Tissue-specific extracellular matrix plays an important role in promoting tissue regeneration and repair. We hypothesized that decellularized annular fibrosus matrix may be an appropriate scaffold for annular fibrosus tissue engineering. We aimed to determine the optimal decellularization method suitable for annular fibrosus. Annular fibrosus tissue was treated with 3 different protocols with Triton X-100, sodium dodecyl sulfate (SDS) and trypsin. After the decellularization process, we examined cell removal and preservation of the matrix components, microstructure and mechanical function with the treatments to determine which method is more efficient. All 3 protocols achieved decellularization; however, SDS or trypsin disturbed the structure of the annular fibrosus. All protocols maintained collagen content, but glycosaminoglycan content was lost to different degrees, with the highest content with TritonX-100 treatment. Furthermore, SDS decreased the tensile mechanical property of annular fibrosus as compared with the other 2 protocols. MTT assay revealed that the decellularized annular fibrosus was not cytotoxic. Annular fibrosus cells seeded into the scaffold showed good viability. The Triton X-100–treated annular fibrosus retained major extracellular matrix components after thorough cell removal and preserved the concentric lamellar structure and tensile mechanical properties. As well, it possessed favorable biocompatibility, so it may be a suitable candidate as a scaffold for annular fibrosus tissue engineering.

Background and Purpose Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. Recently, Dopamine D2 receptor (DRD2) is identified an important component controlling innate immunity and inflammatory response in central nervous system and αB-crystallin (CRYAB) is a potent negative regulator on inflammatory pathways. Here, we sought to investigate the role of DRD2 on neuroinflammation after experimental ICH, and the potential mechanism mediated by CRYAB. Methods Two hundred and twenty-four (224) male CD-1 mice were subjected to intrastriatal infusion of bacterial collagenase or autologous blood. Two DRD2 agonists Quinpirole and Ropinirole were administrated by daily intraperitoneal injection starting at 1 hour post-ICH. DRD2 and CRYAB in vivo knockdown was performed 48 hour before ICH insult. Behavioral deficits and brain water content, western blots, immunofluorescence staining, co-immunoprecipitation assay and proteome cytokine array were evaluated. Results Endogenous DRD2 and CRYAB expression were increased after ICH. DRD2 knockdown aggravated the neurobehavioral deficits and the pronounced cytokines expression. DRD2 activation by Quinpirole and Ropinirole ameliorated neurological outcome, brain edema, IL-1β and MCP-1 expression, as well as microglia/macrophages activation in the perihematomal region. These effects were abolished by pretreated with CRYAB siRNAs. Quinpirole enhanced cytoplasmic binding activity between CRYAB and NF-κB, and decreased nuclear NF-κB expression. Similar therapeutic benefits were observed using autologous blood injection model and intranasal delivery of Quinpirole. Conclusions DRD2 may have anti-inflammatory effects after ICH. DRD2 agonists inhibited neuroinflammation and attenuated brain injury after ICH, which is probably mediated by CRYAB and enhanced cytoplasmic binding activity with NF-κB.

Obligatory blood-triggered reproductive strategy is an evolutionary adaptation of mosquitoes for rapid egg development. It contributes to the vectorial capacity of these insects. Therefore, understanding the molecular mechanisms underlying reproductive processes is of particular importance. Here, we report that microRNA-309 (miR-309) plays a critical role in mosquito reproduction. A spatiotemporal expression profile of miR-309 displayed its blood feeding-dependent onset and ovary-specific manifestation in female Aedes aegypti mosquitoes. Antagomir silencing of miR-309 impaired ovarian development and resulted in nonsynchronized follicle growth. Furthermore, the genetic disruption of miR-309 by CRISPR/Cas9 system led to the developmental failure of primary follicle formation. Examination of genomic responses to miR-309 depletion revealed that several pathways associated with ovarian development are down-regulated. Comparative analysis of genes obtained from the high-throughput RNA sequencing of ovarian tissue from the miR-309 antagomirsilenced mosquitoes with those from the in silico computation target prediction identified that the gene-encoding SIX homeobox 4 protein (SIX4) is a putative target of miR-309. Reporter assay and RNA immunoprecipitation confirmed that SIX4 is a direct target of miR-309. RNA interference of SIX4 was able to rescue phenotypic manifestations caused by miR-309 depletion. Thus, miR-309 plays a critical role in mosquito reproduction by targeting SIX4 in the ovary and serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA. In turn, this microRNA (miRNA)-targeted degradation is required for appropriate initiation of a blood feeding-triggered phase of ovarian development, highlighting involvement of this miRNA in mosquito reproduction.Homeobox protein gene | microRNA | CRISPR/Cas9 | ovary | fast evolution T ransmission of mosquito-borne diseases brings enormous human suffering, with more than 1 million deaths worldwide annually. The yellow fever mosquito Aedes aegypti has reemerged as one of the most dangerous vectors of human diseases, transmitting Dengue fever, Yellow fever, Chikungunya, and Zika virus (1-4). Because of the lack of effective vaccines and increasing drug resistance in pathogens, biological control is considered one of the most promising strategies for preventing disease transmission. In particular, female hematophagous mosquitoes rely on acquisition of blood to initiate a series of physiological events promoting egg development (5, 6). Therefore, understanding the molecular mechanisms underlying ovarian activation is of great significance for the development of effective approaches to control mosquito-borne diseases (7,8).MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level through translational repression or mRNA decay (9). Hence, miRNAs play significant roles in governing multiple functions in animals and plants via integrating sophisticated miRNA-mRNA regulatory networks (10, 11...