Alveolar mimics with periodic strain and its effect on the cell layer formation

We report on the development of a new model of alveolar air–tissue interface on a chip. The model consists of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40–80 µm at the air–liquid interface by application of air pressure in the range of 200–1,000 Pa. With respect to the diameter of the monolayers, that is, 500 µm, this displacement corresponds to a linear strain of 2–10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time of 1–3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz for 1 hr to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.     

Elevated cell-free fetal DNA contributes to placental inflammation and antiangiogenesis via AIM2 and IFI16 during pre-eclampsia

Accumulated evidence has shown that pre-eclampsia (PE) is related to both maternal and utero-placental antiangiogenesis and inflammation. Remarkably, an elevated cell-free fetal DNA (cffDNA) level has been found in maternal circulation; however, it remains unclear whether this DNA can induce activation of cytosolic DNA sensor signaling pathways and lead to the development of PE. In this study, we found that trophoblast cells constitutively expressed the cytosolic DNA sensors, absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16). The cffDNA and pro-inflammatory and antiangiogenic factors were present at higher concentrations in PE compared with the control group and correlated with the severity of PE. DNA stimulation significantly increased the AIM2 and IFI16 levels, consistent with the elevated AIM2 and IFI16 expression in women with PE, and elicited increased production of AIM2-mediated interleukin IL-8 (IL-8), IL-6 and CC chemokine ligand 2 (CCL2) and IFI16-mediated sEndoglin, sFlt-1 and CXCL10. Furthermore, enhancement of the inflammatory response was found to be induced by DNA exposure, but DNA exposure did not induce PE-like symptoms in pregnant mice. It is possible that elevated cffDNA could reflect the degree of placental damage and trigger cytosolic DNA sensor activation, which disrupts the immunity balance and, consequently, contributes to inflammatory and antiangiogenic responses. In conclusion, the results of this study suggest that circulating cffDNA levels are increased in preeclamptic women and act through AIM2 and IFI16 activation to promote the production of pro-inflammatory and antiangiogenic factors, which correlate with the severity of the disease, and may offer insights into the etiology and pathogenesis of PE.     

Thermal manipulation during the middle incubation stage has a repressive effect on the immune organ development of Peking ducklings

Avian embryos are easily influenced by their environment during incubation. Previous studies have demonstrated that incubation temperature changes could influence muscle development and body weight, which subsequently determine the adult phenotype. The objective of this study was to investigate whether the development of immune organs in ducklings could be influenced by thermal manipulation during the middle stage of incubation. To evaluate this hypothesis, a control group was incubated under a normal temperature from E11 to E24, while the incubation temperature of the experimental group was increased by 1 °C. Our results indicated that slight changes in the incubation temperature significantly repressed the bursa of Fabricius index of the duck embryo on E25 (F1, 58=122.51, P<0.0001) and significantly repressed the spleen index of neonatal ducklings (F1, 58=74.38, P<0.0001). At 0 day posthatching (dph) and 14 dph, ducklings hatched from eggs incubated under the higher temperature had a lower percentage of globulin than the control group (F1, 10=19.97, P=0.0111; F1, 10=9.8, P=0.0352). The IFN-γ concentration of ducklings at 14 dph displayed the same trend (F1, 10=284.49, P<0.0001). These results suggested that thermal manipulation during the middle stage of incubation had a repressive effect on the development of immune organs and reduced the concentrations of serum globulin and IFN-γ. These results demonstrated that the subtle alteration of incubation temperature may weaken ducklings' immunity.     

Identification of genetic associations of SP110/MYBBP1A/RELA with pulmonary tuberculosis in the Chinese Han population

Genetic factors play important roles in the development of tuberculosis (TB). SP110 is a promising candidate target for controlling TB infections. However, several studies associating SP110 single nucleotide polymorphisms (SNPs) with TB have yielded conflicting results. This may be partly resolved by studying other genes associated with SP110, such as MYBBP1A and RELA. Here, we genotyped 6 SP110 SNPs, 8 MYBBP1A SNPs and 5 RELA SNPs in 702 Chinese pulmonary TB patients and 425 healthy subjects using MassARRAY and SNaPshot methods. Using SNP-based analysis with Bonferroni correction, rs3809849 in MYBBP1A [Pcorrected (cor) = 0.0038] and rs9061 in SP110 (Pcor = 0.019) were found to be significantly associated with TB. Furthermore, meta-analysis of rs9061 in East Asian populations showed that the rs9061 T allele conferred significant risk for TB [P = 0.002, pooled odds ratio (OR), 1.24, 95 % confidence interval (CI) = 1.08–1.43]. The MYBBP1A GTCTTGGG haplotype and haplotypes CGACCG/TGATTG within SP110 were found to be markedly and significantly associated with TB (P = 2.00E?06, 5.00E?6 and 2.59E?4, respectively). Gene-based analysis also demonstrated that SP110 and MYBBP1A were each associated with TB (Pcor = 0.011 and 0.035, respectively). The logistic regression analysis results supported interactions between SP110 and MYBBP1A, indicating that subjects carrying a GC/CC genotype in MYBBP1A and CC genotype in SP110 possessed the high risk of developing TB (P = 1.74E?12). Our study suggests that a combination of SP110 and MYBBP1A gene polymorphisms may serve as a novel marker for identifying the risk of developing TB in the Chinese Han population.     

The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.     

Apobec-1 Increases Cyclooxygenase-2 and Aggravates Injury in Oxygen-Deprived Neurogenic Cells and Middle Cerebral Artery Occlusion Rats

Given that cyclooxygenase-2 (COX-2) plays a crucial role during cerebral ischemia and Apobec-1 is a critical regulator of COX-2 mRNA stabilization in gastrointestinal settings, the correlation of COX-2 and Apobec-1 was investigated in neurogenic cells and rat model of cerebral ischemia. After neurogenic SH-SY5Y, NG108-15 and PC12 cells were exposed to oxygen-glucose deprivation, cell viability, LDH leakage and Apobec-1 expression were determined. The effect of Apobec-1 overexpression on injury severity of oxygen-glucose deprivation, COX-2 expression, C-to-U editing of COX-2 mRNA were measured in vitro. Then the correlation of Apobec-1 level and injury severity was analyzed in cells with oxygen-glucose deprivation and in rats with middle cerebral artery occlusion. Apobec-1 expression was elevated along with upregulation of COX-2 and injury severity of oxygen-glucose deprivation in the three cell lines. Apobec-1 overexpression aggravated injury of oxygen-glucose deprivation in vitro and could be correlated to injury severity in vivo. Meanwhile, Apobec-1 increased COX-2 expression and COX-2 mRNA stabilization in neurogenic cells, and failed to catalyze C-to-U editing of COX-2 mRNA. Apobec-1 could upregulate COX-2 expression in neurogenic cells by stabilizing COX-2 mRNA, and might aggravate injury of oxygen-glucose deprivation in neurogenic cells as well as in rats with cerebral ischemia.     

The Expression Pattern of Classical MHC Class I Molecules in the Development of Mouse Central Nervous System

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D^b mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D^b in P15 mice. At E10.5, the earliest stage we examined, H2-D^b was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D^b expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D^b mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D^b in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K^b signal paralleled that of H2-D^b and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D^b at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D^b in the developing CNS.     

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli)

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.