Are we choosing wisely in elderly females with breast cancer?

BACKGROUND: The Choosing Wisely Organization and the American College of Surgeons have issued recommendations for patients >70 with breast cancer involving screening and use of radiation therapy (RT) and sentinel lymph node biopsies (SNLB) in early stage tumors. This study evaluated compliance and implementation of these recommendations. METHODS: A database of patients undergoing breast cancer surgery was retrospectively queried from 2002 to 2017. Patients were divided into cohorts before and after the year of each guideline publication. RESULTS: The rate of presentation on mammography was not different before 2009 (65%) vs. after 2009 (66%). RT was given to 57% of patients with T1 ER + Her2-prior to 2013 vs. 27% after (p=<0.001). SLNB was performed in 91% of patients with T1, grade1/2, ER + Her2-tumors prior to 2016 vs. 56% after (p=<0.001). CONCLUSION: Rates of mammography detected breast cancer have not decreased but adjuvant RT and SLNB are less frequently performed in low risk breast cancer in the elderly.

Prevalence of Shiga toxin-producing Shigella species isolated from French travellers returning from the Caribbean: an emerging pathogen with international implications.

Shiga toxins (Stxs) are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Stx-producing Escherichia coli or Shigella dysenteriae serotype 1, and infections with these strains can lead to haemolytic-uraemic syndrome. Over the past decade, there has been increasing recognition that Stx is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage, and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study, we further explored the epidemiological link to this region by utilizing the French National Reference Centre for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travellers returning from the Caribbean. Approximately 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travellers who were infected with Stx-producing Shigella had recently travelled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing showed that the toxin genes were encoded by a prophage that was highly identical to the phage that we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups.

Pulse compression in a time variant system with application to ultrasonic vibrometry.

Pulse compression is normally applied only to time-invariant systems, as the variation of a system's properties during its interrogation violates assumptions of the compression process. However, there is an exact solution to the pulse-compression problem when the time variance satisfies two criteria, which are the same as those required for the operation of an ultrasonic vibrometer in the context of a tissue elastography system. One is that the variations be very small in comparison with the wavelength of the interrogating ultrasound. The other is that the bandwidth of the variations be within one Nyquist band as sampled by the periodic interrogation signal. The solution to this problem involves a step-wise interpolation of the static pulse-compression transfer function in the frequency domain. This technique, in conjunction with the selection of an appropriate interrogation signal, offers significant advantages in measurement time or measurement resolution for an ultrasonic vibrometer limited by additive noise at the receiver. The characteristics of optimal interrogation signals for this technique are the signal's crest factor, spectral energy distribution, and phasing. These relate to the intended compression pulse, the noise, and the static response of the system. The technique has been demonstrated analytically, experimentally, and with numerical models.

Range discrimination in ultrasonic vibrometry: theory and experiment.

A technique has been developed to demodulate periodic broadband ultrasonic interrogation signals that are returned from multiple scattering sites to simultaneously determine the low-frequency displacement time histories of each individual site. The technique employs a broadband periodic transmit signal. The motions of scattering sites are separately determined from the echoed receive signal by an algorithm involving comb filtering and pulse synthesis. This algorithm permits spatial resolution comparable to pulse-echo techniques and displacement sensitivities comparable to pure-tone techniques. A system based on this technique was used to image transient audio-frequency displacements on the order of 1-10 µm peak (≥ 50 nm/√Hz) that were produced by propagating shear waves in a tissue phantom. The system used concentric transmitting and receiving transducers and a carrier signal centered at 2.5 MHz with an 800 kHz bandwidth. The system was self-noise-limited and capable of detecting motions of strongly reflecting regions on the order of 1 nm/√Hz. System performance is limited by several factors including signal selection, component hardware, and ultrasonic propagation within the media of interest.

The Werner syndrome protein is involved in RNA polymerase II transcription.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO.

Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.

Werner syndrome protein. I. DNA helicase and dna exonuclease reside on the same polypeptide.

Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3' --> 5' DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A. , Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100-103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease.

The human FE65 gene: genomic structure and an intronic biallelic polymorphism associated with sporadic dementia of the Alzheimer type.

The FE65 protein binds to the intracellular domain of the beta-amyloid precursor protein (betaPP) and may modulate the internalization of betaPP. This gene is highly expressed in regions of the brain specifically affected in dementia of the Alzheimer type (DAT). As a prelude to further investigations of the role of FE65 in the metabolism of betaPP and in the pathogenesis of DAT, we have determined the entire genomic structure and sequence of human FE65 and have discovered several polymorphisms in this gene. Human FE65 contains 14 exons ranging in size from 6 to 735 bp. All splice sites conform to consensus sequences except for the donor site of intron 10. The 5' end of FE65 mRNA was identified by rapid amplification of the cDNA 5' end and is 31 bp longer than the previously published cDNA sequence. The 5'-flanking region of this gene is TATA-less and is very GC-rich with at least five putative Sp1 binding sites. In comparison to the genomic rat FE65 sequence, the human FE65 5'-untranslated region is 134 bp longer and has an extra exon (exon 1, 86 bp). To identify mutations/polymorphisms of the coding regions of this gene, we performed blinded analysis of 457 Caucasian case-control samples from a large epidemiological study of sporadic DAT. Screening was conducted by single-strand conformation polymorphism. Four minor variants were found within the coding region, with frequencies between 0.002 and 0.015; two of the four result in amino acid substitutions. The more informative biallelic polymorphism (a trinucleotide deletion and a single base substitution) was found within intron 13 (84 bp), which interrupts two exons encoding the betaPP binding site. The frequency of the minor allele in this intron was 0.097 in DAT cases and 0.161 in controls (chi2=7.78, P=0.0054). Having at least one copy of the minor allele was associated with a decreased risk for DAT (chi2=9.20, P<0.005, odds ratio=0.49, 95% CI 0.31-0.77). Multivariate analysis showed that this association was independent of the APOE genotype. These results suggest that either FE65 itself or a closely linked gene influences the pathogenesis of sporadic DAT. The interaction of FE65 with betaPP and the association of a FE65 polymorphism with DAT lend credence to the hypothesis that the metabolism of betaPP is central to the pathogenesis of common sporadic forms of DAT.

Werner helicase is localized to transcriptionally active nucleoli of cycling cells.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS), a "caricature of aging." We have localized the Werner protein (WRNp) to the nucleoli of replicating mammalian cells, where its appearance is associated with transcriptional activity. A dramatic reduction of the nucleolar signal and of [3H]uridine incorporation occurred when cultures were made quiescent or were exposed to 4-nitroquinoline-1-oxide (4NQO), to which WS cells are particularly susceptible. Total cellular levels of WRNp, however, did not change, and virtually all WRNp was in the nuclear fractions, consistent with translocation to the nucleoplasm and/or masking of the epitopes. The 4NQO-induced altered state of WRNp was prevented by Na3VO4, but not by okadaic acid, suggesting that WRNp localization/function is partially regulated by kinases/phosphatases for Tyr substrates on WRNp or interacting proteins. The repression of rDNA transcription by 4NQO was not reversed by Na3VO4. We suggest that physiological states and genotoxic agents modulate the interaction of WRNp with rDNA, consistent with a role of WRNp in rDNA transcription.

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

A 'fail-safe' screening programme for diabetic retinopathy.

OBJECTIVE: To improve screening for diabetic retinopathy in a hospital diabetic clinic through the use of the audit process. DESIGN: Comparison of an existing system of screening for diabetic retinopathy (a specialist optometrist using ophthalmoscopy alone) with a new system in which a specialist optometrist examined retinal Polaroid photographs taken through pharmacologically dilated pupils and combined this with ophthalmoscopy in all cases except when the photographs were perfect and definitely showed no retinopathy. In this new system, the optometrist could discuss cases of uncertainty with a diabetes physician while the patient was still in the clinic with eyes dilated. SETTING: Inner city hospital diabetes clinic. SUBJECTS: 289 hospital diabetic clinic patients not already attending an ophthalmologist; a consecutive series of 144 such patients for the first audit, 145 for the repeat audit. MAIN OUTCOME MEASURES: Assessment of each screening system against a gold standard. For the first audit this was agreement by two of four diabetes physicians, who combined examination of the photographs with the findings from dilated ophthalmoscopy, on the classification of the retinae of each patient, guided by standard European criteria. For the second audit, the gold standard was enhanced by discussing the photographs and findings of all patients with an independent ophthalmologist. For patients requiring referral, a second ophthalmologist also commented on the case. RESULTS: The addition of retinal photography to universal pupil dilatation, and the availability of diabetes physician backup to discuss cases of uncertainty, greatly increased the optometrists' detection rate. Sensitivities for the first (ophthalmoscopy only) and second (ophthalmoscopy plus photography plus diabetologist back-up) audits were, respectively, 71.4% vs 100% for sight-threatening retinopathy, 33% vs 100% for retinopathy requiring six-month review, and 40.3% vs 97.2% for any retinopathy (p = 0.002). CONCLUSIONS: Optometrists specialising in diabetic retinopathy using Polaroid retinal photography and ophthalmoscopy, both through dilated pupils, backed up by experienced diabetologists to discuss cases of uncertainty, could form the basis of a retinopathy screening service that accurately identifies and categorises retinopathy and does not miss sight-threatening cases.

The Werner syndrome protein is a DNA helicase.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein.

Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.

5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.

An evaluation of screening programs for the detection of brucellosis in dairy herds.

Data were collected from approximately 1000 dairy herds, initially blood tested for brucellosis in 1977, in each of southeastern and southwestern Ontario. These data were used to evaluate three brucellosis screening programs, the area recertification program, the market cow program and the milk test program. The milk test program was the most efficient program at detecting brucellosis, 29.5% of the herds tested were classified as infected, but lacked the ability to detect a large proportion of "infected" herds (relative sensitivity = 24%). The market cow program was more efficient than the area recertification program at finding infected herds, 3.9% of the herds tested under the market cow program were infected, but had a low relative sensitivity of 12%. The area recertification program was least efficient, 2.3% of herds tested under the area recertification program were infected, but had the highest relative sensitivity (53%). The relative efficiency (predictive value) of the programs was not affected significantly by location of the herds, season of the initial test or herd size. The relative sensitivity of the milk test program was significantly higher in eastern than western Ontario and tended to decrease as herd size increased. The market cow program tended to be more sensitive in the summer months. The relative specificity of the milk test program (0.997) was higher than that of the market cow program (0.960) and the area recertification program (0.884).

Effects of maternal interference on the attachment and exploratory behavior of one-year-olds.

Effects of maternal interference on social behavior toward mother and exploratory play were examined in a laboratory experimental paradigm. Subjects were 40 1-year-olds and their mothers. Mothers of the 20 interference-group infants were instructed periodically to physically interfere with their child's independent object play during the first half of the observation session. A postinterference free-play period immediately followed. The 20 control-group infants were permitted by mother to play freely throughout the session. Groups were matched for exposure to play materials. Despite its aversiveness, interference had no subsequent effect on infant social initiatives to mother, responsiveness to mother's social bids, or exploratory play.