Direct Comparison of Alere i and cobas Liat Influenza A and B Tests for Rapid Detection of Influenza Virus Infection.

We compared two rapid, point-of care nucleic acid amplification tests for detection of influenza A and B viruses (Alere i [Alere] and cobas Liat [Roche Diagnostics]) with the influenza A and B virus test components of the FilmArray respiratory panel (BioFire Diagnostics) using 129 respiratory specimens collected in universal viral transport medium (80 influenza A virus and 16 influenza B virus positive) from both adult and pediatric patients. The sensitivities of the Alere test were 71.3% for influenza A virus and 93.3% for influenza B virus, with specificities of 100% for both viruses. The sensitivities and specificities of the Liat test were 100% for both influenza A and B viruses. The poor sensitivity of the Alere test for detection of influenza A virus was likely due to a study set that included many low-positive samples that were below its limit of detection.

Reduced susceptibility to all neuraminidase inhibitors of influenza H1N1 viruses with haemagglutinin mutations and mutations in non-conserved residues of the neuraminidase.

OBJECTIVES: We characterized human H1N1 influenza isolate A/Hokkaido/15/02, which has haemagglutinin and neuraminidase mutations that reduce drug susceptibility to oseltamivir, zanamivir and peramivir. METHODS: One wild-type and three mutant viruses were isolated by plaque purification. Viruses were tested in MUNANA-based enzyme assays, cell culture and receptor binding assays. RESULTS: Two viruses had a neuraminidase Y155H mutation that reduced susceptibility in the enzyme inhibition assay to all inhibitors by 30-fold to >100-fold. The Y155H mutation reduced plaque size and affected the stability, Km and pH activity profile of the enzyme. In contrast to previous mutants, this neuraminidase demonstrated a slower rate of inhibitor binding in the IC50 kinetics assay. One virus had both the Y155H mutation and a haemagglutinin D225G mutation that rescued the small-plaque phenotype of the Y155H virus and affected receptor binding and drug susceptibility in cell culture and binding assays. We also isolated a third mutant virus, with both neuraminidase V114I and haemagglutinin D225N mutations, which affected susceptibility in the enzyme inhibition assay and receptor binding, respectively, but to lesser extents than the Y155H and D225G mutations. CONCLUSIONS: Neither Y155 nor V114 is conserved across neuraminidase subtypes. Furthermore, Y155 is not conserved even among avian and swine N1 viruses. Structurally, both residues reside far from the neuraminidase active site. D225 forms part of the receptor binding site of the haemagglutinin. We believe this is the first demonstration of a specific haemagglutinin mutation correlating with reduced drug susceptibility in plaque assays in both Madin Darby Canine Kidney and SIAT cells.

In vitro passaging of a pandemic H1N1/09 virus selects for viruses with neuraminidase mutations conferring high-level resistance to oseltamivir and peramivir, but not to zanamivir.

OBJECTIVES: Pandemic H1N1/09 viruses with the neuraminidase H274Y mutation have emerged in untreated patients or following oseltamivir therapy or prophylaxis. There have been no reports yet of zanamivir-resistant H1N1/09 viruses in previously healthy patients. We wanted to determine whether we could select for neuraminidase mutations conferring high-level resistance to zanamivir by in vitro passage of the virus. We also wanted to investigate if passaging in a combination of zanamivir and oseltamivir could prevent the emergence of the H274Y mutation. METHODS: An H1N1/09 virus was passaged in cell culture in increasing concentrations of either zanamivir or a combination of zanamivir and oseltamivir. RESULTS: Passage in zanamivir selected a virus with N146S neuraminidase and G158E haemagglutinin mutations. The neuraminidase mutation only reduced drug susceptibility by 2-fold in enzyme assays. The haemagglutinin mutation conferred drug dependence and drug resistance in cells to oseltamivir and zanamivir and reduced binding to red blood cells. After four passages in zanamivir and oseltamivir, virus with a D198G neuraminidase mutation was selected with around 10-fold reduced susceptibility to oseltamivir, zanamivir and peramivir in the enzyme assay. Further passaging selected a virus with both D198G and H274Y mutations that was highly resistant to oseltamivir and peramivir, but not zanamivir. All mutations affected growth in cell culture and decreased affinities of the neuraminidases for substrate. CONCLUSIONS: We did not select a virus with a neuraminidase mutation conferring high-level resistance to zanamivir. Dual exposure to zanamivir and oseltamivir was not sufficient to prevent selection of the H274Y mutation.

A state-wide partnership to promote safe and supportive schools: the PBIS Maryland Initiative.

Schools continue to be an important context for preventive interventions targeting a range of behavioral and mental health problems. Yet competing demands on teachers and shifting priorities in response to federal legislation have posed some unique challenges to prevention researchers working in school settings. This paper summarizes an approach to prevention partnerships developed over a decade and centered on the three-tiered Positive Behavioral Interventions and Supports (PBIS) model. A state-wide initiative was formed and led through a partnership between the Maryland State Department of Education, Sheppard Pratt Health System, and Johns Hopkins University, which focused on implementing evidence-based practices and conducting prevention research in Maryland public schools. Drawing on a community-based participatory research framework for developing research partnerships, we highlight the importance of forming and sustaining authentic relationships to support school-based prevention research and implementation of evidence-based programs. We also discuss how these relationships have been used to disseminate PBIS and rigorously test its effectiveness. We describe some lessons learned from the partnership and identify potential areas for future research on the prevention partnership model. We conclude with a discussion of the implications for both researchers and community partners engaged in translational research in school settings.

Crowder-directed interactions and conformational dynamics in multistimuli-responsive intrinsically disordered protein.

The consequences of crowding on the dynamic conformational ensembles of intrinsically disordered proteins (IDPs) remain unresolved because of their ultrafast motion. Here, we report crowder-induced interactions and conformational dynamics of a prototypical multistimuli-responsive IDP, Rec1-resilin. The effects of a range of crowders of varying sizes, forms, topologies, and concentrations were examined using spectroscopic, spectrofluorimetric, and contrast-matching small- and ultrasmall-angle neutron scattering investigation. To achieve sufficient neutron contrast against the crowders, deuterium-labeled Rec1-resilin was biosynthesized successfully. Moreover, the ab initio "shape reconstruction" approach was used to obtain three-dimensional models of the conformational assemblies. The IDP revealed crowder-specific systematic extension and compaction with the level of macromolecular crowding. Last, a robust extension-contraction model has been postulated to capture the fundamental phenomena governing the observed behavior of IDPs. The study provides insights and fresh perspectives for understanding the interactions and structural dynamics of IDPs in crowded states.

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes.

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.

Noninvasive index of cryorecovery and growth potential for human follicles in vitro.

Cryopreservation of oocytes and embryos is commonly used to preserve fertility. However, women undergoing cancer treatment may not have the time or may not be good candidates for these options. Ovarian cortical tissue cryopreservation and subsequent tissue transplant has been proven successful yet inefficient in preserving larger secondary follicles, and is not recommended as a fertility preservation option for women with certain cancers. We evaluated cryopreservation of individual follicles as an alternative option in rodents, nonhuman primates, and human primates. Under optimal conditions, cryopreserved mouse secondary follicles were able to reestablish granulosa cell-oocyte interactions, which are essential for subsequent follicle growth. Individual secondary follicles survived cryopreservation, were able to be cultured in a three-dimensional alginate hydrogel matrix to the antral stage, and the enclosed oocytes were competent for fertilization. Using a vital imaging technique (pol-scope) employed in many fertility centers, we were able to bioassay the thawed, cultured follicles for the presence of transzonal connections between the somatic and germ cells. Perturbations in these linkages were shown to be reversed when follicles were cryopreserved under optimal freezing conditions. We applied the optimized cryopreservation protocol to isolated rhesus monkey and human secondary follicles, and using the birefringent bioassay, we were able to show good correlation between early follicle growth and healthy somatic cell-oocyte connections. Our results suggest that ovarian follicles can be cryopreserved, thawed, and analyzed noninvasively, making follicle preservation an additional option for young cancer patients.

Cumulus-oocyte complexes from small antral follicles during the early follicular phase of menstrual cycles in rhesus monkeys yield oocytes that reinitiate meiosis and fertilize in vitro.

The stage at which follicle-enclosed cumulus-oocyte complexes achieve developmental competence in primates is unknown. Therefore, studies were designed to characterize the ability of oocytes in small antral follicles present during the menstrual cycle to spontaneously resume meiosis, fertilize, and support early embryo development. Ovaries were removed from adult rhesus monkeys (n = 12) during the early follicular phase (Days 3-4) of spontaneous cycles. Small antral follicles were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5-0.99 mm; group III: 1.0-1.49 mm; group IV: 1.5-1.99 mm; and group V: 2.0-2.5 mm. The cumulus-oocyte complex from healthy small antral follicles (devoid of dark oocytes or granulosa cells) were extracted (n = 199) and cultured for 48 h under different conditions: in TALP (tyrode, albumin, lactate, pyruvate) medium alone, SAGE medium alone, or plus gonadotropins. At 48 h, oocyte meiotic status and diameter were measured after treatment of cumulus-oocyte complexes with hyaluronidase. Cumulus-oocyte complexes derived from follicles of 0.5- to 2-mm diameter contain oocytes that typically reinitiate meiosis in the absence or presence of gonadotropins and fertilize via in vitro fertilization or intracytoplasmic sperm injection. Moreover, the inseminated oocytes can reach the morula stage but arrest. Thus, the ability of these oocytes to complete maturation, as monitored from subsequent embryonic development after fertilization, is suboptimal. Further studies on primate IVM of oocytes from SAFs are warranted in order for them to be considered as an additional, novel source of gametes for fertility preservation in cancer patients.

Cumulus cell contact during oocyte maturation in mice regulates meiotic spindle positioning and enhances developmental competence.

PURPOSE: To investigate the role of cumulus cell contact during oocyte maturation on meiotic spindle assembly and the acquisition of developmental competence. METHODS: Cumulus oocyte complexes isolated from mouse ovaries subjected to in vitro or in vivo maturation were analyzed by confocal microscopy with respect to oocyte somatic cell contacts and for their ability to develop after parthenogenic activation during embryo culture. RESULTS: Cell contact is maintained during maturation in vivo, predisposing oocytes to cortical meiotic spindle assembly and developmental competence acquisition. In contrast, oocytes matured in vitro lose cell contact coincident with central meiotic spindle assembly that results in cleavage delays upon egg activation and failure to form blastocysts. Experimental disruption of cell contact by the actin-depolymerizing agent latrunculin B results in the formation of enlarged meiotic spindles with dispersed chromosomes unlike the compact ordering of chromosomes observed on spindles formed after in vivo maturation, suggesting a link between cell contact and the acquisition of developmental competence. CONCLUSIONS: Somatic cell contact optimizes oocyte quality during meiotic maturation by regulating the spatial organization and function of the meiotic spindle through actin-dependent mechanisms that enhance development.

In vitro grown human ovarian follicles from cancer patients support oocyte growth.

BACKGROUND: Young female adult and adolescent cancer patients facing life-preserving but fertility-threatening chemo- or radiation-therapy are increasingly seeking options to protect their reproductive potential. Ovarian tissue cryopreservation with transplantation is a promising technique to safeguard fertility in cancer patients. However, this method may risk re-introduction of the original cancer to the survivor of the disease. Thus, developing a method for in vitro growth of immature follicles may provide a method for fertility restoration in the future. METHODS: Human secondary follicles were isolated from ovarian tissues obtained from cancer patients and grown in vitro within a bio-engineered culture system for 30 days. RESULTS: Human ovarian follicles became steroidogenically active, and developed from the early secondary to antral stage in vitro. The follicles contained healthy, growing oocytes that were connected by transzonal projections between the somatic cells and oocyte. CONCLUSIONS: Our data support the notion that human follicle development can be achieved in vitro in a bio-engineered culture system. More studies are required to investigate whether the fully sized oocytes obtained from in vitro grown follicle are competent to resume meiosis and be fertilized.

Passaging of an influenza A(H1N1)pdm09 virus in a difluoro sialic acid inhibitor selects for a novel, but unfit I106M neuraminidase mutant.

An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its similarity to the natural substrate, sialic acid, we predicted resistant variants would be difficult to select. Yields of both viruses decreased with passaging, so that after 12 passages both viruses were only growing to low titers. Drug concentrations were decreased for another three passages. There was no difference in NA sensitivity in the MUNANA fluorescence-based assay, nor in plaque assays for the passaged virus stocks. All influenza B plaques were still wild type in all assays. There were isolated small diffuse plaques in the P15 pdm09 stock, which after purification had barely detectable NA or hemagglutinin (HA) activity. These had a novel non-active site I106M substitution in the NA gene, but unexpectedly no HA changes. The I106M may impact NA function through steric effects on the movement of the 150 and 430-loops. The I106M viruses had similar replication kinetics in MDCK cells as wild type viruses, but their ability to bind to and infect CHO-K1 cells expressing high levels of cell-bound mucin was compromised. The I106M substitution was unstable, with progeny rapidly reverting to wild type by three different mechanisms. Some had reverted to I106, some had V106, both with wild type NA and HA properties. A third group retained the I106M, but had a compensating R363K substitution, which regained almost wild type NA properties. These viruses now agglutinated chicken red blood cells (CRBCs) but unlike the I/V106, they rebound after elution at 37 °C. There were no mutations in the HA, but each phenotype correlated with the NA sequence. We propose that the activity in the I106M mutant is insufficient to remove carbohydrates from the virion HA and NA, sterically limiting HA access to CRBC receptors, thus resulting in poor HA binding.

Structural and Functional Analysis of Anti-Influenza Activity of 4-, 7-, 8- and 9-Deoxygenated 2,3-Difluoro- N-acetylneuraminic Acid Derivatives.

Competitive inhibitors of the influenza neuraminidase (NA) were discovered almost 20 years ago, with zanamivir and oseltamivir licensed globally. These compounds are based on a transition state analogue of the sialic acid substrate. We recently showed that 5- N-(acetylamino)-2,3,5-trideoxy-2,3-difluoro-d-erythro-ß-l-manno-2-nonulopyranosonic acid (DFSA) and its derivatives are also potent inhibitors of the influenza NA. They are mechanism based inhibitors, forming a covalent bond between the C2 of the sugar ring and Y406 in the NA active site, thus inactivating the enzyme. We have now synthesized a series of deoxygenated DFSA derivatives in order to understand the contribution of each hydroxyl in DFSA to binding and inhibition of the influenza NA. We have investigated their relative efficacy in enzyme assays in vitro, in cell culture, and by X-ray crystallography. We found loss of the 8- and 9-OH had the biggest impact on the affinity of binding and antiviral potency.

Identification of Indonesian clade 2.1 highly pathogenic influenza A(H5N1) viruses with N294S and S246N neuraminidase substitutions which further reduce oseltamivir susceptibility.

We have tested the in vitro susceptibility to the neuraminidase (NA) inhibitors of 96 highly pathogenic clade 2.1 A(H5N1) viruses from Indonesia, isolated between 2008 and 2011. HPAI virus samples obtained through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia were tested for susceptibility to oseltamivir and zanamivir. The NAs of four viruses were identified as extreme outliers to oseltamivir, based on statistical analysis by box plots, with IC50 values ranging from 46 to 62 nM. The NAs of two of these viruses from Sumatra and Aceh, had an N294S substitution, while one virus from Sulawesi had an S246N NA substitution. The NAs of all four viruses showed a specific loss of slow binding to oseltamivir in an IC50 kinetics assay. As observed in our previous surveillance, there was only a minimal effect on the sensitivity to zanamivir or peramivir for these mutants or any of the other isolates tested. The continued circulation of subtype H5N1 viruses in avian species poses an on-going zoonotic threat. The fact that we continue to identify avian isolates with naturally occurring mutations conferring reduced oseltamivir susceptibility remains a concern, given oseltamivir will be a key antiviral in the event of a new pandemic emerging.

Teaching the medical interview: methods and key learning issues in a faculty development course.

OBJECTIVE: To describe the American Academy on Communication in Healthcare's (AACH) Faculty Development Course on Teaching the Medical Interview and report a single year's outcomes. DESIGN: We delivered a Faculty Development course on Teaching the Medical Interview whose theme was relationship-centered care to a national and international audience in 1999. Participants completed a retrospective pre-post assessment of their perceived confidence in performing interview, clinical, teaching, and self-awareness skills. PARTICIPANTS AND SETTING: A total of 79 participants in the 17th annual AACH national faculty development course at the University of Massachusetts Medical School in June 1999. INTERVENTION: A 5-day course utilized the principles of learner-centered learning to teach a national and international cohort of medical school faculty about teaching the medical interview. MEASUREMENTS AND MAIN RESULTS: The course fostered individualized, self-directed learning for participants, under the guidance of AACH faculty. Teaching methods included a plenary session, small groups, workshops, and project groups all designed to aid in the achievement of individual learning goals. Course outcomes of retrospective self-assessed confidence in interview, clinical, teaching, self-awareness, and control variables were measured using a 7-point Likert scale. Participants reported improved confidence in interview, clinical, teaching, and self-awareness variables. After controlling for desirability bias as measured by control variables, only teaching and self-awareness mean change scores were statistically significant (p < .001). CONCLUSIONS: The AACH Faculty Development course on Teaching the Medical Interview utilized learner-centered teaching methods important to insure learning with experienced course participants. Perceived teaching and self-awareness skills changed the most when compared to other skills.

Periacetabular osteotomy in the treatment of severe acetabular dysplasia. Surgical technique.

BACKGROUND: The optimal treatment of severe acetabular dysplasia with subluxation of the femoral head or the presence of a secondary acetabulum remains controversial. The purpose of this study was to analyze the extent of surgical correction and the early clinical results obtained with the Bernese periacetabular osteotomy for the treatment of severely dysplastic hips in adolescent and young adult patients. METHODS: Sixteen hips in thirteen patients with an average age of 17.6 years (range, 13.0 to 31.8 years) were classified as having severe acetabular dysplasia (Group IV or V according to the Severin classification). Eight hips were classified as subluxated, and eight had a secondary acetabulum. Preoperatively, all patients had hip pain and sufficient hip joint congruency on radiographs to be considered candidates for the osteotomy. All sixteen hips underwent a Bernese periacetabular osteotomy, and six of them underwent a concomitant proximal femoral osteotomy. Postoperatively, the hips were assessed radiographically to evaluate correction of deformity, healing of the osteotomy site, and progression of osteoarthritis. Clinical results and hip function were measured with the Harris hip score at an average of 4.2 years postoperatively. RESULTS: Comparison of preoperative and follow-up radiographs demonstrated an average improvement of 44.6 degrees (from -20.5 degrees to 24.1 degrees) in the lateral center-edge angle of Wiberg, an average improvement of 51.0 degrees (from -25.4 degrees to 25.6 degrees) in the anterior center-edge angle of Lequesne and de Seze, and an average improvement of 25.9 degrees (from 37.3 degrees to 11.4 degrees) in acetabular roof obliquity. The hip center was translated medially an average of 10 mm (range, 0 to 31 mm). All iliac osteotomy sites healed. The average Harris hip score improved from 73.4 points preoperatively to 91.3 points at the time of the latest follow-up. Eleven of the thirteen patients (fourteen of the sixteen hips) were satisfied with the result of the surgery, and fourteen hips had a good or excellent clinical result. Major complications included loss of acetabular fixation, which required an additional surgical procedure, in one patient and overcorrection of the acetabulum and an associated ischial nonunion in another patient. Both patients had a good clinical result at the time of the latest follow-up. There were no major neurovascular injuries or intra-articular fractures. CONCLUSIONS: The periacetabular osteotomy is an effective technique for surgical correction of a severely dysplastic acetabulum in adolescents and young adults. In this series, the early clinical results were very good at an average of 4.2 years postoperatively; the two major complications did not compromise the good clinical results.

Retrospective evaluation of the use of glucagon infusion as adjunctive therapy for hypoglycemia in dogs: 9 cases (2005-2014).

OBJECTIVE: To describe the use of glucagon infusion for adjunctive treatment of hypoglycemia in dogs. DESIGN: Multicenter retrospective case series. SETTING: One university and 1 private veterinary referral hospital. ANIMALS: Dogs were included if they were hospitalized and received glucagon therapy for hypoglycemia, defined as blood glucose concentration (BG) <60 mg/dL. A total of 9 dogs were included from September 2005 to May 2014. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The medical record for each eligible case was reviewed. Data recorded included signalment, presenting complaint, underlying disease process, presenting BG, BG after dextrose supplementation, BG before glucagon administration, maximum BG while receiving glucagon, and BG after discontinuation of glucagon, if available. Adverse reactions to glucagon and outcome of case were recorded if available. The most common causative disease was insulinoma (n = 7). Median serum glucose concentration on presentation was 30 mg/dL (20-41 mg/dL). The median bolus of glucagon was 50 ng/kg followed by a median maximum dose of a glucagon CRI of 15 ng/kg/min. The mean time period on glucagon CRI until normoglycemia (defined as BG > 60 mg/dL) was 7 hours. All hypoglycemic patients had improvement of BGs when glucagon was added. Statistically significant differences (P < 0.05) were found between BG measurements on glucagon CRI compared to BG at presentation, BG after dextrose, and BG prior to glucagon with a Friedman statistic of 17.3. A CRI was found to effectively increase the BG without recurrence of hypoglycemia after weaning. The majority of patients (5/9) survived to discharge. CONCLUSION: Glucagon CRI was accompanied by an increase in BG in hypoglycemic dogs. Glucagon CRI appears to be a safe method and can be readily utilized in most practice settings.

Periacetabular osteotomy for the treatment of severe acetabular dysplasia.

BACKGROUND: The optimal treatment of severe acetabular dysplasia with subluxation of the femoral head or the presence of a secondary acetabulum remains controversial. The purpose of this study was to analyze the extent of surgical correction and the early clinical results obtained with the Bernese periacetabular osteotomy for the treatment of severely dysplastic hips in adolescent and young adult patients. METHODS: Sixteen hips in thirteen patients with an average age of 17.6 years (range, 13.0 to 31.8 years) were classified as having severe acetabular dysplasia (Group IV or V according to the Severin classification). Eight hips were classified as subluxated, and eight had a secondary acetabulum. Preoperatively, all patients had hip pain and sufficient hip joint congruency on radiographs to be considered candidates for the osteotomy. All sixteen hips underwent a Bernese periacetabular osteotomy, and six of them underwent a concomitant proximal femoral osteotomy. Postoperatively, the hips were assessed radiographically to evaluate correction of deformity, healing of the osteotomy site, and progression of osteoarthritis. Clinical results and hip function were measured with the Harris hip score at an average of 4.2 years postoperatively. RESULTS: Comparison of preoperative and follow-up radiographs demonstrated an average improvement of 44.6 degrees (from -20.5 degrees to 24.1 degrees ) in the lateral center-edge angle of Wiberg, an average improvement of 51.0 degrees (from -25.4 degrees to 25.6 degrees ) in the anterior center-edge angle of Lequesne and de Seze, and an average improvement of 25.9 degrees (from 37.3 degrees to 11.4 degrees ) in acetabular roof obliquity. The hip center was translated medially an average of 10 mm (range, 0 to 31 mm). All iliac osteotomy sites healed. The average Harris hip score improved from 73.4 points preoperatively to 91.3 points at the time of the latest follow-up. Eleven of the thirteen patients (fourteen of the sixteen hips) were satisfied with the result of the surgery, and fourteen hips had a good or excellent clinical result. Major complications included loss of acetabular fixation, which required an additional surgical procedure, in one patient and overcorrection of the acetabulum and an associated ischial nonunion in another patient. Both patients had a good clinical result at the time of the latest follow-up. There were no major neurovascular injuries or intra-articular fractures. CONCLUSIONS: The periacetabular osteotomy is an effective technique for surgical correction of a severely dysplastic acetabulum in adolescents and young adults. In this series, the early clinical results were very good at an average of 4.2 years postoperatively; the two major complications did not compromise the good clinical results.

Neuraminidase mutations conferring resistance to laninamivir lead to faster drug binding and dissociation.

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are administered twice daily for 5days for treatment of influenza. Laninamivir is a 7-methoxy derivative of zanamivir, but a single dose is effective when taken as the laninamivir octanoate prodrug. We show here in IC50 kinetics assays and a solid phase reactivation assay that compared to zanamivir laninamivir also demonstrates slow binding to but slower dissociation from multiple wild type NAs. A D197E mutation in an influenza B and an E119G in an N9 neuraminidase which confer 15- and 150-fold resistance to laninamivir result in faster binding and dissociation. Despite similar IC50s our assays demonstrate more rapid dissociation of laninamivir from clade 1 compared to 2 H5N1 NAs.

Catalytic mechanism and novel receptor binding sites of human parainfluenza virus type 3 hemagglutinin-neuraminidase (hPIV3 HN).

The human parainfluenza virus type 3 (hPIV3) hemagglutinin-neuraminidase (HN) has opposing functions of binding sialic acid receptors and cleaving them, facilitating virus release. The crystal structure of hPIV3 HN complexed with the substrate analogue difluorosialic acid (DFSA) revealed that catalysis by HN involves the formation of a covalently linked sialosyl-enzyme intermediate which was trapped along with a transition-state analogue resembling an oxocarbenium ion. This mechanism of enzyme catalysis was also confirmed in the crystal structure of the influenza N9 neuraminidase complexed with DFSA. Additionally, novel secondary receptor binding sites were identified in the hPIV3 HN-DFSA complex including one near the catalytic cavity which upon binding DFSA imposes subtle changes and may help the HN balance the opposing functions. Multiple receptor binding sites may increase avidity to facilitate cell binding and fusion promotion. The secondary receptor binding sites in the paramyxoviruses are so far unique to each virus type.