger age (\u3c 50 years), commercially insured, and self-detected their breast cancer reported pre-operative breast MRI use. In adjusted analysis, pre-operative breast MRI use compared with no use was associated with a small but statistically significantly higher decision quality scores (69.5 vs 64.7, p-value = 0.043). Decision regret did not significantly differ in women who reported pre-operative breast MRI use compared with no use (54.2% v. 48.7%, respectively, p-value = 0.11). Study results did not vary when stratified by breast density for either primary outcome. Conclusions and relevance: Breast MRI use in the diagnostic work-up of breast cancer does not negatively alter women\u27s perceptions of surgical treatment decisions in early survivorship. Clinical trials registration number: NCT03029286

Yeast phylogeny and centromeres.

<p>The centromeres are signified by coloured ovals. Our cloned <i>D</i>. <i>bruxellensis</i> centromeres have features of both “regional” and “point” centromeres, and they appear in <i>green—blue</i>. The “conventional point” centromeres appear in <i>blue</i>; the “unconventional point” centromeres in <i>red</i>; the “regional” centromeres in <i>green</i>. A black oval displays the whole-genome duplication event (WGD). The phylogeny is adapted from Curtin and Pretorius, 2014, and the phylogeny and centromeres are adapted from Kobayashi et al., 2015.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Chromosomal-break assay and association with the centromere histone H3.

<p>(a) Karyotype analysis of <i>D</i>. <i>bruxellensis</i> transformants carrying <i>CEN1</i> and <i>CEN2</i> plasmids integrated into the genome by PFGE. Y879 and Y997 strains were used as controls. Transformants with changed karyotypes are arrowed. (b) Co-immunoprecipitation with Cse4 antibodies. ChIP samples were used as a template for PCR performed at two annealing temperatures (64 and 65°C) using specific primers for <i>CEN1</i> (OP98 and OP99) and the sub-cloned fragment of <i>CEN2</i> (<i>CEN2-5</i>, primers SW9 and SW10). Samples: 1) 1 kb ladder; 2) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 3) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 4) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers OP98 and OP99); 5) no antibody, negative control with Y879 chromatin (primers OP98 and OP99); 6) 1 kb ladder; 7) input of ChIP assay, Y879 chromatin (primers OP98 and OP99); 8) 1 kb ladder; 9) ChIP Ca antibodies with Y879 chromatin (primers SW9 and SW10); 10) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 11) 1 kb ladder; 12) input of ChIP assay, Y879 chromatin (primers SW9 and SW9); 13) input of ChIP assay, Y879 chromatin (primers OP98 and OP99); 14) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers SW9 and SW10); 15) no antibody, negative control with Y879 chromatin (primers SW9 and SW10); 16) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers OP98 and OP99); 17) no antibody, negative control with Y879 chromatin (primers OP98 and OP99); 18) 1kb ladder.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p