Spray-Dried and Spray-Freeze-Dried Powder Formulations of an Anti-Interleukin-4Rα Antibody for Pulmonary Delivery.

OBJECTIVE: The therapeutic options for severe asthma are limited, and the biological therapies are all parenterally administered. The purpose of this study was to formulate a monoclonal antibody that targets the receptor for IL-4, an interleukin implicated in the pathogenesis of severe asthma, into a dry powder intended for delivery via inhalation. METHODS: Dehydration was achieved using either spray drying or spray freeze drying, which exposes the thermolabile biomacromolecules to stresses such as shear and adverse temperatures. 2-hydroxypropyl-beta-cyclodextrin was incorporated into the formulation as protein stabiliser and aerosol performance enhancer. The powder formulations were characterised in terms of physical and aerodynamic properties, while the antibody was assessed with regard to its structural stability, antigen-binding ability, and in vitro biological activity after drying. RESULTS: The spray-freeze-dried formulations exhibited satisfactory aerosol performance, with emitted fraction exceeding 80% and fine particle fraction of around 50%. The aerosolisation of the spray-dried powders was hindered possibly by high residual moisture. Nevertheless, the antigen-binding ability and inhibitory potency were unaffected for the antibody in the selected spray-dried and spray-freeze-dried formulations, and the antibody was physically stable even after one-year storage at ambient conditions. CONCLUSIONS: The findings of this study establish the feasibility of developing an inhaled dry powder formulation of an anti-IL-4R antibody using spray drying and spray freeze drying techniques with potential for the treatment of severe asthma.

Theory-driven classification of reading difficulties from fMRI data using Bayesian latent-mixture models.

Decades of research have led to several competing theories regarding the neural contributors to impaired reading. But how can we know which theory (or theories) identifies the types of markers that indeed differentiate between individuals with reading disabilities (RD) and their typically developing (TD) peers? To answer this question, we propose a new analytical tool for theory evaluation and comparison, grounded in the Bayesian latent-mixture modeling framework. We start by constructing a series of latent-mixture classification models, each reflecting one existing theoretical claim regarding the neurofunctional markers of RD (highlighting network-level differences in either mean activation, inter-subject heterogeneity, inter-region variability, or connectivity). Then, we run each model on fMRI data alone (i.e., while models are blind to participants' behavioral status), which enables us to interpret the fit between a model's classification of participants and their behavioral (known) RD/TD status as an estimate of its explanatory power. Results from n=127 adolescents and young adults (RD: n=59; TD: n=68) show that models based on network-level differences in mean activation and heterogeneity failed to differentiate between TD and RD individuals. In contrast, classifications based on variability and connectivity were significantly associated with participants' behavioral status. These findings suggest that differences in inter-region variability and connectivity may be better network-level markers of RD than mean activation or heterogeneity (at least in some populations and tasks). More broadly, the results demonstrate the promise of latent-mixture modeling as a theory-driven tool for evaluating different theoretical claims regarding neural contributors to language disorders and other cognitive traits.

Optimization of PEGylated KL4 Peptide for siRNA Delivery with Improved Pulmonary Tolerance.

Pulmonary delivery of small interfering RNA (siRNA) is a promising therapeutic strategy for treating various respiratory diseases but an effective carrier for the delivery of siRNA into the cells of the lungs and a robust gene-silencing effect is still lacking. Previously, we reported that the KL4 peptide, a synthetic cationic peptide with a repeating KLLLL sequence, can mediate effective siRNA transfection in lung epithelial cells but its high hydrophobic leucine content, and hence poor water solubility, limits its application as a delivery vector. Here, we show that the covalent attachment of monodisperse poly(ethylene glycol) (PEG) improves the solubility of KL4 and the uptake of its complex with siRNA into lung epithelial cells, such that very robust silencing is produced. All PEGylated KL4 peptides, with PEG length varying between 6 and 24 monomers, could bind and form nanosized complexes with siRNA, but the interaction between siRNA and peptides became weaker as the PEG chain length increased. All PEGylated KL4 peptides exhibited satisfactory siRNA transfection efficiency on three human lung epithelial cell lines, including A549 cells, Calu-3 cells, and BEAS-2B cells. The PEG12KL4 peptide, which contains 12 monomers of PEG, was optimal for siRNA delivery and also demonstrated a low risk of inflammatory response and toxicity in vivo following pulmonary administration.

Electroencephalography decoding of Chinese characters in primary school children and its prediction for word reading performance and development.

Research on what neural mechanisms facilitate word reading development in non-alphabetic scripts is relatively rare. The present study was among the first to adopt a multivariate pattern classification analysis to decode electroencephalographic signals recorded for primary school children (N = 236) while performing a Chinese character decision task. Chinese is an ideal script for studying the relationship between neural discriminability (i.e., decodability) of the orthography and behavioral word reading skills since the mapping from orthography to phonology is relatively arbitrary in Chinese. This was also among the first empirical attempts to examine the extent to which decoding performance can predict current and subsequent word reading skills using a longitudinal design. Results showed that neural activation patterns of real characters can be distinguished from activation patterns for pseudo-characters, non-characters, and random stroke combinations in both younger and older children. Topography of the transformed classifier weights revealed two distinct cognitive sub-processes underlying single character recognition, but temporal generalization analysis suggested common neural mechanisms between the distinct cognitive sub-processes. Suggestive evidence from correlational and hierarchical regression analyses showed that decoding performance, assessed on average 2 months before the year 2 behavioral testing, predicted both year 1 word reading performance and the development of word reading fluency over the year. Results demonstrate that decoding performance, one indicator of how the neural system is functionally organized in processing characters and character-like stimuli, can serve as a useful neural marker in predicting current word reading skills and the capacity to learn to read.

Event-related potentials during Chinese single-character and two-character word reading in children.

Word reading involves a series of cognitive processes, from lower-level visual processing to word semantic retrieval. To investigate the timing of the underlying neurocognitive processes in reading, the current study examined the electrophysiological correlates of cognitive functions associated with traditional Chinese word reading in Hong Kong children (Age: M = 8.19, SD = 0.82 years old) using event-related potentials (ERPs). A single-character lexical decision task and a two-character word lexical decision task were used to explore different levels of Chinese word processing. One-hundred ten children's data were analyzed. Results revealed that radical level processing was related to a P1 component at as early as 130 ms. Character-level processing was related to an N1 component from 218 ms onwards. Two-character word semantic processing was related to an N400-like component at as early as 450 ms. The results show a similar time course of visual word processing as previously reported in alphabetic languages, consistent with increasing specialization in the ventral visual stream for increasing wordlikeness leading to processing of linguistic aspects in higher-level language regions.

A Three-Year Longitudinal Study of Reading and Spelling Difficulty in Chinese Developmental Dyslexia: The Matter of Morphological Awareness.

In the present study, we used a three-time point longitudinal design to investigate the associations of morphological awareness to word reading and spelling in a small group of those with and without dyslexia taken from a larger sample of 164 Hong Kong Chinese children who remained in a longitudinal study across ages 6, 7 and 8. Among those 164 children, 15 had been diagnosed as having dyslexia by professional psychologists, and 15 other children manifested average reading ability and had been randomly selected from the sample for comparison. All children were administered a battery of tasks including Chinese character recognition, word dictation, morphological awareness, phonological awareness and rapid automatized naming. Multivariate analysis of variance and predictive discriminate analysis were performed to examine whether the dyslexic children showed differences in the cognitive-linguistic tasks in comparison with controls. Results suggested that the dyslexic groups had poorer performance in morphological awareness and RAN across all 3 years. However, phonological awareness was not stable in distinguishing the groups. Findings suggest that morphological awareness is a relatively strong correlate of spelling difficulties in Chinese, but phonological awareness is not. Copyright © 2017 John Wiley & Sons, Ltd.

Predicting the naming of regular-, irregular- and non-phonetic compounds in Chinese.

This study investigated the contributions of different cognitive measures in predicting the three types of Chinese characters: regular phonetic compounds, irregular phonetic compounds and non-phonetic compounds. A total of 246 Grade 3 children (mean age = 8.63 yrs) were tested using a bunch of tasks including phonological processing, orthographic processing, morphological processing, rapid automatised naming and Chinese character naming. Results showed that advanced phonological processing skills contributed largely to regular phonetic compounds naming while other cognitive measures predict the naming of the other two types of characters. The results imply that the skills underlying the learning of regular phonetic compounds are different from that of irregular phonetic compounds and non-phonetic compounds, which are similar. Data from three poor readers whose performances pattern on naming different types of Chinese characters agreed with the pattern predicted by their performances in different cognitive tasks provide further support to the claim. Theoretical and pedagogical implications were discussed.

Global chemical composition of ambient fine particulate matter for exposure assessment.

Epidemiologic and health impact studies are inhibited by the paucity of global, long-term measurements of the chemical composition of fine particulate matter. We inferred PM2.5 chemical composition at 0.1° × 0.1° spatial resolution for 2004-2008 by combining aerosol optical depth retrieved from the MODIS and MISR satellite instruments, with coincident profile and composition information from the GEOS-Chem global chemical transport model. Evaluation of the satellite-model PM2.5 composition data set with North American in situ measurements indicated significant spatial agreement for secondary inorganic aerosol, particulate organic mass, black carbon, mineral dust, and sea salt. We found that global population-weighted PM2.5 concentrations were dominated by particulate organic mass (11.9 ± 7.3 µg/m(3)), secondary inorganic aerosol (11.1 ± 5.0 µg/m(3)), and mineral dust (11.1 ± 7.9 µg/m(3)). Secondary inorganic PM2.5 concentrations exceeded 30 µg/m(3) over East China. Sensitivity simulations suggested that population-weighted ambient PM2.5 from biofuel burning (11 µg/m(3)) could be almost as large as from fossil fuel combustion sources (17 µg/m(3)). These estimates offer information about global population exposure to the chemical components and sources of PM2.5.

Neural specialization to English words in Chinese children: Joint contribution of age and English reading abilities.

N1 tuning to words, a neural marker of visual word recognition, develops by an interaction between age and ability. The development of N1 tuning to a second learnt print is unclear. The present study examined the joint contribution of age and English reading abilities to N1 amplitude and tuning to English print in Chinese children in Hong Kong. EEG signals were recorded from 179 children (six to nine years old) while they were performing a repetition detection task comprised of different print stimuli measuring three types of tuning, i.e., coarse tuning (real word versus false font), fine tuning (real versus nonword), and lexicality effect (real versus pseudo word). Children were assessed in English word reading accuracy (EWR) and English sub-lexical orthographic knowledge (EOK). Results indicated that coarse tuning decreased with age but increased with EWR and EOK. Fine tuning uniquely increased with EOK, and the lexicality effect increased with EWR. At last, higher EWR was linked to less right-lateralized coarse tuning in younger children. Taken together, the findings support the visual perceptual expertise account in the L2 context, in that N1 coarse tuning, fine tuning, and lexicality effect are driven by skill improvement.

A data-driven computational model for engineered cardiac microtissues.

Engineered heart tissues (EHTs) present a potential solution to some of the current challenges in the treatment of heart disease; however, the development of mature, adult-like cardiac tissues remains elusive. Mechanical stimuli have been observed to improve whole-tissue function and cardiomyocyte (CM) maturation, although our ability to fully utilize these mechanisms is hampered, in part, by our incomplete understanding of the mechanobiology of EHTs. In this work, we leverage experimental data, produced by a mechanically tunable experimental setup, to introduce a tissue-specific computational modeling pipeline of EHTs. Our new modeling pipeline generates simulated, image-based EHTs, capturing ECM and myofibrillar structure as well as functional parameters estimated directly from experimental data. This approach enables the unique estimation of EHT function by data-based estimation of CM active stresses. We use this experimental and modeling pipeline to study different mechanical environments, where we contrast the force output of the tissue with the computed active stress of CMs. We show that the significant differences in measured experimental forces can largely be explained by the levels of myofibril formation achieved by the CMs in the distinct mechanical environments, with active stress showing more muted variations across conditions. The presented model also enables us to dissect the relative contributions of myofibrils and extracellular matrix to tissue force output, a task difficult to address experimentally. These results highlight the importance of tissue-specific modeling to augment EHT experiments, providing deeper insights into the mechanobiology driving EHT function. STATEMENT OF SIGNIFICANCE: Engineered heart tissues (EHTs) have the potential to revolutionize the way heart disease is treated. However, developing mature cardiomyocytes (CM) in these tissues remains a challenge due, in part, to our incomplete understanding of the fundamental biomechanical mechanisms that drive EHT development. This work integrates the experimental data of an EHT platform developed to study the influence of mechanics in CM maturation with computational biomechanical models. This approach is used to augment conclusions obtained in-vitro - by measuring quantities such as cell stress and strain - and to dissect the relevance of each component in the whole tissue performance. Our results show how a combination of specialized in-silico and in-vitro approaches can help us better understand the mechanobiology of EHTs.

Matrix architecture and mechanics regulate myofibril organization, costamere assembly, and contractility of engineered myocardial microtissues.

The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, we established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM-mimetic synthetic, fiber matrices and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), enabling real-time contractility readouts, in-depth structural assessment, and tissue-specific computational modeling. We find that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC-CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell-ECM interactions in myofibril assembly and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC-CM tissue models with controllable architecture and mechanics can inform the design of translatable regenerative cardiac therapies.

Quantifying the regularities between orthography and semantics and their impact on group- and individual-level behavior.

Statistical views of reading highlight the link between proficient literacy and the assimilation of various regularities embedded in writing systems, including those in the mapping between print and meaning. Still, orthographic-semantic (O-S) regularities remain relatively understudied, with open questions regarding 3 issues: (a) how O-S regularities should be quantified, (b) how they impact the behavior of proficient readers, and (c) whether individual differences in sensitivity to these regularities predict reading skills. The goal of the current article is to address these questions. We start by reviewing previous studies estimating print-meaning regularities, where orthography-to-semantics consistency (OSC) is defined as the mean semantic similarity between a word and its orthographic neighbors. While we adopt this general strategy, we identify a potential confound in previous operational definitions. We therefore offer a modified measure, which we use to examine group-level OSC effects in available data sets of single word recognition and reading for comprehension. Our findings validate the existence of OSC effects but reveal variation across tasks, with OSC effects emerging more strongly in tasks involving a direct mapping of print to meaning. Next, we present a reanalysis of word naming data from 399 second through fifth graders, where we examine individual differences in reliance on O-S regularities and their relation to participants' reading skills. We show that early readers whose naming accuracy is more influenced by OSC (i.e., those who rely more on O-S) have better passage comprehension abilities. We conclude by discussing the role of O-S regularities in proficient reading and literacy acquisition. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-dependent Signaling Pathways in the Granule Cell Precursor.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Inhalable Protein Powder Prepared by Spray-Freeze-Drying Using Hydroxypropyl-ß-Cyclodextrin as Excipient.

The prospect of inhaled biologics has garnered particular interest given the benefits of the pulmonary route of administration. Pertinent considerations in producing inhalable dry powders containing biological medicines relate to aerosol performance and protein stability. Spray-freeze-drying (SFD) has emerged as an established method to generate microparticles that can potentially be deposited in the lungs. Here, the SFD conditions and formulation composition were evaluated using bovine serum albumin (BSA) as a model protein and 2-hydroxypropyl-beta-cyclodextrin (HPßCD) as the protein stabilizer. A factorial design analysis was performed to investigate the effects of BSA content, solute concentration of feed solution, and atomization gas flow rate on dispersibility (as an emitted fraction), respirability (as fine particle fraction), particle size, and level of protein aggregation. The atomization gas flow rate was identified as a significant factor in influencing the aerosol performance of the powder formulations and protein aggregation. Nonetheless, high atomization gas flow rate induced aggregation, highlighting the need to further optimize the formulation. Of note, all the formulations exhibited excellent dispersibility, while no fragmentation of BSA occurred, indicating the feasibility of SFD and the promise of HPßCD as an excipient.

Resting state EEG network modularity predicts literacy skills in L1 Chinese but not in L2 English.

EEG network modularity, as a proxy for cognitive plasticity, has been proposed to be a more reliable neural marker than power and coherence in predicting learning outcomes. The present study examined the associations between resting state EEG network modularity and both L1 Chinese and L2 English literacy skills among 90 Hong Kong first to fifth graders. The modularity indices of different frequency bands were highly correlated with one another. An exploratory factor analysis, performed to extract a general modularity index, explained 77.1% of the total variance. The modularity index was positively associated with Chinese word reading, Chinese phonological awareness, Chinese morphological awareness, and Chinese reading comprehension but was not significantly correlated with English word reading or English morphological awareness. Findings suggest that resting state EEG network modularity is likely to serve as a reasonable, reliable, and cost-effective neural marker of the development of first language but not second language literacy skills.

Modification of KL4 Peptide Revealed the Importance of Alpha-Helical Structure for Efficient siRNA Delivery.

A safe and effective delivery system is considered a key to the success of nucleic acid therapeutics. It has been reported that pulmonary surfactants or their components could facilitate the uptake of small interfering RNA (siRNA) into the lung epithelial cells. Previously, our group investigated the use of KL4 peptide, a synthetic cationic peptide that simulates the structural properties of surfactant protein B (SP-B), as siRNA delivery vector. Although KL4 peptide exhibits good in vitro siRNA transfection efficiency on lung epithelial cells, its therapeutic potential is limited by its poor aqueous solubility due to the presence of a high proportion of hydrophobic leucine residues. In this study, we aim to address the solubility issue, designing five different modified peptides by replacing the hydrophobic leucine with alanine or valine, and assess their potential as siRNA delivery vectors. While the modified peptides retain the overall cationic property, their siRNA binding is also affected and their transfection efficiency is inferior to the parent KL4 peptide. A closer examination of the conformation of these peptides by circular dichroism shows that substitution of leucine residues leads to the change of the secondary structure from α-helical content to either ß-sheet or more disordered, ß-turn conformations. Relatively conservative amino acid substitutions, in terms of hydrophobicity bulk, lead to substantial conformational alteration, heavily impacting siRNA binding and release, cellular uptake, and transfection efficiency. Although the peptide modification strategy employed in this study was unsuccessful in developing an improved version of KL4 peptide for siRNA delivery, it highlights the importance of the α-helical conformation for efficient siRNA transfection, providing useful insights for future development of peptide-based RNA delivery system.

Effect of formulation and inhaler parameters on the dispersion of spray freeze dried voriconazole particles.

Spray freeze drying is a particle engineering technique that allows the production of porous particles of low density with excellent aerosol performance for inhalation. There are a number of operating parameters that can be manipulated in order to optimise the powder properties. In this study, a two-fluid nozzle was used to prepare spray freeze dried formulation of voriconazole, a triazole antifungal agent for the treatment of pulmonary aspergillosis. A full factorial design approach was adopted to explore the effects of drug concentration, atomisation gas flow rate and primary drying temperature. The aerosol performance of the spray freeze dried powder was evaluated using the next generation impactor (NGI) operated with different inhaler devices and flow rates. The results showed that the primary drying temperature played an important role in determining the aerosol properties of the powder. In general, the higher the primary drying temperature, the lower the emitted fraction (EF) and the higher the fine particle fraction (FPF). Formulations that contained the highest voriconazole concentration (80% w/w) and prepared at a high primary drying temperature (-10 °C) exhibited the best aerosol performance under different experimental conditions. The high concentration of the hydrophobic voriconazole reduced surface energy and cohesion, hence better powder dispersibility. The powders produced with higher primary drying temperature had a smaller particle size after dispersion and improved aerosol property, possibly due to the faster sublimation rate in the freeze-drying step that led to the formation of less aggregating or more fragile particles. Moreover, Breezhaler®, which has a low intrinsic resistance, was able to generate the best aerosol performance of the spray freeze dried voriconazole powders in terms of FPF.

Usefulness of stellate ganglion block for refractory angina pectoris.

Chronic refractory angina pectoris (AP) affects 600,000 to 1,800,000 Americans, with approximately 50,000 new cases annually. A recent study revealed long-term mortality of refractory AP to be lower than previously reported, with >70% of patients living >9 years. Treating AP can improve quality of life. We describe a patient with refractory AP who underwent a successful stellate ganglion block for symptom control.

Cognitive-linguistic profiles of Chinese typical-functioning adolescent dyslexics and high-functioning dyslexics.

Dyslexia is a developmental disability affecting the acquisition of reading and writing skills, and its developmental nature makes longitudinal research of great importance. This study therefore investigated the cognitive-linguistic profiles of the typical-functioning dyslexics and high-functioning dyslexics with longitudinal cohorts of Chinese-speaking adolescents diagnosed with childhood dyslexia. These two dyslexic groups of fifty students (with 25 typical-functioning dyslexics) were assessed in Grade 2 (Time 1) and in Grade 8 (Time 2), whereas 25 typically developing controls were assessed at Time 2. Students were administered measures of phonological awareness, morphological skills, visual-orthographic knowledge, rapid naming, verbal working memory, and literacy skills. Results showed that, at Time 2, both dyslexic groups performed less well than the control group on most of the measures. Deficits in rapid naming were particularly salient in both dyslexic groups. Comparing the two dyslexic groups, the typical-functioning dyslexics had more multiple deficits than the high-functioning dyslexics. Findings highlight the importance of rapid naming deficits as potential universal causes of dyslexia and the utility of targeting visual-orthographic knowledge and morphological skills in supporting the development of dyslexic adolescents.

Management of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a relatively rare, chronic, and debilitating condition that significantly impacts the patient's quality of life. There is an overall paucity of literature addressing the management of CRPS in immunocompromised patients. We define features of CRPS, outline its treatment options, and describe a course of CRPS management for a 35-year-old patient who had heart transplantation requiring immunosuppressive medications.