Delivery and Diffusion of Retinal in Dermis and Epidermis Through The Combination of Prodrug Nanoparticles and Detachable Dissolvable Microneedles

To minimize fast chemical degradation of retinal, we convert this aldehyde into proretinal nanoparticles (PRNs) by forming retinylidene moieties on chitosan and allowing the grafted polymers to assemble into nanoparticles, and then load the obtained PRNs into detachable microneedles made of 1:1 (by weight) hyaluronic acid/maltose. An embedment of the PRNs in the solid matrix of microneedles helps improving chemical stability of the grafted retinal; the loaded device can be kept at 25 °C for three months (longest time experimented) with less than 30% degradation of the retinylidene moieties. The presence PRNs in the hyaluronic acid-maltose matrix also help improving mechanical strength of the needles. Administration of PRN-loaded detachable microneedles on fresh porcine ear skin results in complete deposition of an array of microneedles in the skin tissue at the dept that spans both epidermis and dermis, as observed by stereomicroscopic and confocal fluorescence microscopic analyses of the cross-sectioned tissue pieces. Obvious diffusion of the PRNs from the originally embedded site of the needles in the skin tissue to the nearby location can be observed, and even distribution in the tissue is reached at 4 h post administration. Rats administered with single dose of PRN-loaded microneedles show significant increased epidermal thickness as compared to rats administered with unloaded microneedles. Both PRN-loaded microneedles and unloaded microneedles produce no skin irritation in rats.

242 Divergent Selection for Early Puberty Impacts KNDy Neuron Gene Expression in Gilts

Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in pre- and postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 µm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P < 0.05. We determined mRNA expression for kisspeptin was not different between groups (P > 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P < 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P < 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P < 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin.

Maintenance of Deep Lung Architecture and Automated Airway Segmentation for 3D Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is a technique for mapping the spatial distributions of molecules in sectioned tissue. Histology-preserving tissue preparation methods are central to successful MSI studies. Common fixation methods, used to preserve tissue morphology, can result in artifacts in the resulting MSI experiment including delocalization of analytes, altered adduct profiles, and loss of key analytes due to irreversible cross-linking and diffusion. This is especially troublesome in lung and airway samples, in which histology and morphology is best interpreted from 3D reconstruction, requiring the large and small airways to remain inflated during analysis. Here, we developed an MSI-compatible inflation containing as few exogenous components as possible, forgoing perfusion, fixation, and addition of salt solutions upon inflation that resulted in an ungapped 3D molecular reconstruction through more than 300 microns. We characterized a series of polyunsaturated phospholipids (PUFA-PLs), specifically phosphatidylinositol (-PI) lipids linked to lethal inflammation in bacterial infection and mapped them in serial sections of inflated mouse lung. PUFA-PIs were identified using spatial lipidomics and determined to be determinant markers of major airway features using unsupervised hierarchical clustering. Deep lung architecture was preserved using this inflation approach and the resulting sections are compatible with multiple MSI modalities, automated interpretation software, and serial 3D reconstruction.

Coexpression and Accumulation of Ubiquitin +1 and ZZ Proteins in Livers of Children with α1-Antitrypsin Deficiency

The ZZ variant of α1-antitrypsin deficiency (AATD) is well known to cause liver damage and cirrhosis in some affected children. Ubiquitin abnormality was recently shown to be significant in AATD in childhood cirrhosis. Molecular misreading (MM), defined as faulty transcription of genomic information from DNA into mRNA, as well as its translation into mutant proteins, has been documented in many pathologic processes where aggregation of abnormal proteins occurs. The misread protein, ubiquitin-B+1 (UBB+1), was recently identified in the hallmarks of various neurological disorders. The objective of this study was to determine whether MM of ubiquitin occurs in AATD. Twelve explanted liver specimens from AATD-affected children with cirrhosis were retrieved from archival sources, along with 10 control liver specimens obtained from autopsies of age-matched children with no clinical, gross anatomic, or histologic evidence of liver disease. Double immunofluorescence studies using rabbit polyclonal antibodies against UBB+1 and AAT were performed on consecutively sectioned tissue. UBB+1 immunoreactivity was colocalized with AAT in all cirrhotic AATD livers. The control livers were consistently negative. Ubiquitin MM is prominent in AATD-affected cirrhotic livers. This indicates that for children with AATD and cirrhosis, ubiquitin MM is a necessary cofactor to the aggregation of mutant ZZ isoform of AATD.