Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates

An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.     

Reliability of Rehabilitative Ultrasound Imaging of the Transversus Abdominis and Lumbar Multifidus Muscles

Koppenhaver SL, Hebert JJ, Fritz JM, Parent EC, Teyhen DS, Magel JS. Reliability of rehabilitative ultrasound imaging of the transversus abdominis and lumbar multifidus muscles.

Objectives

To evaluate the intraexaminer and interexaminer reliability of rehabilitative ultrasound imaging (RUSI) in obtaining thickness measurements of the transversus abdominis (TrA) and lumbar multifidus muscles at rest and during contractions.

Design

Single-group repeated-measures reliability study.

Setting

University and orthopedic physical therapy clinic.

Participants

A volunteer sample of adults (N=30) with current nonspecific low back pain (LBP) was examined by 2 clinicians with minimal RUSI experience.

Interventions

Not applicable.

Main Outcome Measures

Thickness measurements of the TrA and lumbar multifidus muscles at rest and during contractions were obtained by using RUSI during 2 sessions 1 to 3 days apart. Percent thickness change was calculated as thickness_contracted-thickness_rest/thickness_rest. Intraclass correlation coefficients (ICC) were used to estimate reliability.

Results

By using the mean of 2 measures, intraexaminer reliability point estimates (ICC_3,2) ranged from 0.96 to 0.99 for same-day comparisons and from 0.87 to 0.98 for between-day comparisons. Interexaminer reliability estimates (ICC_2,2) ranged from 0.88 to 0.94 for within-day comparisons and from 0.80 to 0.92 for between-day comparisons. Reliability estimates comparing measurements by the 2 examiners of the same image (ICC_2,2) ranged from 0.96 to 0.98. Reliability estimates were lower for percent thickness change measures than the corresponding single thickness measures for all conditions.

Conclusions

RUSI thickness measurements of the TrA and lumbar multifidus muscles in patients with LBP, when based on the mean of 2 measures, are highly reliable when taken by a single examiner and adequately reliable when taken by different examiners.     

Medical literature and vena cava filters: so far so weak

STUDY OBJECTIVE: With the development of percutaneous inferior vena cava (IVC) filters, IVC interruption has become a widely used procedure in patients with or at risk for venous thromboembolism. In an attempt at clarifying the indications for filter placement, a systematic literature review was undertaken. DESIGN: Bibliographic search and analysis. MEASUREMENTS AND RESULTS: A systematic MEDLINE search about vena cava filters produced a total of 568 references with abstracts between 1975 and 2000 inclusively. Each reference was analyzed according to predetermined criteria. Nearly two thirds (65.0%) of these publications were retrospective studies or case reports (33.3 and 31.7%, respectively), 12.9% were animal or in vitro studies, 7.4% were prospective studies, 6.7% were reviews, and 8.1% reported on miscellaneous related topics. Among the prospective studies, only 16 studies included > or = 100 patients, only 1 study was a randomized controlled trial (0.02% of 568 references), and heterogeneity among series precluded any relevant comparison. In a similar search about heparin and venous thromboembolism, 47.4% of 531 references were randomized controlled trials. CONCLUSIONS: Until more relevant data become available, literature reviews about vena cava filters will remain narrative, and many if not most indications for filter placement will remain a matter of opinion.     

The effects of melatonin on Ca(2+) homeostasis in endothelial cells

The effect of melatonin on the Ca(2+) signaling process in bovine aortic endothelial cells (BAE) and in primary cultured vascular endothelial cells from normotensive Sprague Dawley (SDR) and genetically hypertensive (SHR) rats was investigated using the Ca(2+) indicator Fura-2. Acute applications of melatonin failed to initiate a Ca(2+) response in the three cell types considered. However, preincubating SHR aortic endothelial cells with exposure to melatonin increased the internal Ca(2+) release triggered by bradykinin (BK) and ATP while stimulating the related agonist-evoked Ca(2+) entry. This effect appeared specific for SHR cells, as a similar incubation period failed to alter the Ca(2+) responses in BAE and SDR cells. Because of the known overproduction of free radicals in SHR cells, the effect of melatonin on Ca(2+) signaling was also tested in SDR and BAE cells exposed to the superoxide anion radical. Melatonin reversed the deleterious action of free radicals on Ca(2+) signaling in both cases, suggesting that its stimulatory effect in SHR was linked to its antioxidative properties. Finally, experiments where melatonin was applied between successive BK stimulation periods showed an enhancement of the agonist-evoked Ca(2+) entry in BAE and SDR cells. This effect appeared to be independent of the production of second messengers as no specific binding sites for melatonin, including MT1, MT2 and MT3 receptors, could be detected in BAE cells. We conclude that melatonin improves Ca(2+) signaling in dysfunctional endothelial cells characterized by an overproduction of free radicals while stimulating the agonist-evoked Ca(2+) entry in normal endothelial cells through a mechanism not related to its antioxidative properties.     

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.     

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks

Necrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P?netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P?C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.     

Researching Children’s Adjustment in Stepfamilies: How is it Studied? What Do we Learn?

The probability that children whose parents have separated will become members of a stepfamily has increased considerably in the last decades. This situation has encouraged researchers to document the impact of this family transition on children’s adjustment. The present article examines empirical publications on this subject between 2000 and 2015. Screening and eligibility assessment based on inclusion and exclusion criteria yielded a final sample of 130 studies. The theoretical models used by the authors are described and discussed, with particular attention to how theories are relied upon in this field of research. Second, an examination of the methodology applied allowed us to take a critical look at the way children’s outcomes were examined. The studies’ main results were then analyzed so as to draw up a contemporary portrait of how stepfamily children adjust. Finally, an examination of the studies’ limitations and theoretical foundations points to avenues for future research.     

Bigger,Faster, Stronger,More Ethical

Consider four elite female runners who trained hard for a 1500‐meter race. Runner 1 took extra‐strength aspirin before the race. Runner 2 has a genetic condition that results in greater levels of testosterone in her body than the typical range for a woman. Runner 3 has been on a carefully scheduled regimen of the hormone erythropoietin (EPO), which has increased her red blood cell count. Runner 4 has a team of diet, sleep, and exercise experts who ensured that she coordinated the exactly right balance of carbs and protein with her rest and workouts. Which of them cheated? Thomas Murray's Good Sport: Why Our Games Matter—and How Doping Undermines Them does not answer this question, but it provides tools to figure it out, and it argues that trying to answer is important.