Exploring the feasibility of validating early developmental screening tools for American Indian and Alaska Native children.

Screening children from birth through age 5 is critical to early identification of challenges and referral to intervention to support optimal development. Screening of American Indian and Alaska Native (AIAN) children lags behind that of other children, partly due to the lack of screening tools validated for this population. This study tested the feasibility of an online data collection strategy for use in a future study of the validity of existing screening instruments for AIAN children. Parents of AIAN children in four communities were recruited to complete screeners, provide demographic information, and provide feedback on experiences with online data collection. Participants were given the option of receiving screening results from the local early childhood program through which they were recruited. 240 participants began the process, 183 were enrolled in a partner program and reported a birthdate for at least one AIAN child, 157 had an age-eligible child, 81 began the consent process, 62 consented, and 39 fully completed data collection. Most participants were female and AIAN, the majority reported that online data collection was easy. Collecting screener validation data on a large sample of AIAN children may be able to utilize online data collection tools, with in-person support to facilitate participation.

Un examen de detección en los niños a partir del nacimiento hasta la edad de 5 años es esencial para la temprana identificación de retos y la referencia a intervenciones como apoyo a un desarrollo óptimo. El examen de detección en el caso de niños del grupo Indio Americano y Nativo de Alaska (AIAN) está muy por debajo del de otros niños, en parte debido a la falta de herramientas de detección validadas para esta población. Este estudio puso a prueba la posibilidad de una estrategia electrónica de recolección de datos para uso en un estudio futuro acerca de la validez de los existentes instrumentos de detección para niños AIAN. Se reclutaron progenitores de niños AIAN en cuatro comunidades para completar los exámenes de detección, proveer información demográfica, así como proveer información sobre las experiencias con la recolección electrónica de datos. A los participantes se les dio la opción de recibir los resultados de la detección de parte del programa local para la temprana niñez a través del cual habían sido reclutados. 240 participantes comenzaron el proceso; 183 estaban matriculados en un programa paralelo y reportaron la fecha de nacimiento de por lo menos un niño AIAN; 157 tenían un niño elegible según la edad; 81 comenzaron el proceso de consentimiento; 62 consintieron; 39 completaron en su totalidad la recolección de datos. La mayoría de los participantes eran mujeres y AIAN; la mayoría reportó que la recolección electrónica de datos fue fácil. La recolección de información de validación de la detección en un grupo muestra grande de niños AIAN pudiera ser capaz de utilizar herramientas electrónicas de recolección de datos, con un apoyo presencial para facilitar la participación.

Le dépistage des enfants de la naissance à l'âge de 5 ans est critique pour l'identification précoce des défis et problèmes et l'orientation vers l'intervention afin de soutenir le développement optimal. Le dépistage des enfants d'amérindiens des Etats-Unis et des autochtones d'Alaska est en retard par rapport à celui des autres enfants, en partie du fait du manque d'outils de dépistage validés pour cette population. Cette étude a testé la fiabilité de la stratégie de collecte de données en ligne pour son utilisation pour une étude à venir sur la validité d'instruments de dépistage existants pour les enfants AIAN. Les parents d'enfants AIAN de quatre communautés ont été recrutés afin de remplir des dépistages, d'offrir des renseignements démographiques, et d'offrir des commentaires sur les expériences de collecte de données en ligne. Les participants ont reçu l'option de recevoir les résultats de dépistage d'un programme de petite enfance local au travers duquel ils avaient été recrutés. 240 participants ont commencé le processus. 183 ont été inscrits dans un programme partenaire et ont fait état de la date de naissance d'au moins un enfant AIAN. 157 avait un enfant admissible par l'âge. 81 ont commencé le processus de consentement. 62 ont consenti. 39 ont fini la collecte de données en ligne. La collecte de données de validation du filtre de recherche sur un grand échantillon d'enfants AIAN pourrait utiliser des outils de collecte de données en ligne avec un soutien en personne afin de faciliter la participation.

Nerve and Tendon Transfer Surgery in Cervical Spinal Cord Injury: Individualized Choices to Optimize Function.

Background: Recent adaption of nerve transfer surgery to improve upper extremity function in cervical spinal cord injury (SCI) is an exciting development. Tendon transfer procedures are well established, reliable, and can significantly improve function. Despite this, few eligible surgical candidates in the United States undergo these restorative surgeries. Evidence Acquisition: The literature on these procedures was reviewed. Results: Options to improve function include surgery to restore elbow extension, wrist extension, and hand opening and closing function. Tendon transfers are reliable and well tolerated but require weeks of immobilization and limits on extremity use. The role of nerve transfers is still being established. Early results indicate variable return of meaningful function with less immobilization but longer periods (up to years) required to gain appreciable function. Conclusion: Nerve and tendon transfer surgery sacrifice an expendable donor to restore a missing and more critical function. These procedures are well described in hand surgery; are reliable, well tolerated, and covered by insurance; and should be part of the SCI rehabilitation discussion.

Non-invasive UPR monitoring system and its applications in CHO production cultures.

Unfolded protein response (UPR) is the primary signaling network activated in response to the accumulation of unfolded and/or misfolded protein in the endoplasmic reticulum (ER). The expression of high levels of recombinant proteins in mammalian cell cultures has been linked to the increased UPR. However, the dynamics of different UPR-mediated events and their impact on cell performance and recombinant protein secretion during production remain poorly defined. Here, we have created a non-invasive UPR-responsive, fluorescence-based reporter system to detect and quantify specific UPR-mediated transcriptional activation of different intracellular signaling pathways. We have generated stable antibody-expressing CHO clones containing this UPR responsive system and established FACS-based methods for real-time, continuous monitoring of the endogenous UPR activation in live cultures. The results showed that the UPR activation is dynamically regulated during production culture. The clones differed in their UPR patterns; both the timing and the degree of UPR-induced transcriptional activation were linked to cell performance, such as growth, and viability. In addition, the cell culture environment, such as media composition and osmolarity, significantly impacted endogenous UPR activation. Taken together, these data demonstrate a utility of this UPR monitoring system in recombinant protein production processes and the observations increase our understanding of the critical role of UPR in regulating diverse phenotypes of the cells including growth, survival and recombinant protein secretion under different culture environments and processing conditions.

PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosa isolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance, P. aeruginosa strains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-function phoQ alleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations in phoP were identified in some cystic fibrosis strains with resistance-conferring phoQ mutations, suggesting that additional loci can be involved in polymyxin resistance in P. aeruginosa. Disruption of chromosomal phoQ in the presence of an intact phoP allele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of the pmrH (arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate that phoQ loss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

A high-throughput UPLC method for the characterization of chemical modifications in monoclonal antibody molecules.

Development of high-throughput release and characterization assays is critical for the effective support of the rapidly growing biologics pipeline for biotherapeutics. Clipping of polypeptide chains is commonly monitored during process optimization, formulation development, and stability studies. A reduced capillary electrophoresis-sodium dodecyl sulfate (rCE -SDS) method is often used as a purity release assay for monitoring clips in monoclonal antibodies (mAbs); however, it has a cycle time of approximately 40 min, which is not suited for high-throughput screening. Additionally, the characterization of clips and variants from electropherograms is not straightforward and takes significant time. Reduced reversed-phase (RP) chromatography has been a popular assay for the characterization and identification of clips and variants because it can be directly coupled with online mass spectrometric analysis. However, the high-column temperature and low pH required for RP assays can induce on-column cleavage and therefore skew the results. To minimize on-column degradation, we have developed a high-throughput method with a significantly shorter cycle time of 5 min. The short cycle time was achieved using an ultra-high-pressure liquid chromatography (UPLC) system with a 1.7 µm phenyl column. This UPLC method allowed quantitation of hinge clipping in an IgG1 molecule and acid induced aspartic acid/proline (D/P) clip in an IgG2 molecule. The results from the UPLC method were comparable to those obtained with rCE-SDS. Additionally, the phenyl column offered partial resolution of oxidation and other chemical modifications, making this technique an attractive assay for high-throughput process characterization and formulation screens.

Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody.

A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency.

Colistin susceptibility testing: evaluation of reliability for cystic fibrosis isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia.

OBJECTIVES: Antibiotic susceptibility methods that are commonly used to test bacterial isolates from patients with cystic fibrosis are of uncertain reliability for the polymyxins. To assess the reliability of four standard testing methods, this pilot study used a challenge set that included polymyxin-resistant isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia. METHODS: Twenty-five P. aeruginosa and 12 S. maltophilia isolates were tested for susceptibility to colistin (polymyxin E). Repeatability (concordance of replicates performed concurrently), reproducibility (concordance of replicates performed over time) and comparability (concordance of different methods) of agar dilution, broth microdilution, Etest and disc diffusion were assessed through the use of descriptive statistics and scatterplot analyses. RESULTS: All four methods displayed excellent repeatability (overall concordance rate of 99%). However, analysis of reproducibility revealed substantially lower rates of concordance (74% for agar dilution, 84% for broth microdilution and Etest, and 91% for disc diffusion). In addition, comparability to agar dilution of the three other methods was generally poor, with overall rates of very major error ranging from 12% for broth microdilution to 18% for Etest and disc diffusion. CONCLUSIONS: Compared with agar dilution, other susceptibility testing methods give high rates of apparent false polymyxin susceptibility for cystic fibrosis isolates of P. aeruginosa and S. maltophilia. Prospective study of the correlation between in vitro susceptibility and clinical response is needed to clarify whether these discrepancies reflect oversensitivity of the agar dilution method or insensitivity of the other methods.

Substantial formation of hydrates and hemiacetals from pyridinium ketones.

Pyridinium ketones have been found to exist as hydrates and hemiacetals in considerable amount in aqueous and alcoholic solutions, respectively. The relative position of the pyridinium positive charge has a large effect on the equilibrium constants. The polar substituent constants, sigma*, of the pyridinium group substituted at different positions can be estimated from the hydration constants.

Excellent correlation between substituent constants and pyridinium N-methyl chemical shifts.

Substituents on the pyridinium ring of N-methylpyridinium derivatives, especially those on the 2- or 4-positions, have a large effect on the (1)H and (13)C NMR chemical shifts of the N-methyl group. Reasonable correlations between the chemical shift changes and the resonance substituent constants are observed. The dual substituent parameter approach provides an excellent correlation when a combination of polar and resonance substituent constants is employed.