Papillomavirus found in anorectal squamous carcinoma, not in colon adenocarcinoma.

We used polymerase chain reaction DNA amplification methods for the detection and typing of genital human papillomaviruses in paraffin-embedded tissue sections of five patients with anorectal squamous cell carcinoma and 22 patients with colonic adenocarcinoma. The cases were further tested by in situ hybridization with biotin-labeled probes specific for human papillomavirus types 6/11, 16/18, and 31/33/35. By polymerase chain reaction, human papillomavirus DNA was demonstrated in all of the cases of anorectal squamous cell carcinoma and in none of the cases of colonic adenocarcinoma for which analyzable DNA was available. Tumor cell nuclei stained for human papillomavirus DNA by in situ hybridization in four of the five cases of squamous cell carcinoma and in none of the cases of colonic adenocarcinoma. We conclude that human papillomavirus types usually associated with malignant transformation are uniformly present in anorectal squamous cell carcinoma but are absent from adenocarcinoma of the colon.     

Evaluation of four immunoassay screening kits for the detection of benzodiazepines in urine

The identification of benzodiazepines (BZD) in biological fluids can be a challenging process. The large number of various BZD in pharmaceutical distribution, with similar core structures, and individual metabolic profiles all contribute to a complicated and time-consuming analysis. The purpose of the current study was to evaluate the performance of four commercially available immunoassay urine screening kits for use in a forensic urine analysis testing program. The four kits included the Roche Benzodiazepine Plus KIMS assay, Microgenics CEDIA Benzodiazepine assay, Microgenics CEDIA high sensitivity assay with beta-glucuronidase, and Microgenics DRI reagent ready Benzodiazepine assay. Each kit was evaluated for linearity, precision, accuracy, carryover, reagent specificity, and confirmation rates. BZD reagent specificity was compared by analysis of 55 structurally dissimilar compounds to BZD. Negative responses to all 55 compounds were elicited by all four reagent assays. Cross-reactivity for the assays was demonstrated by detecting 27 structurally similar BZD. In addition, greater than 10,000 randomly collected urine samples were screened at a 200 ng/mL cutoff for each assay. Positive samples were confirmed by gas chromatography-mass spectrometry using a panel of 13 BZD confirmation standards. The Microgenics CEDIA high sensitivity assay demonstrated the highest positive screening rate as well as the highest confirmation rate of the four assays.     

Depression, physical activity, energy consumption, and quality of life in OSA patients before and after CPAP treatment

Background

A variety of studies have demonstrated improvement in quality of life and depressive symptoms in obstructive sleep apnea (OSA) patients after continuous positive airway pressure (CPAP) treatment. However, very little is known about the effect of OSA treatment on physical activity and energy consumption.

Objectives

The aim of this study was to evaluate the changes in depression, physical activity, energy expenditure, and quality of life (QoL) in OSA patients before and after CPAP therapy.

Methods

Forty-one patients with OSA as revealed by polysomnography, were included to the study. They responded to the generic World Health Organization Quality of Life (WHOQoL) questionnaire, to the specific-disease Quebec Sleep Questionnaire, and to Center for Epidemiologic Studies Depression Scale (CES-D) in order to evaluate QoL and the incidence of depression. In addition, all patients wore an accelerometer which measured physical activity and energy expenditure during a week. At least 6 months after initiation of CPAP treatment (mean time, 9 months) we re-examined 24 patients who met the compliance with the treatment criteria.

Results

Patients after CPAP therapy had significantly higher scores in all domains of the Quebec Sleep Questionnaire and in the domains of physical health/level of independence and psychological health/spirituality of the WHOQoL. Depression scores were also better in CES-D after treatment. However, despite the improvement in QoL and psychological status, CPAP therapy had no impact on physical activity and energy expenditure.

Conclusions

CPAP therapy improves QoL and lessens depressive symptoms in our group of well-treated OSA patients. However, physical activity and energy expenditure did not present statistically significant improvement in the same group of OSA patients.     

Spectral indicators for salinity effects in crops: a comparison of a new green indigo ratio with existing indices

Spectroradiometric measurements were taken from eggplant (Solanum melongena) and cauliflower (Brassica oleracea) plants in a controlled experiment treated with sodium chloride (NaCl) concentrations between 30 and 150 mmol. Assessment of seven vegetation indices indicated potential difference between three levels of salinity in effects, with the green indigo ratio (GIR of reflectance of 554 and 436 nm) yielding the most consistent results. Salinity effects started from the second week, but the highest differences were obtained in the fourth week for cauliflower and in the third and sixth weeks for eggplant.     

An imidazopiperidine series of CCR5 antagonists for the treatment of HIV: the discovery of N-{(1S)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]propyl}acetamide (PF-232798)

Preventing entry of HIV into human host cells has emerged as an attractive approach to controlling viral replication. Maraviroc 1 is an approved antagonist of the human CCR5 receptor which prevents the entry of HIV. Herein, we report the design and discovery of a series of imidazopiperidine CCR5 antagonists which retain the attractive antiviral profile and window over hERG activity of maraviroc 1, combined with improved absorption profiles in rat and dog. Furthermore, this series of compounds has been shown to retain activity against a laboratory generated maraviroc-resistant HIV-1 strain, which indicates an alternative resistance profile to that of maraviroc 1. Compound 41f (PF-232798) was selected as a clinical candidate from the imidazopiperidine series and is currently in phase II clinical trials.     

The acute effect of aerobic exercise on brachial artery endothelial function in renal transplant recipients

This study compared the effect of a 30-minute walk on brachial artery endothelial vasodilatation in kidney transplant (KT) recipients and healthy controls (HCs). Endothelial-dependent vasodilatation was measured by ultrasound before and after exercise. The HCs experienced a significant increase in vasodilatation after exercise 1 minute postocclusion when compared with the KT recipients (22%+/-13% vs 3%+/-4%; P<.05). Also, the HCs had a significantly higher vasodilatation from pre-treadmill walk to post-treadmill walk (1 minute postocclusion) when compared with KT recipients (from 3%+/-6% to 22%+/-13% vs 1%+/-3% to 3%+/-4%; P<.05). This acute vasodilatory response observed in the HCs may be related to the immediate release of nitric oxide and the combined response to shear stress and exercise. The KT recipients had several coronary artery disease risk factors that may have adversely affected endothelial function.     

Restless legs syndrome in Multiple Sclerosis patients: a contributing factor for fatigue,impaired functional capacity,and diminished health-related quality of life

Objectives Restless legs syndrome (RLS) symptoms are common in Multiple Sclerosis (MS) patients. The aim of the current study was to examine for the first time whether RLS could affect the functional capacity and various contributing parameters related to quality of life and fatigue in MS patients.

Methods According to their RLS status, 50 relapsing-remitting MS patients were divided into the RLS (n = 10) and non-RLS groups (n = 40). Specific questionnaires were used in order to assess the health-related quality of life (HRQoL), fatigue levels, sleep quality, daily sleepiness, and depression symptoms of the patients. Functional capacity was examined using a battery of functional tests. Total body and visceral fat levels were assessed via bioelectrical impedance analyzers.

Results Sleep quality, depression, fatigue, and HRQoL levels were found to be significantly worse in the patients with RLS compared to their free-RLS counterparts (P < 0.05). In addition, patients with RLS were found to exhibit further impairments in their performance in various functional tests related mainly with strength levels of lower extremities (P < 0.05). Finally, the patients with RLS were found to have significantly higher both total and trunk fat levels compared to patients without RLS (P < 0.05). A strong correlation was observed between the severity of RLS symptoms, sleep quality, fatigue, and QoL levels.

Discussion It seems that RLS contributes even further to impairments on sleep quality, fatigue, functional capacity, and therefore HRQoL levels in relapsing-remitting MS patients, whilst for the first time a link between high fat levels has been revealed.     

New Small-Molecule Inhibitor Class Targeting Human Immunodeficiency Virus Type 1 Virion Maturation

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.Despite the wide-ranging success of highly active retroviral therapies against human immunodeficiency virus type 1 (HIV-1), there still remain several powerful drivers to discover and develop new classes of HIV inhibitors. First and foremost is the continued acquisition of HIV-1 resistance to currently administered antiretroviral drugs. The discovery of compounds that inhibit the replication of HIV-1 via new mechanisms offers the best hope of generating drugs that are active against all HIV-1 variants in the clinic. In principle, viral mutations conferring resistance to any existing drug classes would not confer cross-resistance to drugs targeting a new mechanism. Also, as the current standard of care requires lifelong therapy for HIV-infected individuals, it is imperative that highly active retroviral therapy-related adverse effects and toxicity are minimized with new drugs.Recently, two new classes of antiretroviral medications have been approved for use in patients for whom previous HIV treatment regimens have failed; the CCR5 [chemokine (C-C motif) receptor 5] inhibitor maraviroc (9, 11) and the HIV-1 integrase (IN) inhibitor raltegravir (22). Additional novel mechanism drug classes will add similar value to the treatment of HIV-1 by further expanding the possibilities for combination regimens and providing a wider range of options for treatment of experienced patients failing first- and second-line therapies.Virion maturation represents one new antiviral mechanism that is currently being evaluated in the clinic (20, 21). During maturation of the HIV-1 viral particle, the Gag polyprotein undergoes several cleavage events mediated by the viral protease that yield the individual structural proteins, matrix (MA), capsid (CA), nucleocapsid (NC), and the p6 protein. In addition, there are spacer peptides that must be excised from between CA and NC (spacer peptide 1 [SP1]) and from between NC and p6 (SP2). Although the functions of the spacer peptides have not been clearly delineated, recent evidence suggests that SP1 stabilizes CA hexamers (25). Gag cleavage events occur in a sequential fashion as dictated by the relative kinetics of proteolysis at each site. The last of these proteolytic events cleaves at the CA-SP1 junction to convert p25 Gag to the mature p24 CA protein. This is a critical step in the replication cycle of HIV-1, as only in its mature form is CA capable of forming the higher-order complexes that comprise the mature viral core and in the absence of this maturation, viral particles remain noninfectious. Currently, two HIV-1 inhibitors [3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB/PA-457/bevirimat) and MPC-9055] targeting HIV-1 virion maturation are being evaluated in clinical trials (3, 4, 14, 16). The hallmark of this class of inhibitors is the accumulation of the CA/SP1 (p25) precursor in cells and HIV-1 viral particles in the presence of the compounds. However, to date, all of the Gag maturation inhibitors reported belong to the same general chemical class of betulinic acid derivatives.In this report, we describe a specific small-molecule inhibitor of HIV-1 Gag maturation, PF-46396 {1-[2-(4-tert-butylphenyl)-2-(2,3-dihydro-1H-inden-2-ylamino)ethyl]-3-(trifluoromethyl)pyridin-2(1H)-one}. This pyridone-based compound was first discovered as a hit from our HIV-1 high-throughput full-replication screen that incorporates all of the HIV-1 targets required for viral replication in cell culture (8). This approach allowed us to screen for multiple targets in the context of a full replication cycle and to identify compounds directed against new HIV-1 mechanisms. We demonstrate that PF-46396 is a late stage (postintegration) HIV-1 inhibitor by showing that the compound inhibits the production of infectious viral particles by HIV-1 producer cells and that the compound does not inhibit early events in HIV-1 replication. We present mechanism-of-action studies demonstrating that PF-46396 does not inhibit HIV protease but interferes specifically with the cleavage of the CA/SP1 (p25) Gag precursor to the mature CA (p24) protein and show a correlation between this mechanism and the antiretroviral activity of the compound. Consistent with these mechanistic data, we show that selection of HIV-1 isolates that are resistant to PF-46396 yields a mutation in the C-terminal region of HIV-1 CA. This single mutation is sufficient to cause significant loss of susceptibility to the compound and displays cross-resistance to the previously reported HIV-1 Gag maturation inhibitor, DSB. Conversely, we demonstrate that a mutation selected for resistance to DSB is cross-resistant to PF-46396.Our data show that PF-46396 is a specific Gag maturation inhibitor that is clearly differentiated from previously reported compounds that share this mechanism of action. The structure and, in particular, the low molecular weight of PF-46396 show that this mechanism of action can be prosecuted through a more diverse set of chemical structures than previously represented in the literature.