Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs.

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.

Shoulder Conditions: Diagnosis and Treatment Guideline.

The following guideline covers a wide array of shoulder conditions seen in the workers' compensation, as well as the nonworkers' compensation, population. The guideline is intended to help establish work relatedness and aid in making the diagnosis of shoulder injuries and degenerative conditions. It also provides a nonoperative and operative guideline for the treatment of several shoulder conditions, not limited to rotator cuff tears, subacromial impingement syndrome, acromioclavicular arthritis and dislocations, as well as glenohumeral arthritis.

Diagnosis and Treatment of Cervical Radiculopathy and Myelopathy.

This article is a guideline covering a wide array of cervical conditions seen in the workers' compensation, as well as the nonworkers' compensation, population. The guideline is intended to provide a diagnostic and treatment algorithm to commonly seen cervical conditions such as single-level and multilevel radiculopathies and myelopathies.

Progesterone receptor A predominance is a discriminator of benefit from endocrine therapy in the ATAC trial.

Progesterone receptor (PR) function, while essential in normal human breast, is also implicated in breast cancer risk. The two progesterone receptors, PRA and PRB, are co-expressed at equivalent levels in normal breast, but early in carcinogenesis normal levels of PRA:PRB are frequently disrupted, and predominance of one isoform, usually PRA, results. In model systems, PRA and PRB have different activities, and altering the PRA:PRB ratio in cell lines alters PR signaling. The purpose of this study was to determine whether hormonal or reproductive factors contribute to imbalanced PRA:PRB expression in breast tumors and the impact of PRA:PRB imbalance on disease outcome. The relative expression of PRA and PRB proteins was determined by dual immunofluorescence histochemistry in archival breast tumors and associations with clinical and reproductive history assessed. PRA:PRB expression was not influenced by reproductive factors, whereas exogenous hormone use (menopausal hormone treatment, MHT) favored PRB expression (p < 0.035). The PRA:PRB ratio may be a discriminator of response to endocrine therapy in the TransATAC sample collection, with high PRA:PRB ratio predicting earlier relapse for women on tamoxifen, but not anastrozole (mean lnPRA:PRB ratio; HR (95 % CI) tamoxifen 2.45 (1.20-4.99); p value 0.02; anastrozole 0.80 (0.36-1.78); p value 0.60). The results of this study show that PRA:PRB imbalance in breast cancers is not associated with lifetime endogenous endocrine and reproductive factors, but is associated with MHT use, and that PRA predominance can discriminate those women who will relapse earlier on tamoxifen treatment. These data support a role for imbalanced PRA:PRB expression in breast cancer progression and relative benefit from endocrine treatment.

ERp57 does not require interactions with calnexin and calreticulin to promote assembly of class I histocompatibility molecules, and it enhances peptide loading independently of its redox activity.

ERp57 is a thiol oxidoreductase that catalyzes disulfide formation in heavy chains of class I histocompatibility molecules. It also forms a mixed disulfide with tapasin within the class I peptide loading complex, stabilizing the complex and promoting efficient binding of peptides to class I molecules. Since ERp57 associates with the lectin chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, we examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells. Moreover, ERp57 formed a mixed disulfide with tapasin and promoted efficient peptide loading in the absence of interactions with calnexin and calreticulin. These findings suggest that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin chaperones. We also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide binding site in substrate recognition. Finally, by inactivating the redox-active sites of ERp57, we demonstrate that its enzymatic activity is dispensable in stabilizing the peptide loading complex and in supporting efficient peptide loading. Thus, ERp57 appears to play a structural rather than catalytic role within the peptide loading complex.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

BACKGROUND: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. METHODS: The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories. RESULTS: Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty. CONCLUSIONS: Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.

What features do patients notice that help to distinguish between benign pigmented lesions and melanomas?: the ABCD(E) rule versus the seven-point checklist.

The ABCD(E) rule and the seven-point checklist are diagnostic aids that have proven to be useful in the hands of physicians; however, little is known of their value to patients with respect to aiding self-detection. The objective of this study was to investigate features that patients notice when identifying melanomas and to explore how well these features correspond to the ABCD(E) rule and the seven-point checklist. A retrospective, modified, case-control study involving patient interviews was performed. All interviews were conducted through the private consulting rooms of a Melbourne dermatologist (JWK) and a Newcastle plastic surgeon (CH) prior to the result of pathology being known to the patients and the interviewers. Sixty-seven patients with benign pigmented skin lesions and 46 patients with melanomas were included. Using a logistic regression model, the change in size/new lesion and change in colour (major criteria, seven-point checklist) were most useful in differentiating between melanomas and benign pigmented lesions in the hands of patients [odds ratio (OR), 4.74; 95% confidence interval (CI), 1.85-12.19; P=0.001; OR, 4.27; 95% CI, 1.62-11.26; P=0.003, respectively). The ABCD(E) rule failed to discriminate between melanoma and other benign pigmented skin lesions. It can be concluded that, of the patients' observations, changes in size or colour were most important in distinguishing between benign pigmented lesions and melanomas. Such features therefore deserve emphasis in public education campaigns. Medical professionals should also remember to seek a history of change in assessing pigmented skin lesions.

Methodologies for detection of hemoglobin-based oxygen carriers.

Blood substitutes based on hemoglobin or hemoglobin-based oxygen carriers (HBOCs) are oxygen-carrying therapeutic agents developed for use in operations and emergencies in place of donated blood. Increased oxygen-carrying capacity through the use of blood substitutes could help elite athletes to lengthen endurance capacity and improve their performance. As blood substitutes become more readily available, it is essential that a qualitative detection method for their abuse in sport is available. Ideally, such a method would be simple and inexpensive. This study investigates methods that could be used as screening procedures to easily detect HBOCs in plasma and develops tests that can unequivocally confirm their presence. The investigation into the screening method indicates that the direct visual screening of plasma discoloration is the most appropriate with detection limits of less than 1% HBOC in plasma. Two methods are shown to confirm the presence of exogenous hemoglobin in plasma samples, size-exclusion chromatography with photodiode array detection and high-performance liquid chromatography analysis of enzymatic digests with detection by electrospray mass spectrometry. This work emphasizes the need for cooperation between drug developers and sports testing laboratories to ensure that methods for the detection of putative doping agents are available prior to product release.

Second-generation blood tests to detect erythropoietin abuse by athletes.

BACKGROUND AND OBJECTIVES: We previously developed blood tests that were introduced at the Sydney 2000 Olympic Games to identify athletes injecting recombinant human erythropoietin (rHuEPO). The aim of this study was to re-analyse our existing database to develop models with heightened sensitivity, using wherever possible blood parameters measurable with appropriate standards of analytical performance. DESIGN AND METHODS: The principal database for this study was derived from a double-blind trial in which 57 recreational athletes were administered either rHuEPO or placebo. Standard discriminant analysis was used to derive two ON models (ON-hes and ON-he) and two OFF models (OFF-hr and OFF-hre) sensitive to accelerated and decelerated erythropoiesis respectively, utilising concentrations of hemoglobin (h), erythropoietin (e) and serum transferrin receptor (s), as well as percent reticulocytes (r). The ability of our models to detect rHuEPO administration was assessed by comparing model scores of subjects in the administration trial with the model scores of 1152 elite athletes from 12 countries. RESULTS: The ability of the new models to detect rHuEPO administration was generally higher than that of our previous models, particularly during phases when low doses of rHuEPO were used, and after injections had ceased. INTERPRETATION AND CONCLUSIONS. The increased stability of the new blood parameters facilitates transport of samples to central laboratories, and the heightened sensitivity of the new models makes them better than existing models for federations wishing to screen samples for urine testing and to identify and target suspect athletes for out-of-competition testing. However procedures should be incorporated that respect an elevated model score caused by genetic, health or environmental circumstances.

Development of reference ranges in elite athletes for markers of altered erythropoiesis.

BACKGROUND AND OBJECTIVES: Our previous research developed two statistical models that are useful indicators of current (ON-model) or recently discontinued (OFF-model) recombinant human erythropoietin (rHuEPO) use by athletes. The component variables of the ON-model are hematocrit (Hct), reticulocyte hematocrit (RetHct), serum erythropoietin (EPO), percent macrocytes (%Macro), and soluble transferrin receptor (sTfr), whilst the OFF-model uses only the first three variables. Genetics and training modalities of elite athletes may conceivably produce unusual values for blood parameters related to erythropoiesis. The aims of this study were to develop reference ranges in elite athletes for key hematologic parameters as well as ON- and OFF-models scores, and to evaluate the effect of ethnicity, gender, residence at moderate altitude (approximately 2000 m) and within-individual variation on the variables and model scores. DESIGN AND METHODS: Over a period of three weeks, 413 female and 739 male elite athletes from 12 countries visited laboratories to provide three blood samples for analysis of blood parameters sensitive to erythropoiesis. For each parameter and for the ON- and OFF-model scores, we used mixed modeling to establish the range within which we could be 95% certain that the value for a randomly chosen athlete would fall, taking into account various random effects (variation within and between subjects and laboratories) and fixed effects (means for different levels of ethnicity, age, sport, altitude of residency). We performed similar analyses for changes in the ON- and OFF-model scores between the three visits. RESULTS: Most fixed effects were accompanied by clear-cut, small to moderate differences in several parameters. However, residency at moderate altitude was accompanied by a much higher hematocrit than residency nearer sea level, with the mean (and 95% confidence limits) for the difference being 2.3 (0.9 to 3.7) and 1.8 (0.1 to 3.5) units for males and females, respectively. Males at altitude also demonstrated a moderately higher ON-model score. Otherwise the influence of these effects was small for ON-, OFF- and changes in model scores. INTERPRETATION AND CONCLUSIONS: Assessment of an athlete's blood parameters and ON- and OFF-model scores may need adjustment for training modalities and other characteristics of the subject. Changes in model scores (together with monitoring of urine samples for the presence of rHuEPO) provide a promising approach to detection of rHuEPO abuse, because they are less sensitive to subject characteristics and less variable than raw model scores.

Physiological and pharmacological regulation of 20-kDa growth hormone.

The 20-kDa growth hormone (GH) is generated from alternative splicing of the primary transcript of full-length 22-kDa GH. We have studied the regulation of 20-kDa GH over a range of pathophysiological conditions and in response to pharmacological stimulation using isoform-specific enzyme-linked immunosorbent assays (ELISAs). Mean 24-h levels of 20- and 22-kDa GH were higher in acromegaly and lower in GH deficiency than in normal subjects, with the 20-to-22-kDa ratio not different between the three groups. In normal subjects, 20-kDa GH was secreted in a pulsatile manner throughout the day, with peaks coinciding with those of 22-kDa GH. However, the half-life of 20-kDa GH (18.7 +/- 0.8 min) was significantly longer than that of 22-kDa GH (14.7 +/- 0.8 min; P < 0.02). Insulin-induced hypoglycemia, androgen, and oral estrogen caused a parallel and proportionate increase in both isoforms. Octreotide suppressed 20-kDa less rapidly than 22-kDa GH in blood. Administration of recombinant 22-kDa GH in normal subjects rapidly reduced the 20-kDa GH levels. In conclusion, 20-kDa GH is cosecreted with and circulates at a constant proportion of 22-kDa GH. The 20-kDa GH level is reduced by administration of exogenous 22-kDa GH, suggesting rapid negative feedback regulation on pituitary release.

Un nuevo método que utiliza marcadores de eritropoyesis alterada para la detección de abuso de eritropoyetina recombinante humana en deportistas / A novel method utilizing markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes

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