Mussel inspired sequential protein delivery based on self-healing injectable nanocomposite hydrogel.

Polysaccharide based self-healing and injectable hydrogels with reversible characteristics have widespread potential in protein drug delivery. However, it is a challenge to design the dynamic hydrogel for sequential release of protein drugs. Herein, we developed a novel mussel inspired sequential protein delivery dynamic polysaccharide hydrogel. The nanocomposite hydrogel can be fabricated through doping polydopamine nanoparticles (PDA NPs) into reversible covalent bond (imine bonds) crosslinked polymer networks of oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CEC), named PDA NPs@OHA-l-CEC. Besides multiple capabilities (i.e., injection, self-healing, and biodegradability), the nanocomposite hydrogel can achieve sustained and sequential protein delivery of vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA). PDA NPs doped in hydrogel matrix serve dual roles, acting as secondary protein release structures and form dynamic non-covalent interactions (i.e., hydrogen bonds) with polysaccharides. Moreover, by adjusting the oxidation degree of OHA, the hydrogels with different crosslinking density could control overall protein release rate. Analysis of different release kinetic models revealed that Fickian diffusion drove rapid VEGF release, while the slower BSA release followed a Super Case II transport mechanism. The novel biocompatible system achieved sequential release of protein drugs has potentials in multi-stage synergistic drug deliver based on dynamic hydrogel.

ADP receptor P2y12 prevents excessive primitive hematopoiesis in zebrafish by inhibiting Gata1.

In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), ß-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.

Ca(2+) Ion and Autophagy.

Controlled by a strict mechanism, intracellular calcium (Ca(2+)) is closely related to various cellular activities, including the regulation of autophagy. Researchers believed that under normal or stress state, Ca(2+) has a positive or negative regulation effect on autophagy, the mechanisms of which are different. This bidirectional role of Ca(2+), promotive or suppressing in the regulation of autophagy under different conditions remains controversial, so as the potential mechanisms. Several studies reported that Ca(2+) promotes autophagy through plenty of ways, like inositol 1,4,5-trisphosphate receptor (IP3R) and beclin1 pathway, calmodulin-dependent kinase kinase beta (CaMKKß)-AMPK-mTOR pathway, mitochondrial energy metabolism-related Ca(2+) uptake, lysosome's regulation of Ca(2+) signal, and so on. Others thought Ca(2+) may inhibit autophagy through IP3R and beclin1-Bcl-2 complex and the AMPK-mTOR pathway, either. It seems to be still a long way to thoroughly understand the truth of Ca(2+) and autophagy.

Autophagy in Development and Differentiation.

Autophagy is crucial in the differentiation and development of both mammals and invertebrates, as a rapid response to environmental and hormonal stimuli. Autophagy is also important for intracellular renewal, maintaining the health of terminally differentiated cells. Studies of Drosophila, Caenorhabditis elegans, and other species revealed abnormal autophagy lead to developmental and differential abnormality, including those in salivary glands and midgut development, protein aggregation, removal of apoptotic cell corpses, and development of dauer and synapse. Autophagy also participates in the development of mammalian embryos before implantation into the uterus, adaption to the nascent hunger environment, blood cells production, and cell differentiation in adipogenesis. Autophagy found in various stem cells, like hematopoietic stem cells, bone marrow mesenchymal stem cells and neural stem cells (NSCs), is tightly associated with their self-renewal, directed differentiation, and senescence.

Heat shock protein DNAJA1 stabilizes PIWI proteins to support regeneration and homeostasis of planarian Schmidtea mediterranea.

PIWI proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how PIWI proteins themselves are regulated remains largely unknown. To identify candidate proteins that interact with PIWI proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea We show that DNAJA1, a heat shock protein 40 family member, interacts with the PIWI protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and PIWI-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PIWIL1) and is required for PIWIL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between PIWI and DNAJA1 that is essential for PIWI protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of proteins involved in stem cell regulation.

Neoblast-enriched zinc finger protein FIR1 triggers local proliferation during planarian regeneration.

Regeneration, relying mainly on resident adult stem cells, is widespread. However, the mechanism by which stem cells initiate proliferation during this process in vivo is unclear. Using planarian as a model, we screened 46 transcripts showing potential function in the regulation of local stem cell proliferation following 48 h regeneration. By analyzing the regeneration defects and the mitotic activity of animals under administration of RNA interference (RNAi), we identified factor for initiating regeneration 1 (Fir1) required for local proliferation. Our findings reveal that Fir1, enriched in neoblasts, promotes planarian regeneration in any tissue-missing context. Further, we demonstrate that DIS3 like 3'-5' exoribonuclease 2 (Dis3l2) is required for Fir1 phenotype. Besides, RNAi knockdown of Fir1 causes a decrease of neoblast wound response genes following amputation. These findings suggest that Fir1 recognizes regenerative signals and promotes DIS3L2 proteins to trigger neoblast proliferation following amputation and provide a mechanism critical for stem cell response to injury.

Forkhead containing transcription factor Albino controls tetrapyrrole-based body pigmentation in planarian.

Pigmentation processes occur from invertebrates to mammals. Owing to the complexity of the pigmentary system, in vivo animal models for pigmentation study are limited. Planarians are capable of regenerating any missing part including the dark-brown pigments, providing a promising model for pigmentation study. However, the molecular mechanism of planarian body pigmentation is poorly understood. We found in an RNA interference screen that a forkhead containing transcription factor, Albino, was required for pigmentation without affecting survival or other regeneration processes. In addition, the body color recovered after termination of Albino double stranded RNA feeding owing to the robust stem cell system. Further expression analysis revealed a spatial and temporal correlation between Albino and pigmentation process. Gene expression arrays revealed that the expression of three tetrapyrrole biosynthesis enzymes, ALAD, ALAS and PBGD, was impaired upon Albino RNA interference. RNA interference of PBGD led to a similar albinism phenotype caused by Albino RNA interference. Moreover, PBGD was specifically expressed in pigment cells and can serve as a pigment cell molecular marker. Our results revealed that Albino controls planarian body color pigmentation dominantly via regulating tetrapyrrole biogenesis. These results identified Albino as the key regulator of the tetrapyrrole-based planarian body pigmentation, suggesting a role of Albino during stem cell-pigment cell fate decision and provided new insights into porphyria pathogenesis.

Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.

AIM: MicroRNAs (miRNAs) play key roles in inflammatory responses of macrophages. However, the function of miRNAs in macrophage-derived foam cell formation is unclear. Here, we investigated the role of miRNAs in macrophage-derived foam cell formation and atherosclerotic development. METHODS AND RESULTS: Using quantitative reverse transcription-PCR (qRT-PCR), we found that the level of miR-155 expression was increased significantly in both plasma and macrophages from atherosclerosis (ApoE(-/-)) mice. We identified that oxidized low density lipoprotein (oxLDL) induced the expression and release of miR-155 in macrophages, and that miR-155 was required to mediate oxLDL-induced lipid uptake and reactive oxygen species (ROS) production of macrophages. Furthermore, ectopic overexpression and knockdown experiments identified that HMG box-transcription protein1 (HBP1) is a novel target of miR-155. Knockdown of HBP1 enhanced lipid uptake and ROS production in oxLDL-stimulated macrophages, and overexpression of HBP1 repressed these effects. Furthermore, bioinformatics analysis identified three YY1 binding sites in the promoter region of pri-miR-155 and verified YY1 binding directly to its promoter region. Detailed analysis showed that the YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress oxLDL-induced foam cell formation. Importantly, inhibition of miR-155 by a systemically delivered antagomiR-155 decreased clearly lipid-loading in macrophages and reduced atherosclerotic plaques in ApoE(-/-) mice. Moreover, we observed that the level of miR-155 expression was up-regulated in CD14(+) monocytes from patients with coronary heart disease. CONCLUSION: Our findings reveal a new regulatory pathway of YY1/HDACs/miR-155/HBP1 in macrophage-derived foam cell formation during early atherogenesis and suggest that miR-155 is a potential therapeutic target for atherosclerosis.

Heterochromatin protein 1 promotes self-renewal and triggers regenerative proliferation in adult stem cells.

Adult stem cells (ASCs) capable of self-renewal and differentiation confer the potential of tissues to regenerate damaged parts. Epigenetic regulation is essential for driving cell fate decisions by rapidly and reversibly modulating gene expression programs. However, it remains unclear how epigenetic factors elicit ASC-driven regeneration. In this paper, we report that an RNA interference screen against 205 chromatin regulators identified 12 proteins essential for ASC function and regeneration in planarians. Surprisingly, the HP1-like protein SMED-HP1-1 (HP1-1) specifically marked self-renewing, pluripotent ASCs, and HP1-1 depletion abrogated self-renewal and promoted differentiation. Upon injury, HP1-1 expression increased and elicited increased ASC expression of Mcm5 through functional association with the FACT (facilitates chromatin transcription) complex, which consequently triggered proliferation of ASCs and initiated blastema formation. Our observations uncover an epigenetic network underlying ASC regulation in planarians and reveal that an HP1 protein is a key chromatin factor controlling stem cell function. These results provide important insights into how epigenetic mechanisms orchestrate stem cell responses during tissue regeneration.