Development and Validation of Two Screening Assays for the Hepatitis C Virus NS3 Q80K Polymorphism Associated with Reduced Response to Combination Treatment Regimens Containing Simeprevir.

Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate an NS3 clinical sequencing assay to detect the Q80K polymorphism and potentially other HCV NS3 drug resistance mutations. HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, and sequenced using Sanger and/or next-generation (MiSeq) methods. Sanger chromatograms were analyzed using in-house software (RECall), and nucleotide mixtures were called automatically. MiSeq reads were iteratively mapped to the H77 reference genome, and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision, and sensitivity for detecting the Q80K polymorphism were assessed in 70 samples previously sequenced by an external laboratory. A comparison of the sequences generated by the Sanger and MiSeq methods with those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of the Q80K polymorphism. The results were both highly repeatable and reproducible (>99.7% nucleotide concordance and 100% Q80K concordance). The limits of detection (>2 and ∼5 log10 IU/ml for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically infected treatment-naive persons. No systematic bias in the under- or overamplification of minority variants was observed. Coinfection with other viruses (e.g., HIV and hepatitis B virus [HBV]) did not affect the assay results. The two independent HCV NS3 sequencing assays with the automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations.

A pilot study: are dental hygienists in Texas ready for the elderly population explosion?

PURPOSE: At the beginning of the 20th century, people 65 and older comprised 4.1% of the population. By the year 2030, it is estimated that people 65 and older, the 'Baby Boomer' generation, will comprise more than 20% of the population. This will have a profound effect on the practice of dentistry and on society as a whole. The purpose of this study was to determine whether dental hygienists in Texas felt prepared and willing to treat the elderly in alternative practice settings such as nursing homes. METHODS: After institutional review board approval, a questionnaire was mailed to 500 hygienists. A 5% systematic sample of dental hygiene graduates was taken from four dental hygiene schools in Texas, United States of America (USA). Of these, 175 were returned for a 35% response rate. Questions asked were degree held, how prepared the participants felt to treat the special needs of the elderly, if participants were willing to work in alternative practice settings such as a nursing home and if they felt additional education was needed. Frequency distributions, correlations and chi square were used to analyse the data. RESULTS: Results revealed 86.5% of the respondents felt prepared to somewhat prepared to treat the special needs of the elderly based on education; equally, 86.5% felt more education was needed to better prepare them to treat the elderly. Over half of the respondents would not be willing to work in alternative practice settings such as nursing homes. CONCLUSION: The average respondents do not feel fully prepared to treat the elderly with special needs, and they think more education is needed to better prepare them to treat this important target population.

Streaked sub-ps-resolution x-ray line shapes and implications for solid-density plasma dynamics (invited).

A high-resolution x-ray spectrometer was coupled with an ultrafast x-ray streak camera to produce time-resolved line shape spectra measured from hot, solid-density plasmas. A Bragg crystal was placed near laser-produced plasma to maximize throughput; alignment tolerances were established by ray tracing. The streak camera produced single-shot, time-resolved spectra, heavily sloped due to photon time-of-flight differences, with sufficient reproducibility to accumulate photon statistics. The images are time-calibrated by the slope of streaked spectra and dewarped to generate spectra emitted at different times defined at the source. The streaked spectra demonstrate the evolution of spectral shoulders and other features on ps timescales, showing the feasibility of plasma parameter measurements on the rapid timescales necessary to study high-energy-density plasmas.

Larval aquatic and terrestrial mites infesting parthenogenetic Ischnura hastata (Odonata: Coenagrionidae) from the Azores islands.

We report here the prevalence of parasitism by water mites (Arrenurus sp.) and terrestrial mites (Leptus killingtoni) on parthenogenetic Ischnura hastata (Odonata: Coenagrionidae) from the Azores islands. Leptus killingtoni was only found on the island of Pico, and the prevalence of infestation was highly variable among the different ponds studied, ranging from 0 to 41%. Leptus killingtoni was observed on three of the four odonate species from the archipelago: I. hastata, I. pumilio, and Sympetrum fonscolombii, all of them new hosts for this species. Aquatic mites have been found parasitizing I. hastata females on the island of São Miguel. The prevalence of mite parasitism by Arrenurus sp. on I. hastata was very low, ranging from 12% (2003) to 1% (2008), and in most of the studied ponds, no mites were found attached to females. Although I. hastata coexists with a sexual congener species in the Azores (I. pumilio), they are syntopic in only a small fraction of ponds. Therefore, a comparison between I. hastata and I. pumilio was insufficient to test the predictions of the Red Queen Hypothesis, and further research on parasitism rates in both species needs to be done. In any case, the low prevalence of mite parasitism found in the Azores, coupled with the fact that most of the populations in the archipelago are almost free from competitors and predators, could explain the persistence of these I. hastata parthenogenetic populations, despite their low levels of genetic variation.

Widespread Wolbachia infection in an insular radiation of damselflies (Odonata, Coenagrionidae).

Wolbachia is one of the most common endosymbionts found infecting arthropods. Theory predicts symbionts like Wolbachia will be more common in species radiations, as host shift events occur with greatest frequency between closely related species. Further, the presence of Wolbachia itself may engender reproductive isolation, and promote speciation of their hosts. Here we screened 178 individuals belonging to 30 species of the damselfly genera Nesobasis and Melanesobasis - species radiations endemic to the Fiji archipelago in the South Pacific - for Wolbachia, using multilocus sequence typing to characterize bacterial strains. Incidence of Wolbachia was 71% in Nesobasis and 40% in Melanesobasis, and prevalence was also high, with an average of 88% in the Nesobasis species screened. We identified a total of 25 Wolbachia strains, belonging to supergroups A, B and F, with some epidemic strains present in multiple species. The occurrence of Wolbachia in both males and females, and the similar global prevalence found in both sexes rules out any strong effect of Wolbachia on the primary sex-ratio, but are compatible with the phenotype of cytoplasmic incompatibility. Nesobasis has higher species richness than most endemic island damselfly genera, and we discuss the potential for endosymbiont-mediated speciation within this group.

Role of bcl-2 in growth factor triggered signal transduction.

The role of bcl-2 in the signal transduction pathway was assessed by studying the inositol phospholipid metabolism in fibroblasts transfected by this oncogene. The rate of accumulation of water soluble inositol phosphates in response to several growth factors was much higher in bcl-2 transfected NIH3T3 clones than in untransfected control. Moreover, bcl-2 transfected clones express elevated levels of phosphatidic acid, a phospholipid produced during receptor stimulated breakdown of phosphoinositides. Our findings suggest that the expression of bcl-2 in NIH3T3 fibroblasts leads to the coupling of growth factor receptors to stimulate inositol phosphate production, henceforth establishing its role in the growth factor receptor mediated signal transduction pathway.

Cellular localization of the bcl-2 protein and response to glucocorticoid stress.

We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.

Aging: effects on the prostaglandin production by skeletal muscle of male rhesus monkeys (Macaca mulatta).

Prostaglandin (PG) production from [1-14C] arachidonic acid was measured in homogenates of sartorius muscle of aged (more than 20 years) and young adult (7 to 11 years) rhesus monkeys. Total production by the aged series (3.21 +/- 0.31 (S.E.) nmol PG per g N per min) was about two times that of the young series (1.76 +/- 0.14 (S.E.) nmol per g N per min). Epinephrine-stimulated PG production was also twice as great in the aged muscle series as in the young adult series (7.22 +/- 0.74 (S.E.) and 3.54 +/- 0.52 (S.E.) nmol per g N per min). This difference was mainly due to greater production of 6-keto-PGF1 alpha and PGF2 alpha. In both series, the addition of 500 microM epinephrine to the assay media significantly increased production of all types of PGs measured (6-keto-PGF1 alpha, PGF2 alpha, PGE2 plus thromboxane B2 [TXB2], the stable breakdown product of TXA2, and PGD2). The absolute increases in all types of PGs studied in the presence of epinephrine were significantly greater in the aged muscle, with the exception of PGF2 alpha. The distribution patterns of the various types of PGs studied were similar in all series, except for 6-keto-PGF1 alpha; this PG accounted for a greater percentage of the total production by aged muscle than by young adult muscle (p less than 0.05).

Effect of BAEP monitoring on hearing preservation during acoustic neuroma resection.

We measured the effect of brainstem auditory evoked potential (BAEP) monitoring on hearing preservation in acoustic neuroma resection in 90 consecutive patients with monitoring compared with 90 historical controls matched for tumor size and preoperative hearing status. In small tumors (less than 2 cm), BAEP monitoring was associated with a higher rate of hearing preservation and a greater chance that the hearing preserved was clinically useful. Changes in the BAEP intraoperatively showed a good correlation with postoperative hearing status.

Effects of ADP and epinephrine on macaque and human platelets. Implications for studies on human atherosclerosis.

The ranges for near-threshold ADP concentrations for the aggregation of macaque and human citrated platelets overlapped. The minimum concentrations of epinephrine, 0.05 microM to 1.0 microM, that at least doubled the aggregation response at threshold ADP concentrations were comparable for macaque and human citrated platelets. Epinephrine (1.0 microM to 10 mM) alone never aggregated macaque citrated platelets. Biphasic aggregation occurred with both macaque and human citrated platelets. The addition of heparin to a final concentration of 2.2 units/ml had no effect on the threshold ADP concentrations or the sensitivity of macaque or human citrated platelets to epinephrine. One microM phentolamine eliminated the potentiating effect of 1 microM epinephrine on ADP-induced aggregation of macaque and human citrated platelets. The threshold concentrations of ADP for macaque platelets were sharply reduced when heparin was used as an anticoagulant rather than citrate. However, epinephrine induced a similar increase in aggregability with both citrated and heparinized platelets, 0.55 +/- 0.09 SEM% and 0.44 +/- 0.09 SEM%, respectively. These data indicate that macaque and human platelets behave in a similar manner in response to ADP and that epinephrine potentiates the ADP-induced aggregation of macaque and human platelets equally well.

Metabolism of arachidonic acid by macaque platelets. Implications for studies on atherosclerosis.

The metabolism of [1-14C]arachidonic acid [( 1-14C]AA) by washed platelets from macaques and human subjects was investigated. The results were as follows: At substrate levels of 1 microM, similar amounts of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), and thromboxane A2 (TXA2), measured as thromboxane B2 (TXB2), were produced from [1-14C]AA by platelets from rhesus, Celebes black, and cynomolgus macaques and humans. An increase in the AA concentration from 1 microM to 20 microM decreased the TXB2: PGD2 ratio (aggregator: antiaggregator) from greater than 5 to less than 2 in all series. In the human series, the ratio decrease was due to an increase in PGD2 production; in the macaque series, PGD2 production increased and TXB2 production decreased. Under basal conditions and at 1 microM AA concentrations, the amounts of prostaglandins and thromboxanes produced by platelets from male and female rhesus macaques were the same. An increase in substrate concentration from 1 microM to 20 microM AA decreased TXB2 production and increased PGD2 production to the same extent in platelets from male and female rhesus macaques. Imidazole increased prostaglandin production and decreased TXB2 production by platelets from both male and female rhesus macaques. The TXB2: PGD2 ratios were reduced below 1.5; there was no difference between the ratios in the two series. In the presence of 1 mM imidazole, greater amounts of prostaglandins and thromboxanes were produced in the male than in the female series. These data indicate that macaque's platelets are a suitable model for the study of AA metabolism in human platelets.

Stereotactic radiosurgery using the gamma knife for acoustic neuromas.

PURPOSE: To assess the efficacy and toxicity of stereotactic radiosurgery using the gamma knife for acoustic neuromas. METHODS AND MATERIALS: Between January 1990 and January 1993, 36 patients with acoustic neuromas were treated with stereotactic radiosurgery using the gamma knife. The median maximum tumor diameter was 21 mm (range: 6-32 mm). Tumor volumes encompassed within the prescribed isodose line varied from 266 to 8,667 mm3 (median: 3,135 mm3). Tumors < or = 20 mm in maximum diameter received a dose of 20 Gy to the margin, tumors between 21 and 30 mm received 18 Gy, and tumors > 30 mm received 16 Gy. The dose was prescribed to the 50% isodose line in 31 patients and to the 45%, 55%, 60%, 70%, and 80% isodose line in one patient each. The median number of isocenters per tumor was 5 (range: 1-12). RESULTS: At a median follow-up of 16 months (range: 2.5-36 months), all patients were alive. Thirty-five patients had follow-up imaging studies. Nine tumors (26%) were smaller, and 26 tumors (74%) were unchanged. No tumor had progressed. The 1- and 2-year actuarial incidences of facial neuropathy were 52.2% and 66.5%, respectively. The 1- and 2-year actuarial incidences of trigeminal neuropathy were 33.7% and 58.9%, respectively. The 1- and 2-year actuarial incidence of facial or trigeminal neuropathy (or both) was 60.8% and 81.7%, respectively. Multivariate analysis revealed that the following were associated with the time of onset or worsening of facial weakness or trigeminal neuropathy: (a) patients < age 65 years, (b) dose to the tumor margin, (c) maximum tumor diameter > or = 21 mm, (d) use of the 18 mm collimator, and (e) use of > five isocenters. The 1- and 2-year actuarial rates of preservation of useful hearing (Gardner-Robertson class I or II) were 100% and 41.7% +/- 17.3, respectively. CONCLUSION: Stereotactic radiosurgery using the gamma knife provides short-term control of acoustic neuromas when a dose of 16 to 20 Gy to the tumor margin is used. Preservation of useful hearing can be accomplished in a significant proportion of patients.

Usher's syndrome with unusual otologic manifestations.

Usher's syndrome is characterized by a congenital hearing loss and retinitis pigmentosa. Ocular symptoms and signs are usually established at adolescence, and the hearing loss, the onset of which is at a young age, generally remains stable. The following case is of interest, therefore, because the hearing loss that had been present since birth progressed suddenly to total bilateral deafness in adulthood. There were no ocular symptoms at any time; the classic findings of retinitis pigmentosa were disclosed only on routine examination of the eyes.

Childhood sinusitis.

Childhood sinusitis is difficult to diagnose. It is classified on the basis of duration of inflammation--acute or chronic--and cause of inflammation--infectious or noninfectious. Infectious sinusitis is often a result of obstruction of the osteomeatal complex. Inflammation in noninfectious sinusitis is similar to the inflammatory changes detected in respiratory mucosa of patients with asthma. Acute sinusitis is primarily an infectious process similar to a prolonged infection of the upper respiratory tract. Plain radiography has limited value for the diagnosis of acute sinusitis in children. The most effective treatment of acute sinusitis is administration of a beta-lactamase-resistant antibiotic. Chronic sinusitis may be infectious, noninfectious, or both. Coronal computed tomography of the sinuses and nasal endoscopy are the preferred methods for determining the presence of chronic sinusitis. When physicians prescribe therapy for chronic sinusitis, they need to consider whether the underlying cause is infectious, noninfectious, or both. Treatment of chronic infectious sinusitis is most effective when a beta-lactamase-resistant antibiotic is administered. Chronic noninfectious sinusitis may respond to topically intranasally applied corticosteroids. If medical treatment fails to resolve the disease within 3 months, surgical intervention may be necessary. Finally, although an association between asthma and sinusitis exists, a cause-and-effect relationship has not been established.

Improved preservation of facial nerve function with use of electrical monitoring during removal of acoustic neuromas.

Continuous spontaneous electromyographic activity and responses to electrical stimulation of the facial nerve in the surgical field were monitored in 48 patients undergoing primary removal of an acoustic neuroma. The operative and postoperative results in these patients were compared with the results in 48 patients who were matched for age and size of tumor and who underwent the same surgical procedure without intraoperative monitoring. Eighty-three percent of the patients had preoperative evidence of facial neuropathy, which was more severe with larger tumors. Postoperative facial nerve function was most accurately predicted on the basis of the extent of facial neuropathy on preoperative electrophysiologic testing. Anatomic preservation of the facial nerve in patients with large tumors was substantially improved in the monitored patients (67%) in comparison with those without monitoring (33%). No difference was noted in facial nerve function in the two groups of patients immediately postoperatively. By 3 months, the degree of improvement in the monitored group exceeded that in those who were not monitored, particularly in patients with medium-sized and large tumors.

Predictors of response to transantral orbital decompression in severe Graves' ophthalmopathy.

OBJECTIVE: To identify factors that may help predict the outcome after transantral orbital decompression in Graves' ophthalmopathy. DESIGN: A retrospective study was conducted of 428 patients who had undergone an initial transantral orbital decompression for severe Graves' ophthalmopathy at the Mayo Clinic between November 1969 and May 1989. MATERIAL AND METHODS: With use of logistic regression analysis, we assessed the preoperative characteristics, the early postoperative results, and the follow-up questionnaire data (obtained a median of 9.5 years postoperatively) from 304 female and 124 male patients with Graves' ophthalmopathy who had undergone transantral orbital decompression at a median age of 53 years. RESULTS: On multivariate stepwise regression analysis, young age, male sex, and long duration of eye symptoms were predictors of severe initial proptosis (P < 0.001). The only independent predictors of greater postoperative recession of proptosis were severity of initial proptosis and longer interval between operation and postoperative examination (P < 0.001). Patients with the most reduction of proptosis had the greatest improvement in visual acuity but more chance for postoperative development of continuous diplopia. Failure of prior corticosteroid or orbital radiation therapy did not affect the degree of recession of proptosis or improvement in visual acuity. On multivariate analysis for predictors of long-term overall patient satisfaction, only young age of the patient was of borderline significance (P = 0.05), and the only significant predictor of satisfaction with the postoperative eye appearance was an operation done primarily for cosmetic purposes (P = 0.012). CONCLUSION: Although various factors may influence the outcome of orbital decompression in patients with Graves' ophthalmopathy, this study showed that the more pronounced the initial proptosis, the greater the degree of recession postoperatively. A higher degree of reduction of proptosis is associated with better visual acuity but also a greater likelihood of development of continuous diplopia.

Benign positional vertigo: incidence and prognosis in a population-based study in Olmsted County, Minnesota.

A retrospective review of our population-based medical records linkage system for residents of Olmsted County, Minnesota, revealed 53 patients (34 women and 19 men; mean age, 51 years) with newly diagnosed benign positional vertigo in 1984. The age- and sex-adjusted incidence was 64 per 100,000 population per year (95% confidence interval, 46 to 81 per 100,000). The incidence of benign positional vertigo increased by 38% with each decade of life (95% confidence interval, 23 to 54%). One patient had an initial stroke during follow-up; thus, the relative risk for new stroke associated with benign positional vertigo was 1.62 (95% confidence interval, 0.04 to 8.98) in comparison with the expected occurrence based on incidence rates for an age- and sex-adjusted control population. The observed survival among the 53 Olmsted County residents with benign positional vertigo diagnosed in 1984 was not significantly different from that of an age- and sex-matched general population. Patients with benign positional vertigo seem to have a good prognosis.

The canalith repositioning procedure for the treatment of benign paroxysmal positional vertigo: a randomized controlled trial.

OBJECTIVE: To compare the canalith repositioning procedure (CRP) with a sham maneuver for the treatment of benign paroxysmal positional vertigo. PATIENTS AND METHODS: We recruited 50 patients with a history of positional vertigo and unilateral positional nystagmus on physical examination (Dix-Hallpike maneuver). Patients were randomized to either the CRP (n = 24) or a sham maneuver (n = 26). Measured outcomes included resolution of vertigo and positional nystagmus at follow-up examination. RESULTS: The mean duration of follow-up was 10 days for both groups. Resolution of symptoms was reported by 12 (50%) of the 24 patients in the CRP group and by 5 (19%) of the 26 patients in the sham group (P = .02). The results of the Dix-Hallpike maneuver were negative for positional nystagmus in 16 (67%) of 24 patients in the CRP group and in 10 (38%) of 26 patients in the sham group (P = .046). CONCLUSION: The CRP is effective treatment of benign paroxysmal positional vertigo, and this procedure can be performed by general internists on outpatients with this disorder.

BCL-2 alpha encodes a novel small molecular weight GTP binding protein.

Despite little sequence homology other than the GDP/GTP binding region, bcl-2 and ras proteins behave in similar fashion in many physiological and biochemical aspects. Both of them are toxin insensitive, small Mr G-proteins attached to the inner surface of the cell membrane with autophosphorylation activity and can cooperate with c-myc in cell transformation. In the case of bcl-2, however, the mechanism of activation is still unclear. One possibility is that, following antigen or mitogen stimulation, the bcl-2 protein is activated by nucleotide exchange; then the activated bcl-2 protein may interact with some other protein in the signal transduction pathway leading to cell proliferation. Dissection of the role of bcl-2 in the regulation of B-cell proliferation will be an important step in understanding its role in the pathogenesis of B-cell neoplasia.