Controlling cleaning agent residues in pharmaceutical manufacturing: A harmonized scientific strategy.

This paper proposes a scientifically justified and harmonized strategy to control cleaning agent ingredients' (CAIs) residues in pharmaceutical manufacturing. Firstly, we demonstrate that worst-case cleaning validation calculations on CAI residues with representative GMP standard cleaning limits (SCLs) are enough to control CAI residues of low concern to safe levels. Secondly, a new harmonized strategy for the toxicological assessment of CAI residues is presented and validated. The results establish a framework applicable to cleaning agent mixtures based on hazard and exposure considerations. This framework is primarily based on the hierarchy of a single CAI's critical effect, where the lowest resulting limit may become the driver of the cleaning validation process. The six critical effect groups are: (1) CAIs of low concern based on safe exposure reasoning; (2) CAIs of low concern based on the mode of action reasoning; (3) CAIs with local concentration-dependent critical effects; (4) CAIs with dose-dependent systemic critical effects for which a route-specific PDE should be calculated; (5) poorly characterized CAIs with unknown critical effect for which a default value of 100 µg/day is proposed; (6) poorly characterized CAIs which should be avoided because of potential mutagenicity and/or potency.

PDE concept for controlling cleaning agent residues in pharmaceuticals- A critical analysis.

Cleaning agents (CAs) are used in multipurpose facilities to control carryover contamination of active pharmaceutical ingredients (APIs) to scientifically justified limits. While this is often done with the PDE methodology used for API impurities, it is unclear if it is justifiable and necessary for cleaning agents, which generally represent a comparatively lower health risk. Comparing calculated oral PDE values for CA ingredients (CAIs) from four companies with PDEs of a selected number of small-molecule APIs showed that the toxicity of CAIs is several orders of magnitude lower. Furthermore, a critical review of the toxicity and everyday exposure to the general population of the main CAIs functional groups showed that the expected health risks are generally negligible. This is particularly true if the associated mode of actions cause local toxicity that is usually irrelevant at the concentration of potential residue carryover. This work points towards alternative approaches to the PDE concept to control CAIs' contamination and provides some guidance on grouping and identifying compounds with lower health risks based on exposure and mode of action reasoning. In addition, this work supports the concept that limit values should only be set for CAIs of toxicological concern.

Preinterventional Third-Molar Assessment Using Robust Machine Learning.

Machine learning (ML) models, especially deep neural networks, are increasingly being used for the analysis of medical images and as a supporting tool for clinical decision-making. In this study, we propose an artificial intelligence system to facilitate dental decision-making for the removal of mandibular third molars (M3M) based on 2-dimensional orthopantograms and the risk assessment of such a procedure. A total of 4,516 panoramic radiographic images collected at the Center of Dental Medicine at the University of Zurich, Switzerland, were used for training the ML model. After image preparation and preprocessing, a spatially dependent U-Net was employed to detect and retrieve the region of the M3M and inferior alveolar nerve (IAN). Image patches identified to contain a M3M were automatically processed by a deep neural network for the classification of M3M superimposition over the IAN (task 1) and M3M root development (task 2). A control evaluation set of 120 images, collected from a different data source than the training data and labeled by 5 dental practitioners, was leveraged to reliably evaluate model performance. By 10-fold cross-validation, we achieved accuracy values of 0.94 and 0.93 for the M3M-IAN superimposition task and the M3M root development task, respectively, and accuracies of 0.9 and 0.87 when evaluated on the control data set, using a ResNet-101 trained in a semisupervised fashion. Matthew's correlation coefficient values of 0.82 and 0.75 for task 1 and task 2, evaluated on the control data set, indicate robust generalization of our model. Depending on the different label combinations of task 1 and task 2, we propose a diagnostic table that suggests whether additional imaging via 3-dimensional cone beam tomography is advisable. Ultimately, computer-aided decision-making tools benefit clinical practice by enabling efficient and risk-reduced decision-making and by supporting less experienced practitioners before the surgical removal of the M3M.

Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial.

BACKGROUND: B-type CpG oligodeoxynucleotides (ODN) is currently used in clinical trials because of its prolonged half-life, which is due to its phosphorothioate backbone. A-type CpG ODN is a stronger inducer of IFN but has an unstable phosphodiester backbone that has so far prohibited its clinical use. However, upon association with virus-like particles (VLP) consisting of the bacteriophage Qbeta coat protein, A-type CpG ODN can be stabilized and can become an efficient adjuvant in mice. Therefore, the phase I/IIa study presented represents the first test of A-type CpGs in humans. OBJECTIVE: To test the safety, tolerability and clinical efficacy of QbG10 as an adjuvant for subcutaneous immunotherapy with a house dust mite (HDM) allergen extract in allergic patients. METHODS: A single centre, open-label phase I/IIa study evaluated the safety, tolerability and clinical efficacy of QbG10 as an adjuvant to immunotherapy with a subcutaneous HMD allergen extract in 20 patients suffering from HDM allergy. Twenty-one patients were enrolled between March and July 2005. Individual immunotherapy lasted 10 weeks. Clinical end-points included questionnaires, conjunctival provocation, skin prick tests and the measurement of allergen-specific IgG and IgE. RESULTS: QbG10 was well tolerated. Almost complete tolerance to the allergen was observed in conjunctival provocation testing after treatment with QbG10, and symptoms of rhinitis and allergic asthma were significantly reduced. Within 10 weeks of therapy, patients were nearly symptom-free and this amelioration lasted for at least 38 weeks post-treatment. Following injections of QbG10 and HDM allergen extract, allergen-specific IgG increased, while there was a transient increase in allergen-specific IgE titres. Skin reactivity to HDM was reduced. CONCLUSION: The subcutaneous application of HDM allergen, together with A-type CpG ODN packaged into VLP, was safe. All patients achieved practically complete alleviation of allergy symptoms after 10 weeks of immunotherapy. This promising clinical outcome calls for larger placebo-controlled phase II studies.

Laser Doppler distance sensor using phase evaluation.

This paper presents a novel optical sensor which allows simultaneous measurements of axial position and tangential velocity of moving solid state objects. An extended laser Doppler velocimeter setup is used with two slightly tilted interference fringe systems. The distance to a solid state surface can be determined via a phase evaluation. The phase laser Doppler distance sensor offers a distance resolution of 150 nm and a total position uncertainty below 1 microm. Compared to conventional measurement techniques, such as triangulation, the distance resolution is independent of the lateral surface velocity. This advantage enables precise distance and shape measurements of fast rotating surfaces.

Renal tolerability of intermittent intravenous ibandronate treatment for patients with postmenopausal osteoporosis: a review.

While intravenous (IV) bisphosphonates are well established in managing metastatic bone disease and hypercalcemia of malignancy, oral bisphosphonates are the primary treatment for postmenopausal osteoporosis. The availability of a well-tolerated, effective, IV bisphosphonate regimen for postmenopausal osteoporosis would increase physicians' options, allowing treatment of patients who cannot tolerate oral therapy, for whom oral bisphosphonates should be avoided or patients who are unable to comply with the oral dosing recommendations. Ibandronate is a potent, nitrogen-containing bisphosphonate, with proven efficacy and good tolerability when administered intermittently either orally or intravenously. Preclinical experience in animal models with IV ibandronate indicated that it had good renal tolerability. These data are supported by clinical pharmacology studies. Prolonged follow-up of patients receiving intermittent IV 15-30 second injections of 0.5-3 mg IV ibandronate has demonstrated no clinical evidence of renal toxicity in patients with postmenopausal osteoporosis. What is seen in controlled studies is not always the case in uncontrolled studies, however, no reports of renal failure have been received in post-marketing surveillance of >500,000 patients receiving IV ibandronate infusions in various indications including metastatic breast and prostate cancer. The good renal tolerability of IV ibandronate in patients with osteoporosis with glomerular filtration rates >30 mL/minute and without renal co-morbid conditions is reassuring.

Parathyroid hormone increases the concentration of insulin-like growth factor-I and transforming growth factor beta 1 in rat bone.

Intermittent treatment with parathyroid hormone (PTH) increases bone mass in experimental animals and humans. In vitro studies have suggested that the anabolic effect of PTH may be mediated by local growth factors. However, the relevance of these findings to in vivo situations remains unclear. In this study, we examined a time course of daily s.c. injections of hPTH (1-34) on the skeletal concentration of insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor beta (TGF-beta) in the proximal tail vertebrae of male rats. PTH caused a time and dose-dependent increase in the bone mineral density of the lumbar spine. This anabolic effect on bone mass was accompanied by progressive increases in bone matrix-associated IGF-I and TGF-beta 1. Increases in IGF-I and TGF-beta 1 became apparent after four and eight weeks of PTH treatment respectively and persisted through week 12. PTH had no effect on circulating IGF-I, suggesting that the increase of bone matrix IGF-I was due to the local effect of PTH on bone tissue directly rather than to an increase of circulating IGF-I. These data are consistent with the hypothesis that IGF-I and TGF-beta 1 may play a role as local mediators of the anabolic effects of PTH on bone metabolism.

[Linkage to care after first hospitalisation for psychosis]. / Insertion dans les soins après une première hospitalisation dans un secteur pour psychose.

BACKGROUND: First hospitalisation for a psychotic episode causes intense distress to patients and families, but offers an opportunity to make a diagnosis and start treatment. However, linkage to outpatient psychiatric care remains a notoriously difficult step for young psychotic patients, who frequently interrupt treatment after hospitalisation. Persistence of symptoms, and untreated psychosis may therefore remain a problem despite hospitalisation and proper diagnosis. With persisting psychotic symptoms, numerous complications may arise: breakdown in relationships, loss of family and social support, loss of employment or study interruption, denial of disease, depression, suicide, substance abuse and violence. Understanding mechanisms that might promote linkage to outpatient psychiatric care is therefore a critical issue, especially in early intervention in psychotic disorders. OBJECTIVE: To study which factors hinder or promote linkage of young psychotic patients to outpatient psychiatric care after a first hospitalisation, in the absence of a vertically integrated program for early psychosis. Method. File audit study of all patients aged 18 to 30 who were admitted for the first time to the psychiatric University Hospital of Lausanne in the year 2000. For statistical analysis, chi2 tests were used for categorical variables and t-test for dimensional variables; p<0.05 was considered as statistically significant. RESULTS: 230 patients aged 18 to 30 were admitted to the Lausanne University psychiatric hospital for the first time during the year 2000, 52 of them with a diagnosis of psychosis (23%). Patients with psychosis were mostly male (83%) when compared with non-psychosis patients (49%). Furthermore, they had (1) 10 days longer mean duration of stay (24 vs 14 days), (2) a higher rate of compulsory admissions (53% vs 22%) and (3) were more often hospitalised by a psychiatrist rather than by a general practitioner (83% vs 53%). Other socio-demographic and clinical features at admission were similar in the two groups. Among the 52 psychotic patients, 10 did not stay in the catchment area for subsequent treatment. Among the 42 psychotic patients who remained in the catchment area after discharge, 20 (48%) did not attend the scheduled or rescheduled outpatient appointment. None of the socio demographic characteristics were associated with attendance to outpatient appointments. On the other hand, voluntary admission and suicidal ideation before admission were significantly related to attending the initial appointment. Moreover, some elements of treatment seemed to be associated with higher likelihood to attend outpatient treatment: (1) provision of information to the patient regarding diagnosis, (2) discussion about the treatment plan between in- and outpatient staff, (3) involvement of outpatient team during hospitalisation, and (4) elaboration of concrete strategies to face basic needs, organise daily activities or education and reach for help in case of need. CONCLUSION: As in other studies, half of the patients admitted for a first psychotic episode failed to link to outpatient psychiatric care. Our study suggests that treatment rather than patient's characteristics play a critical role in this phenomenon. Development of a partnership and involvement of patients in the decision process, provision of good information regarding the illness, clear definition of the treatment plan, development of concrete strategies to cope with the illness and its potential complications, and involvement of the outpatient treating team already during hospitalisation, all came out as critical strategies to facilitate adherence to outpatient care. While the current rate of disengagement after admission is highly concerning, our finding are encouraging since they constitute strategies that can easily be implemented. An open approach to psychosis, the development of partnership with patients and a better coordination between inpatient and outpatient teams should therefore be among the targets of early intervention programs. These observations might help setting up priorities when conceptualising new programs and facilitate the implementation of services that facilitate engagement of patients in treatment during the critical initial phase of psychotic disorders.

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.

Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects.

The catalytic subunit of protein kinase A (PKAc) was purified to apparent homogeneity from two species of cold-hardy goldenrod gall insects, Epiblema scudderiana and Eurosta solidaginis. Final specific activity for both enzymes was approximately 74.5 nmol of phosphate transferred per minute per milligram protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. K(m) values at 24 degrees C for the artificial substrate, Kemptide, were 38.1+/-4.9 and 3.67+/-0.11 microM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K(m) Mg-ATP values were 61.1+/-6.9 and 30.7+/-4.1 microM. Assay at 4 degrees C lowered the K(m) for Kemptide of E. scudderiana PKAc by 55% and addition of 1M glycerol further lowered the K(m). Low assay temperature also enhanced holoenzyme dissociation in both species with the K(a) value for cyclic 3'5'-monophosphate at 4 degrees C lowered to just 13-18% of the value at 24 degrees C. Low temperature did not affect affinity for Mg-ATP or inhibition by PKA inhibitors (PKAi, H7, H89) but increased inhibition by some salts. PKAc from both species showed a break in the Arrhenius relationship at approximately 10 degrees C which suggests a conformational change at low temperature; activation energies (E(a)) were 2.2-3 fold higher for the lower (<10 degrees C) versus higher (>10 degrees C) range. Addition of naturally occurring polyols, 1M glycerol or 0.4M sorbitol, affected E(a) in some cases. Temperature dependent regulation of holoenzyme dissociation and PKAc kinetic properties may have an role in regulating the enzymes involved in polyol synthesis in cold-hardy insects.

Characteristics of the poliovirus replication complex.

In the infected cell, the poliovirus replication complex (RC) is found in the center of a rosette formed by many virus-induced vesicles. The RC is attached to the vesicular membranes and contains a compact central part which encloses the replication forks of the replicative intermediate and all proteins necessary for strand elongation. The growing plus strands of the replicative intermediate protrude from the central part of the RC, but are still enclosed by membraneous structures of the rosette. After completion, progeny 36S RNA is set free at the surface of the rosette. In an in vitro transcription system, isolated replication complex-containing rosettes are active in initiation, elongation and maturation (release) of plus strand progeny RNA. Full functionality of the RC depends on an intact structural framework of all membraneous components of the rosette.

Determination of renal tissue ibandronate levels in rats with normal and mildly impaired renal function.

BACKGROUND: After entering the blood, bisphosphonates are immediately bound to bone or excreted unchanged by the kidney. During renal excretion about 0.5% of administrated dosage remains in kidney tissue. The renal tissue level of bisphosphonates (RTL) decreases over time and remains at about 0.15% after 3weeks, but the influence of renal insufficiency (RI) is unclear. METHOD: We investigated the influence of mild to moderate RI on RTL of ibandronate (IBD). First a method for determination of RTL was implemented and validated. We measured RTL in rats with normal renal function (SHAM) and after unilateral nephrectomy (UNX). In each case one SHAM and one UNX groups received one or alternatively 9 times every 3weeks a dosage of 1.5mg/kg IBD. After the last dosage the rats were sacrificed and RTL of IBD were determined. RESULTS: In SHAM-rats IBD concentrations increased from 272.7ng/g kidney after one injection to 428.9ng/g kidney after nine injections (p<0.0001). RTL in UNX rats likewise increased significantly (p<0.0001) from 289.9ng/g kidney to 520.2ng/g kidney. CONCLUSION: Our study found a 1.6 fold increase of RTL in SHAM rats and a 1.8 fold increase of RTL in UNX rats after nine versus one injection. As steady state is generally reached after five half-lives we anticipate no further accumulation on continued treatment.

NANOG modulates stemness in human colorectal cancer.

NANOG is a stem cell transcription factor that is essential for embryonic development, reprogramming normal adult cells and malignant transformation and progression. The nearly identical retrogene NANOGP8 is expressed in multiple cancers, but generally not in normal tissues and its function is not well defined. Our postulate is that NANOGP8 directly modulates the stemness of individual human colorectal carcinoma (CRC) cells. Stemness was measured in vitro as the spherogenicity of single CRC cells in serum-free medium and the size of the side population (SP) and in vivo as tumorigenicity and experimental metastatic potential in NOD/SCID mice. We found that 80% of clinical liver metastases express a NANOG with 75% of the positive metastases containing NANOGP8 transcripts. In all, 3-62% of single cells within six CRC lines form spheroids in serum-free medium in suspension. NANOGP8 is translated into protein. The relative expression of a NANOG gene increased 8- to 122-fold during spheroid formation, more than the increase in OCT4 or SOX2 transcripts with NANOGP8 the more prevalent family member. Short hairpin RNA (shRNA) to NANOG not only inhibits spherogenicity but also reduces expression of OCT4 and SOX2, the size of the SP and tumor growth in vivo. Inhibition of NANOG gene expression is associated with inhibition of proliferation and decreased phosphorylation of G2-related cell-cycle proteins. Overexpression of NANOGP8 rescues single-cell spherogenicity when NANOG gene expression is inhibited and increases the SP in CRC. Thus, NANOGP8 can substitute for NANOG in directly promoting stemness in CRC.

[The ultrastructure of psoriatic nail changes]. / Die Feinstruktur psoriatischer Nagelveränderungen.

The various and subsequent psoriatic nail changes can only be understood on the basis of parakeratotic changes. If parakeratosis is localized in the nail matrix specific changes in the nail will result depending on localization and spreading in the matrix. Punctate indentations (pits) may develop; if there is a ribbon-like, square-dimensioned localization we see streaks and ridges (psoriasis unguis striata). If parakeratotic cells invade the whole of the matrix the so-called psoriasis unguis crateriformis will develop. Well-known to the dermatologist is the irregular salmon-colored patch--a type of psoriasis involving the nail-bed. This salmon-colored patch may finally cause onycholysis semilunaris as the nail plate is not fixed to the nail-bed any longer. There may be onycholysis, too, if the cuticle is involved in the course of the psoriatic change. A long-standing psoriasis of the nails is characterized by para- or hyperkeratotic masses ("Krümelnagel").

Polypeptide p41 of a Norwalk-like virus is a nucleic acid-independent nucleoside triphosphatase.

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family Caliciviridae. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication.

Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication.

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.

Poliovirus subviral particles associated with progeny RNA in the replication complex.

The poliovirus replication complex (RC), the site of genomic 36S RNA synthesis, was previously shown to contain subviral particles of 5S protomer and 14S pentamer antigenicity. The present investigation demonstrates that 5S/14S antigenic subviral particles can be cross-linked to viral RNA by UV irradiation of a subcellular fraction containing the poliovirus RC. Each capsid protein of the subviral particles, i.e. VP0, VP1 and VP3, was cross-linked to viral RNA. SDS-PAGE analysis of the cross-linked capsid proteins revealed a bandshift for VP1, whereas VP0 migrated in several bands, which were interpreted to be multimers of VP0 linked by short stretches of RNA. It was found that 36S RNA rather than replicative intermediate RNA was cross-linked to capsid proteins. Our results indicate that encapsidation of poliovirus RNA starts in the RC and is initiated by 14S pentamers.

[Scanning electron microscopic studies of diseases fingernails by means of a casting technic]. / Raster-elektronenmikroskopische Untersuchungen am kranken Nagel mittels Abdruck-Verfahren.

By a cast method (silicone resins) which allows a form-fitting reprint of nail-plate relief are made scanning electron microscopic studies of healthy and sick nails. In this way a form-fitting restitution of the surface-relief is guaranteed up to magnifications of 5000:1. Thus it is possible to get a better impression of the affected nail-structure. The exactitude of this nail-structure-representation cannot be reached by normal light microscopy. This method is very simple and quickly to use. Nail-changes in onychoschizia, subungual hematoma and in senile nails are shown.

Effects of music on exercise and perceived symptoms in patients with chronic obstructive pulmonary disease.

BACKGROUND: The purpose of this study was to examine the effects of music on exercise tolerance and perceived symptoms during treadmill walking in patients with chronic obstructive pulmonary disease (COPD). METHODS: Nineteen patients with COPD recruited from a pulmonary rehabilitation program participated in treadmill walking sessions on three separate days. The first session consisted of a practice walk. The second and third walking sessions were 6-minute timed tests in which the subjects were asked to walk as fast as possible under two conditions: a control walk and a music walk. Subjective assessments of perceived dyspnea and effort were obtained during the walks by the modified Borg scale. RESULTS: Distances walked were 331m +/- 19 (SE) for the music walk and 321m +/- 21 (SE) for the control walk (P = .25). Within each condition, ratings of perceived exertion and dyspnea increased from minute 1 to minute 6 (P < 0.05). CONCLUSION: There were no statistically significant differences observed between treatment conditions for distance walked, perceived dyspnea or ratings of perceived exertion. It should be noted that 60% of the subjects voluntarily commented that they enjoyed listening to music while they exercised.

Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes.

Polioviral RNA was localized by electron microscopic in situ hybridization on sections of poliovirus-infected HEp-2 cells. Viral plus-strand RNA was found accumulated in the close surroundings of the membrane-bound replication complex. Two different regions of the viral genome were detected with the same frequency, which indicates the predominant presence of full-length genomic RNA. Viral proteins of the P2 and the P3 genomic region were detected mainly over the core of the replication complex, whereas the hybridization signal was present rather at the peripheral parts of the complex. A more than 100-fold excess of viral plus- over minus-strand RNA was found by strand-specific hybridization to RNA extracted from isolated replication complexes. These findings support the idea of a pool of viral plus-strand RNA set free from the replication complex and accumulating in the close vicinity of the replication complex possibly before encapsidation.