Citations

Background— The KCNH2 or human ether-a-go-go related gene ( hERG ) encodes the Kv11.1 α-subunit of the rapidly activating delayed rectifier K + current ( I Kr ) in the heart. Type 2 congenital long-QT syndrome (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function. Several mechanisms have been identified, including disruption of Kv11.1 channel synthesis (class 1), protein trafficking (class 2), gating (class 3), or permeation (class 4). For a few class 2 LQT2-Kv11.1 channels, it is possible to increase surface membrane expression of Kv11.1 current ( I Kv11.1 ). We tested the hypotheses that (1) most LQT2 missense mutations generate trafficking-deficient Kv11.1 channels, and (2) their trafficking-deficient phenotype can be corrected. Methods and Results— Wild-type (WT)-Kv11.1 channels and 34 missense LQT2-Kv11.1 channels were expressed in HEK293 cells. With Western blot analyses, 28 LQT2-Kv11.1 channels had a trafficking-deficient (class 2) phenotype. For the majority of these mutations, the class 2 phenotype could be corrected when cells were incubated for 24 hours at reduced temperature (27°C) or in the drugs E4031 or thapsigargin. Four of the 6 LQT2-Kv11.1 channels that had a wild-type–like trafficking phenotype did not cause loss of Kv11.1 function, which suggests that these channels are uncommon sequence variants. Conclusions— This is the first study to identify a dominant mechanism, class 2, for the loss of Kv11.1 channel function in LQT2 and to report that the class 2 phenotype for many of these mutant channels can be corrected. This suggests that if therapeutic strategies to correct protein trafficking abnormalities can be developed, it may offer clinical benefits for LQT2 patients.

ABSTRACT Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being d -mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of ≤1 μg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of “farcy.” By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense . Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing ≥98.4% identity with each other and ≥98.6% identity with the sequence of five bovine strains of M. senegalense . There was ≤97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense . This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.

A number of recently developed and promising approaches to antitumoral immunotherapy are being investigated as potential treatments for advanced prostate cancer. These approaches largely revolve around strategies to increase antigen-specific T-cell activation against prostate tumors as well as precise manipulations of critical co-regulatory receptors that help to maintain and prolong the activity of antigen-presenting cells and T cells that are capable of mediating tumor regression. Herein, we describe the experience with the most recent and promising approaches pertaining to prostate cancer immunotherapy. Additionally, we discuss the mechanistic basis for these approaches as well as current limitations that must still be addressed in order to propel immunotherapy into the forefront of prostate cancer treatment.

The goal of “Practice Abstracts” is to present interesting, topical, and novel applications of operations research methodology to a wide range of industrial applications. “Practice Abstracts” are intended to provide Interfaces readers with short (2–4 page) descriptions of the most relevant aspects of operations research-based projects, in a form that is accessible to academics and practitioners in other organizations. Contributions should be sent for evaluation to the editor of “Practice Abstracts,” Brian T. Denton, Division of Health Care Policy and Research, Pavilion 3, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, denton.brian@mayo.edu.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3′ end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9–exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10 – tau RNA into exon 10 + tau RNA could be achieved with ≈34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.

This investigation examined listener transcriptions of phrases produced by speakers with mild to severe hypokinetic dysarthria to examine individual strategies for the identification of word boundaries in connected speech. It was hypothesized that the most efficient listeners would use syllabic strength information to guide their parsing of the continuous acoustic stream. Six-hundred transcribed phrases (10 listeners ×60 phrases) were coded independently by two judges to identify (1) correct word transcriptions, (2) evidence of accurate lexical parsing, regardless of exact word identification, and (3) the proportions of accurate parsing of strong and weak syllable word onsets. Linear regression analysis of the group data revealed that correct segmentation of strong syllable word onsets predicted listener performance on segmenting weak syllable word onsets [R=0.805, F(1,29)=51.440, p<0.001]. Despite a wide range of listener performance on ``words correct,'' individual strategies for perceptual segmentation were evident only in the transcriptions for the most severe speaker. In this case, the poorest listeners exhibited disproportionate difficulty with the segmentation weak syllable word onsets. Results suggest that a listener's ability to use syllabic strength information in lexical parsing determines, in part, their ability to recognize word onsets. [Work supported by NIDCD, NIH.]

Abstract Purpose: To optimize treatment strategies for postmenopausal breast cancer patients, we investigated the efficacy of the steroidal aromatase inhibitor exemestane alone or in combination with the antiestrogen tamoxifen in a xenograft model of postmenopausal breast cancer. We also determined the effects of these agents in sequential second-line therapy and the effect of the nonsteroidal aromatase inhibitor letrozole on tumors that progressed on the above treatments. Experimental: Aromatase-transfected human estrogen receptor-positive breast cancer cells (MCF-7Ca) were grown as tumors in ovariectomized athymic mice. Animals received subcutaneous injection with vehicle, tamoxifen, exemestane, tamoxifen plus exemestane, and letrozole. Tumor volumes were measured weekly. Results: All treatments were effective initially in suppressing tumor growth as first-line therapy compared with vehicle treatment. Exemestane suppressed tumor growth to a greater extent than tamoxifen. However, the combination of tamoxifen plus exemestane was more effective than either drug alone. After tumor volumes doubled on initial treatment, the mice were crossed over to receive exemestane or tamoxifen. Tumor growth slowed briefly in mice treated with tamoxifen and crossed over to exemestane, but tumor growth continued unabated in those changed from exemestane to tamoxifen. However, letrozole was effective in both groups as third-line therapy for a limited period. Letrozole as initial single agent was the best overall treatment in terms of the degree of tumor suppression and the length of effectiveness of treatment. Conclusion: Exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.