In this study, we have employed Digital Light Processing (DLP) printing technology for the fabrication of solid microneedle (MN) arrays. Several arrays with various geometries, such as cones, three-sided pyramids and four-sided pyramids, with different height to aspect ratios of 1:1, 2:1 and 3:1, were printed. Post-processing curing optimizations showed that optimal mechanical properties of the photocurable resin were obtained at 40 °C and 60 min. Ex vivo skin studies showed that piercing forces, penetration depth and penetration width were affected by the MN geometry and height to aspect ratio. Cone-shaped MNs required lower applied forces to penetrate skin and showed higher penetration depth with increasing height to aspect ratio, followed by three-sided and four-sided printed arrays. Cytotoxicity studies presented 84% cell viability of human fibroblasts after 2.5 h, suggesting the very good biocompatibility of the photocurable resin. Overall, DLP demonstrated excellent printing capacity and high resolution for a variety of MN designs.

Development of human leishmaniasis is dependent on the ability of intracellular Leishmania parasites to spread and enter macrophages. The mechanism through which free promastigotes and amastigotes bind and enter host macrophages has been previously investigated; however, little is known about intracellular trafficking and cell-to-cell spreading. In this study, the mechanism involved in the spreading of Leishmania aethiopica and Leishmania mexicana was investigated. A significant increase in phosphatidylserine (PS) exhibition, cytochrome C release, and active caspase-3 expression was detected (P < 0.05) during L. aethiopica, but not L. mexicana spreading. A decrease (P < 0.05) of protein kinase B (Akt) protein and BCL2-associated agonist of cell death (BAD) phosphorylation was also observed. The nuclear factor kappa-lightchain enhancer of activated B cells (NF-kB) signaling pathway and pro-apoptotic protein protein kinase C delta (PKC-d) were downregulated while inhibition of caspase-3 activation prevented L. aethiopica spreading. Overall suggesting that L. aethiopica induces host cell's apoptosis during spreading in a caspase-3-dependent manner. The trafficking of amastigotes within macrophages following cell-to-cell spreading differed from that of axenic parasites and involved co-localization with lysosomal-associated membrane protein 1 (LAMP-1) within 10 min postinfection. Interestingly, following infection with axenic amastigotes and promastigotes, co-localization of parasites with LAMP-1-positive structures took place at 1 and 4 h, respectively, suggesting that the membrane coat and LAMP-1 protein were derived from the donor cell. Collectively, these findings indicate that host cell apoptosis, demonstrated by PS exhibition, caspase-3 activation, cytochrome C release, downregulation of Akt, BAD phosphorylation, NF-kB activation, and independent of PKC-d expression, is involved in L. aethiopica spreading. Moreover, L. aethiopica parasites associate with LAMP-rich structures when taken up by neighboring macrophages.Abbreviations 7AAD, 7-aminoactinomycin D; Akt, protein kinase B; BAD, BCL2-associated agonist of cell death; CF, PKC-d cleaved fragment; CL, cutaneous leishmaniasis; EEA 1, early endosome antigen 1; HI-FBS, heat-inactivated fetal bovine serum; IF1, intermediate form 1; LAMP-1, lysosomal-associated membrane protein 1; NF-kB, nuclear factor kappa-light-chain enhancer of activated B cells; PKC-d, protein kinase C delta; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; RA, retinoic acid; SEM, scanning electron microscope; Ser, serine; Thr, threonine.

De novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/− NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells – two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/− developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

CHD8 (Chromodomain Helicase DNA Binding Protein 8) is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. De novo mutations (DNMs) in CHD8 are strongly associated with a specific subtype of autism characterized by enlarged foreheads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. How CHD8 haploinsufficiency alters the normal developmental trajectory of the human cortex is poorly understood and debated. Previous studies in the mammalian developing cortex have shown progressive lengthening of the G1 phase of the cell cycle as neural stem cells transition from proliferative to neurogenic divisions. G1 length has been proposed to operate as a molecular clock that controls timing of this crucial developmental switch. To determine the influence of CHD8 on cell cycle timing, we disrupted one allele of CHD8 in human embryonic stem cells (hESCs), differentiated these cells into neural precursor cells (NPCs), and imaged cell cycle progression of individual CHD8+/- NPCs, in parallel with their isogenic CHD8+/+ counterparts, during several rounds of cell division. We found a specific and marked decrease in G1 duration in CHD8+/- NPCs, resulting in an overall shortening of the cell cycle. Consistent with faster progression of CHD8+/- NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells; two central signalling pathways involved in S phase entry. Together, our findings show dysregulated proliferation of NPCs in a human stem cell model of CHD8 haploinsufficiency and predict enlargement of the neural progenitor pool in CHD8+/- developing brains, a phenotype that may explain macrocephaly in individuals with CHD8 DNMs. Furthermore, our work provides further evidence for a link between autism and cancer and identifies MAPK signaling as a potential therapeutic target for the treatment of this autism subtype.