Story Generation and Narrative Retells in Children Who Are Hard of Hearing and Hearing Children.

PURPOSE: The primary goal for this study was to compare story generation and narrative retell performance in children who are hard of hearing (CHH) and hearing children. A secondary goal was to determine factors that influence narrative performance. Research on this topic is important because narrative language competency is an essential communication function. METHOD: Participants included 86 CHH and 53 seven-year-old hearing children who had completed a test battery composed of vocabulary, grammar, phonological processing, story generation, and narrative retell tasks. Coders who were blind to hearing status used a scoring rubric to judge the quality of narrative macrostructure in story generation and narrative retells. Data were analyzed using analysis of variance to determine group differences and correlational models to examine individual differences. RESULTS: At 7 years of age, CHH demonstrated significant deficits in narrative macrostructure compared to hearing children, with larger delays in narrative retell than story generation. Vocabulary, grammar, and phonological memory acted as mediators in the relationship between hearing status and story generation; grammar acted as a mediator between hearing status and narrative retell. Auditory access variables accounted for a significant proportion of shared variance in story generation skills for CHH. CONCLUSIONS: School-age CHH are at risk for delays in narrative production, particularly with retelling stories. The results of this study highlight a narrative coding approach and task procedures that are sensitive to differences in language levels and may be clinically useful for professionals working with early school-age children.

A Multilinguistic Spelling Analysis of Children who are Hard of Hearing.

Spelling in writing samples was compared between children who are hard of hearing (HH) (n = 142) and their peers with typical hearing (TH) (n = 72) in second and fourth grade. The same analyses were then conducted comparing groups of children who are HH with different levels of aided audibility. Compared to children with TH, children who are HH produced fewer misspelled words (p = .041, d = .42) at second grade but performed similarly in fourth grade (p = .943, d = .02). Compared to peers with TH, children who are HH demonstrated similar distributions of errors in roots but some differences in the distribution of errors for affixes. Different levels of aided audibility among children who are HH were not associated with significant differences in spelling accuracy at both grades. However, second-grade children with poorer aided audibility produced significantly more phonological omissions (p = .005, r = .32) and orthographic consonant errors for monomorphemic words (p = .001, r = .37), as well as more orthographic consonant errors for and affixes (p = .015, r = .28).

A multilinguistic analysis of spelling among children with cochlear implants.

This study examines the spelling of nine elementary school children with cochlear implants (CIs) who use spoken language, and compares their performance with children who have typical hearing and children who are hard of hearing (HH). Compared to children with typical hearing, children with CIs did not produce a significantly different percentage of misspelled words (p = 0.431, d = 0.38), but their spelling errors comprised significantly lower percentages of homophone substitutions (p = 0.019, r = 0.61) and legal vowel errors (p = 0.011, r = 0.61). Children with CIs and children who are HH did not produce a significantly different percentage of misspelled words (p = 0.521, d = 0.31) or a significantly different distribution of categorical spelling errors. Results suggest that children with CIs utilize similar linguistic strategies as their peers who are HH but different strategies than peers with typical hearing when attempting to spell unfamiliar words.

Service Provision for Children Who Are Hard of Hearing at Preschool and Elementary School Ages.

Purpose: To characterize preschool and school services for children who are hard of hearing (CHH), we described service setting, amount, and configuration and analyzed the relationship between service receipt and student hearing levels and language scores. Characteristics of professionals providing services were described and then used to predict level of comfort with skills supporting listening and spoken language. The amount of provider communication with children's audiologists was also investigated. Method: Participants included parents of CHH (preschool n = 174; school n = 155) and professionals (preschool n = 133; school n = 104) who completed interviews and questionnaires as part of a longitudinal study. Children's hearing, speech, and language data were collected from annual testing and analyzed in relation to service data. Results: A majority (81%) of preschool-age CHH received services. Children were more likely to be in a preschool for children who are deaf or hard of hearing (CDHH) or exceptional children than a general education preschool. By elementary school, 70% received services, nearly all in general education settings. Sessions averaged twice a week for a total of approximately 90 min. Children who no longer received services performed significantly better on speech/language measures than those who received services, regardless of service setting. Professionals were primarily speech-language pathologists (SLPs) and teachers of CDHH. SLPs reported significantly less comfort with skills involving auditory development and hearing technologies and less frequent communication with the child's audiologists than teachers of CDHH. Overall communication with audiologists was more frequent in the preschool years. Conclusions: As preschool-age CHH transition into school, the majority continue to qualify for services. Congruent with national trends, school-age CHH in the Outcomes of Children with Hearing Loss study were most often in general education settings. Without specialized preprofessional or postgraduate training, SLPs and teachers of CDHH did not report comfort with all the skills critical to developing listening and spoken language. This finding supports the need for increased implementation of interprofessional practice among SLPs and teachers of CDHH, as well as audiologists, to best meet the needs unique to this population.

A Case Of Neuroendocrine Carcinoma Expressing Myeloid Markers By Flow Cytometry And Review Of the Literature.

BACKGROUND: Flow cytometric data is often analyzed in isolation, without the benefit of clinical and morphologic context, and the findings must be interpreted with caution when unexpected results are obtained. METHODS: A bone marrow aspirate from a 69-year-old female with incidentally discovered pancytopenia was initially analyzed by flow cytometry alone. The results were subsequently correlated with clinical, morphologic, immunohistochemical, and cytogenetic findings. RESULTS: Morphology and immunohistochemistry confirmed metastatic small cell carcinoma; by flow cytometric analysis, the neoplastic cells were positive for CD56 and showed unexpected expression of CD13 and CD117, raising the possibility of a myeloid neoplasm. CONCLUSIONS: Flow cytometric markers are not entirely sensitive or specific, and aberrant expression or lack of expression of certain markers can complicate interpretation, particularly when this is done in isolation (i.e., in reference laboratories). Whenever possible, clinical and morphologic correlation is strongly recommended. © 2016 International Clinical Cytometry Society.

Factors Affecting Early Services for Children Who Are Hard of Hearing.

PURPOSE: To describe factors affecting early intervention (EI) for children who are hard of hearing, we analyzed (a) service setting(s) and the relationship of setting to families' frequency of participation, and (b) provider preparation, caseload composition, and experience in relation to comfort with skills that support spoken language for children who are deaf and hard of hearing (CDHH). METHOD: Participants included 122 EI professionals who completed an online questionnaire annually and 131 parents who participated in annual telephone interviews. RESULTS: Most families received EI in the home. Family participation in this setting was significantly higher than in services provided elsewhere. EI professionals were primarily teachers of CDHH or speech-language pathologists. Caseload composition was correlated moderately to strongly with most provider comfort levels. Level of preparation to support spoken language weakly to moderately correlated with provider comfort with 18 specific skills. CONCLUSIONS: Results suggest family involvement is highest when EI is home-based, which supports the need for EI in the home whenever possible. Access to hands-on experience with this population, reflected in a high percentage of CDHH on providers' current caseloads, contributed to professional comfort. Specialized preparation made a modest contribution to comfort level.

Outcomes of Children with Hearing Loss: Data Collection and Methods.

OBJECTIVES: The primary objective of this article was to describe recruitment, data collection, and methods for a longitudinal, multicenter study involving children with bilateral mild to severe hearing loss. The goals of this research program were to characterize the developmental outcomes of children with mild to severe bilateral hearing loss during infancy and the preschool years. Furthermore, the researchers examined how these outcomes were associated with the child's hearing loss and how home background and clinical interventions mediated and moderated these outcomes. DESIGN: The participants in this study were children who are hard of hearing (CHH) and children with normal hearing (CNH) who provided comparison data. CHH were eligible for participation if (1) their chronological age was between 6 months and 7 years of age at the time of recruitment, (2) they had a better-ear pure-tone average of 25 to 75 dB HL, (3) they had not received a cochlear implant, (4) they were from homes where English was the primary language, and (5) they did not demonstrate significant cognitive or motor delays. Across the time span of recruitment, 430 parents of potential children with hearing loss made contact with the research group. This resulted in 317 CHH who qualified for enrollment. In addition, 117 CNH qualified for enrollment. An accelerated longitudinal design was used, in which multiple age cohorts were followed long enough to provide overlap. Specifically, children were recruited and enrolled continuously across an age span of 6.5 years and were followed for at least 3 years. This design allowed for tests of time (period) versus cohort age effects that could arise by changes in services and technology over time, yet still allowed for examination of important developmental relationships. RESULTS: The distribution of degree of hearing loss for the CHH showed that the majority of CHH had moderate or moderate-to-severe hearing losses, indicating that the sample undersampled children with mild HL. For mothers of both CHH and CNH, the distribution of maternal education level showed that few mothers lacked at least a high school education and a slight majority had completed a bachelor's degree, suggesting that this sample of research volunteers was more advantaged than the United States population. The test battery consisted of a variety of measures concerning participants' hearing and behavioral development. These data were gathered in sessions during which the child was examined by an audiologist and a speech-language examiner. In addition, questionnaires concerning the child's behavior and development were completed by the parents. CONCLUSION: The Outcomes of Children with Hearing Loss study was intended to examine the relationship between variation in hearing ability across children with normal and mild to severe hearing loss and variation in their outcomes across several domains of development. In addition, the research team sought to document important mediators and moderators that act between the hearing loss and the outcomes. Because the study design provided for the examination of outcomes throughout infancy and early childhood, it was necessary to employ a number of different measures of the same construct to accommodate changes in developmental performance across age. This resulted in a large matrix of measures across variable types and developmental levels, as described in this manuscript.

Language Outcomes in Young Children with Mild to Severe Hearing Loss.

OBJECTIVES: This study examined the language outcomes of children with mild to severe hearing loss during the preschool years. The longitudinal design was leveraged to test whether language growth trajectories were associated with degree of hearing loss and whether aided hearing influenced language growth in a systematic manner. The study also explored the influence of the timing of hearing aid fitting and extent of use on children's language growth. Finally, the study tested the hypothesis that morphosyntax may be at particular risk due to the demands it places on the processing of fine details in the linguistic input. DESIGN: The full cohort of children in this study comprised 290 children who were hard of hearing (CHH) and 112 children with normal hearing who participated in the Outcomes of Children with Hearing Loss (OCHL) study between the ages of 2 and 6 years. CHH had a mean better-ear pure-tone average of 47.66 dB HL (SD = 13.35). All children received a comprehensive battery of language measures at annual intervals, including standardized tests, parent-report measures, and spontaneous and elicited language samples. Principal components analysis supported the use of a single composite language score for each of the age levels (2, 3, 4, 5, and 6 years). Measures of unaided (better-ear pure-tone average, speech intelligibility index) and aided (residualized speech intelligibility index) hearing were collected, along with parent-report measures of daily hearing aid use time. Mixed modeling procedures were applied to examine the rate of change (227 CHH; 94 children with normal hearing) in language ability over time in relation to (1) degree of hearing loss, (2) aided hearing, (3) age of hearing aid fit and duration of use, and (4) daily hearing aid use. Principal components analysis was also employed to examine factor loadings from spontaneous language samples and to test their correspondence with standardized measures. Multiple regression analysis was used to test for differential effects of hearing loss on morphosyntax and lexical development. RESULTS: Children with mild to severe hearing loss, on average, showed depressed language levels compared with peers with normal hearing who were matched on age and socioeconomic status. The degree to which CHH fell behind increased with greater severity of hearing loss. The amount of improved audibility with hearing aids was associated with differential rates of language growth; better audibility was associated with faster rates of language growth in the preschool years. Children fit early with hearing aids had better early language achievement than children fit later. However, children who were fit after 18 months of age improved in their language abilities as a function of the duration of hearing aid use. These results suggest that the language learning system remains open to experience provided by improved access to linguistic input. Performance in the domain of morphosyntax was found to be more delayed in CHH than their semantic abilities. CONCLUSION: The data obtained in this study largely support the predictions, suggesting that mild to severe hearing loss places children at risk for delays in language development. Risks are moderated by the provision of early and consistent access to well-fit hearing aids that provide optimized audibility.

Speech sound production in 2-year-olds who are hard of hearing.

PURPOSE: The purpose of the study was to (a) compare the speech sound production abilities of 2-year-old children who are hard of hearing (HH) to children with normal hearing (NH), (b) identify sources of risk for individual children who are HH, and (c) determine whether speech sound production skills at age 2 were predictive of speech sound production skills at age 3. METHOD: Seventy children with bilateral, mild-to-severe hearing loss who use hearing aids and 37 age- and socioeconomic status-matched children with NH participated. Children's speech sound production abilities were assessed at 2 and 3 years of age. RESULTS: At age 2, the HH group demonstrated vowel production abilities on par with their NH peers but weaker consonant production abilities. Within the HH group, better outcomes were associated with hearing aid fittings by 6 months of age, hearing loss of less than 45 dB HL, stronger vocabulary scores, and being female. Positive relationships existed between children's speech sound production abilities at 2 and 3 years of age. CONCLUSION: Assessment of early speech sound production abilities in combination with demographic, audiologic, and linguistic variables may be useful in identifying HH children who are at risk for delays in speech sound production.

Surgical treatments for otitis media with effusion: a systematic review.

BACKGROUND AND OBJECTIVE: The near universality of otitis media with effusion (OME) in children makes a comparative review of treatment modalities important. This study's objective was to compare the effectiveness of surgical strategies currently used for managing OME. METHODS: We identified 3 recent systematic reviews and searched 4 major electronic databases. Eligible studies included randomized controlled trials, nonrandomized trials, and cohort studies that compared myringotomy, adenoidectomy, tympanostomy tubes (tubes), and watchful waiting. Using established criteria, pairs of reviewers independently selected, extracted data, rated risk of bias, and graded strength of evidence of relevant studies. We incorporated meta-analyses from the earlier reviews and synthesized additional evidence qualitatively. RESULTS: We identified 41 unique studies through the earlier reviews and our independent searches. In comparison with watchful waiting or myringotomy (or both), tubes decreased time with OME and improved hearing; no specific tube type was superior. Adenoidectomy alone, as an adjunct to myringotomy, or combined with tubes, reduced OME and improved hearing in comparison with either myringotomy or watchful waiting. Tubes and watchful waiting did not differ in language, cognitive, or academic outcomes. Otorrhea and tympanosclerosis were more common in ears with tubes. Adenoidectomy increased the risk of postsurgical hemorrhage. CONCLUSIONS: Tubes and adenoidectomy reduce time with OME and improve hearing in the short-term. Both treatments have associated harms. Large, well-controlled studies could help resolve the risk-benefit ratio by measuring acute otitis media recurrence, functional outcomes, quality of life, and long-term outcomes. Research is needed to support treatment decisions in subpopulations, particularly in patients with comorbidities.

Timeliness of service delivery for children with later-identified mild-to-severe hearing loss.

PURPOSE: In this study, the authors examined diagnostic and intervention services for children identified with hearing loss (HL) after the newborn period. METHOD: The authors compared ages at service delivery and length of delays between service delivery steps for 57 later-identified children with HL and 193 children who referred for assessment from the newborn hearing screen (NHS). For only later-identified children, regression models were used to investigate relationships among predictor variables and dependent variables related to service delivery. RESULTS: Children who referred from the NHS received follow-up services at younger ages than later-identified children. Later-identified children had significantly longer delays from HL confirmation to entry into early intervention, compared to children who referred from the NHS. For later-identified children, degree of HL predicted ages at follow-up clinical services. Children with more severe HL received services at younger ages compared to children with milder HL. Gender predicted the length of the delay from confirmation to entry into early intervention, with girls demonstrating shorter delays. CONCLUSIONS: The current results lend support to the need for ongoing hearing monitoring programs after the neonatal period, particularly when children enter early intervention programs because of language/developmental delays.

Clinical practice guideline: tympanostomy tubes in children--executive summary.

The American Academy of Otolaryngology-Head and Neck Surgery Foundation (AAO-HNSF) has published a supplement to this issue featuring the new Clinical Practice Guideline: Tympanostomy Tubes in Children. To assist in implementing the guideline recommendations, this article summarizes the rationale, purpose, and key action statements. The 12 recommendations developed address patient selection, surgical indications for and management of tympanostomy tubes in children. The development group broadly discussed indications for tube placement, perioperative management, care of children with indwelling tubes, and outcomes of tympanostomy tube surgery. Given the lack of current published guidance on surgical indications, the group focused on situations in which tube insertion would be optional, recommended, or not recommended. Additional emphasis was placed on opportunities for quality improvement, particularly regarding shared decision making and care of children with existing tubes.

Clinical practice guideline: Tympanostomy tubes in children.

OBJECTIVE: Insertion of tympanostomy tubes is the most common ambulatory surgery performed on children in the United States. Tympanostomy tubes are most often inserted because of persistent middle ear fluid, frequent ear infections, or ear infections that persist after antibiotic therapy. Despite the frequency of tympanostomy tube insertion, there are currently no clinical practice guidelines in the United States that address specific indications for surgery. This guideline is intended for any clinician involved in managing children, aged 6 months to 12 years, with tympanostomy tubes or being considered for tympanostomy tubes in any care setting, as an intervention for otitis media of any type. PURPOSE: The primary purpose of this clinical practice guideline is to provide clinicians with evidence-based recommendations on patient selection and surgical indications for and management of tympanostomy tubes in children. The development group broadly discussed indications for tube placement, perioperative management, care of children with indwelling tubes, and outcomes of tympanostomy tube surgery. Given the lack of current published guidance on surgical indications, the group focused on situations in which tube insertion would be optional, recommended, or not recommended. Additional emphasis was placed on opportunities for quality improvement, particularly regarding shared decision making and care of children with existing tubes. ACTION STATEMENTS: The development group made a strong recommendation that clinicians should prescribe topical antibiotic eardrops only, without oral antibiotics, for children with uncomplicated acute tympanostomy tube otorrhea. The panel made recommendations that (1) clinicians should not perform tympanostomy tube insertion in children with a single episode of otitis media with effusion (OME) of less than 3 months' duration; (2) clinicians should obtain an age-appropriate hearing test if OME persists for 3 months or longer (chronic OME) or prior to surgery when a child becomes a candidate for tympanostomy tube insertion; (3) clinicians should offer bilateral tympanostomy tube insertion to children with bilateral OME for 3 months or longer (chronic OME) and documented hearing difficulties; (4) clinicians should reevaluate, at 3- to 6-month intervals, children with chronic OME who did not receive tympanostomy tubes until the effusion is no longer present, significant hearing loss is detected, or structural abnormalities of the tympanic membrane or middle ear are suspected; (5) clinicians should not perform tympanostomy tube insertion in children with recurrent acute otitis media (AOM) who do not have middle ear effusion in either ear at the time of assessment for tube candidacy; (6) clinicians should offer bilateral tympanostomy tube insertion to children with recurrent AOM who have unilateral or bilateral middle ear effusion at the time of assessment for tube candidacy; (7) clinicians should determine if a child with recurrent AOM or with OME of any duration is at increased risk for speech, language, or learning problems from otitis media because of baseline sensory, physical, cognitive, or behavioral factors; (8) in the perioperative period, clinicians should educate caregivers of children with tympanostomy tubes regarding the expected duration of tube function, recommended follow-up schedule, and detection of complications; (9) clinicians should not encourage routine, prophylactic water precautions (use of earplugs, headbands; avoidance of swimming or water sports) for children with tympanostomy tubes. The development group provided the following options: (1) clinicians may perform tympanostomy tube insertion in children with unilateral or bilateral OME for 3 months or longer (chronic OME) and symptoms that are likely attributable to OME including, but not limited to, vestibular problems, poor school performance, behavioral problems, ear discomfort, or reduced quality of life and (2) clinicians may perform tympanostomy tube insertion in at-risk children with unilateral or bilateral OME that is unlikely to resolve quickly as reflected by a type B (flat) tympanogram or persistence of effusion for 3 months or longer (chronic OME).

Binding by the hepatitis C virus NS3 helicase partially melts duplex DNA.

Binding of NS3 helicase to DNA was investigated by footprinting with KMnO(4), which reacts preferentially with thymidine residues in single-stranded DNA (ssDNA) compared to those in double-stranded DNA (dsDNA). A distinct pattern of reactivity was observed on ssDNA, which repeated every 8 nucleotides (nt) and is consistent with the known binding site size of NS3. Binding to a DNA substrate containing a partial duplex was also investigated. The DNA contained a 15 nt overhang made entirely of thymidine residues adjacent to a 22 bp duplex that contained thymidine at every other position. Surprisingly, the KMnO(4) reactivity pattern extended from the ssDNA into the dsDNA region of the substrate. Lengthening the partial duplex to 30 bp revealed a similar pattern extending from the ssDNA into the dsDNA, indicating that NS3 binds within the duplex region. Increasing the length of the ssDNA portion of the partial duplex by 4 nt resulted in a shift in the footprinting pattern for the ssDNA by 4 nt, which is consistent with binding to the 3'-end of the ssDNA. However, the footprinting pattern in the dsDNA region was shifted by only 1-2 bp, indicating that binding to the ssDNA-dsDNA region was preferred. Footprinting performed as a function of time indicated that NS3 binds to the ssDNA rapidly, followed by slower binding to the duplex. Hence, multiple molecules of NS3 can bind along a ssDNA-dsDNA partial duplex by interacting with the ssDNA as well as the duplex DNA.

A case of cutaneous Scedosporium infection in an immunocompromised patient.

Scedosporium apiospermum, the asexual stage of Pseudoallescheria boydii, is a fungus ubiquitous in soil as well as organically polluted areas, where nitrogen-containing compounds are abundant. It is an emerging opportunistic pathogen that can range from cutaneous to disseminated infection and can be fatal within months of diagnosis. Here we present a case of disseminated S. apiospermum infection with cutaneous manifestations in a 59-year-old woman with myelodysplastic syndrome, in remission from chronic lymphocytic leukemia, presented with pneumonia and deteriorating mental status. An X-ray computed tomography scan showed three non-contrast-enhancing hypodensities affecting the brain. Many erythematous, indurated skin lesions, measuring 3-5 mm in diameter, were noted on her chest, shoulders and arms. Biopsies were submitted for culture and histology. Histopathologic examination revealed superficial and deep perivascular and periadnexal inflammatory infiltrates of lymphocytes and neutrophils. Scattered collections of fungal organisms were noted near the eccrine glands. The periodic acid Schiff with diastase stain showed the presence of variable sized spores and hyphae with some acute angle branching. Both tissue and blood cultures were positive for a single Scedosporium species. Histologically, eccrine or peri-eccrine involvement by fungi may be an important finding for Scedosporium infection of the skin.

NS3 helicase from the hepatitis C virus can function as a monomer or oligomer depending on enzyme and substrate concentrations.

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro.

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.

RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase.

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.

Structural and biological identification of residues on the surface of NS3 helicase required for optimal replication of the hepatitis C virus.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only approximately 2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.

Trends in age of identification and intervention in infants with hearing loss.

OBJECTIVE: In 1993, 11 hospitals in the United States were known to screen more than 90% of newborns for hearing loss. By 2000, approximately 1,000 hospitals reported screening at least 90% of their babies. This study was designed to identify trends in the age of identification and intervention for infants and young children with hearing loss in light of expanded implementation of newborn hearing screening. DESIGN: Parents of children under 6 yr of age with a confirmed hearing loss were surveyed. The survey instrument was designed to investigate three questions: 1) is the age of identification and intervention earlier for babies whose hearing is screened at birth compared with those whose hearing is not screened; 2) when hearing is screened at birth, do ages of diagnosis of hearing loss and intervention meet the guide-lines established in 2000, by the Joint Committee on Infant Hearing (Reference Note 1), and 3) what are the barriers to timely identification and intervention? Six hundred fifty-seven parents received the mailing. RESULTS: Responses of 151 parents of children with hearing loss, born between 1996 and 2000, were analyzed. Parents from 41 states provided information. Approximately half the children reported on were screened for hearing loss at birth. Age of identification and hearing aid fitting varied substantially based on degree of hearing loss and whether the cause of hearing loss was known or unknown; however, diagnosis and intervention occurred at an earlier age for infants screened at birth. Findings indicate that when hearing is screened at birth, infants with more severe degrees of hearing loss and an unknown cause tend to be identified and receive intervention within the 2000 timelines proposed by the Joint Committee on Infant Hearing. Barriers to timely identification and intervention are discussed. CONCLUSIONS: Before widespread implementation of newborn hearing screening, age of identification and intervention were consistently reported to exceed 2 yr of age. The results reported here indicate a trend toward earlier identification and hearing aid fitting with the implementation of newborn hearing screening. Although limited to literate and English speaking respondents, the study provides supporting evidence that newborn hearing screening lowers the ages of identification and intervention.