The general concept of tissue engineering is to restore biological function by replacing defective tissues with implantable, biocompatible, and easily handleable cell-laden scaffolds. In this study, osteoinductive and osteoconductive super paramagnetic Fe3O4 nanoparticles (MNP) and hydroxyapatite (HAP) nanoparticles were incorporated into a di-block copolymer based thermo-responsive hydrogel, methoxy(polyethylene glycol)-polyalanine (mPA), at various concentrations to afford composite, injectable hydrogels. Incorporating nanoparticles into the thermo-responsive hydrogel increased the complex viscosity and decreased the gelation temperature of the starting hydrogel. Functionally, the integration of inorganic nanoparticles modulated bio-markers of bone differentiation and enhanced bone mineralization. Moreover, this study adopted the emerging method of using either a supplementary static magnetic field (SMF) or a moving magnetic field to elicit biological response. These results demonstrate that combining external (magnet) and internal (scaffold) magnetisms is a promising approach for bone regeneration.

Many members of the public and important stakeholders operating at the upper end of the food chain, may be unfamiliar with how food is produced, including within modern animal production systems. The intensification of production is becoming increasingly common in modern farming. However, intensive systems are particularly susceptible to production diseases, with potentially negative consequences for farm animal welfare (FAW). Previous research has demonstrated that the public are concerned about FAW, yet there has been little research into attitudes towards production diseases, and their approval of interventions to reduce these. This research explores the public's attitudes towards, and preferences for, FAW interventions in five European countries (Finland, Germany, Poland, Spain and the UK). An online survey was conducted for broilers (n = 789), layers (n = 790) and pigs (n = 751). Data were analysed by means of Kruskal-Wallis ANOVA, exploratory factor analysis and structural equation modelling. The results suggest that the public have concerns regarding intensive production systems, in relation to FAW, naturalness and the use of antibiotics. The most preferred interventions were the most "proactive" interventions, namely improved housing and hygiene measures. The least preferred interventions were medicine-based, which raised humane animal care and food safety concerns amongst respondents. The results highlighted the influence of the identified concerns, perceived risks and benefits on attitudes and subsequent behavioural intention, and the importance of supply chain stakeholders addressing these concerns in the subsequent communications with the public.

In gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression.

A decline in malaria morbidity and mortality has been documented in Ethiopia since 2005 following a scale-up of the distribution of long-lasting insecticidal nets (LLINs). However, universal access to LLINs ownership and use has not yet been achieved. This study aimed to determine ownership and use of LLINs over time in south-central Ethiopia.

C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.

Glaucoma, a leading cause of blindness worldwide, often remains undetected until irreversible vision loss has occurred. Treatments focus on lowering intraocular pressure (IOP), the only modifiable and readily measurable risk factor. However, IOP can vary and does not always predict disease progression. MicroRNAs (miRNAs) are promising biomarkers. They are abundant and stable in biological fluids, including plasma and aqueous humor (AqH). We aimed to identify differentially expressed miRNAs in AqH and plasma from glaucoma, exfoliation syndrome (XFS), and control subjects.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453-462.

Neonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion.

Heterozygous de novo mutations in GATA6 are the most frequent cause of pancreatic agenesis in humans. In mice, however, a similar phenotype requires the biallelic loss of Gata6 and its paralog Gata4. To elaborate the human-specific requirements for GATA6, we chose to model GATA6 loss in vitro by combining both gene-edited and patient-derived pluripotent stem cells (hPSCs) and directed differentiation toward β-like cells. We find that GATA6 heterozygous hPSCs show a modest reduction in definitive endoderm (DE) formation, while GATA6-null hPSCs fail to enter the DE lineage. Consistent with these results, genome-wide studies show that GATA6 binds and cooperates with EOMES/SMAD2/3 to regulate the expression of cardinal endoderm genes. The early deficit in DE is accompanied by a significant reduction in PDX1+ pancreatic progenitors and C-PEPTIDE+ β-like cells. Taken together, our data position GATA6 as a gatekeeper to early human, but not murine, pancreatic ontogeny.

Naturally-occurring orange carotenoid protein (OCP) is synthesized in cyanobacteria and red algae for photoprotection. Holo-OCP can be produced with three plasmids in E. coli, which needs two inducers (arabinose and isopropyl β-D-thiogalactoside) to initiate two processes: one for generation of carotenoid and the other for generation of apo-OCP, so takes about two days. Afterwards, a two-plasmid method using two plasmids in E. coli is established, in which E. coli cells are induced only by isopropyl β-D-thiogalactoside, so can yield different holo-OCPs from several cyanobacteria within three days. In this work, we optimized the two-plasmid method as follows: (1) re-organization of the two plasmids, letting carotenoid-generating gene, crtW, be arranged together with apo-OCP-generating gene, ocp, in a single plasmid, which causes that both carotenoid and apo-protein were properly produced, (2) modification of several amino acids at the N-terminus of apo-OCP, in this way increasing the yield and purity of holo-OCP. After these optimizations, we can generate much more amount of holo-OCP within shorter time of only 16 h, and pure holo-OCP be conveniently prepared after routine purification. Comparing with the reported data, the general yield of holo-OCP is increased by ∼10-fold under similar conditions. The high quality of the prepared holo-OCPs is verified by fluorescence quenching of the phycobilisomes.

In China, L-3-n-butylphthalide (L-NBP) showed promising pharmacological actions in stroke treatment. Analyzing the characteristics of L-NBP might provide valuable hints for new drug design. The current study is aimed to determine the effects of L-NBP on neuritogenesis and further to elucidate the neuronal protection against stroke impairment in vitro. L-NBP was applied to rat pheochromocytoma PC12 cells and cultured rat cortical neurons under the normoxic condition and the oxygen-glucose deprivation/reoxygenation (OGD/R) insults, respectively. Immunofluorescence staining, western blot analysis, Sholl analysis, lactate dehydrogenase (LDH) release assay, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay and enzyme-linked immunosorbent assay (ELISA) were performed. L-NBP could concentration-dependently stimulate the development of growth cones, enhance the neuritic branches and synapse formation. It indicated that L-NBP possibly promoted the neuritogenic activity in a stage-dependent manner. Further research proved that L-NBP could promptly activate epidermal growth factor (EGF) receptor, up-regulate the expressions of extracellular signal-regulated kinase1/2 (ERK1/2), cAMP response element-binding protein (CREB) and E-26-like protein 1 (ELK-1). In addition, L-NBP enhanced the sustained expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The inhibition to the receptors of EGF, NGF,BDNF could attenuate L-NBP induced neuritogenic and neuronal survival after the OGD/R toxicity. Basing on these investigations, we concluded that L-NBP might reconstruct the impaired neuronal network and improved the neuronal complexity after the ischemic insults through multiple pathways which at least were via the activations of EGF receptor, BDNF and NGF related signals.

In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2- to 3-nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well defined. RNAP contains a clamp domain that closes around the nucleic acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.

There are conflicting reports on the outcomes after live donor liver transplantation in patients with hepatocellular carcinoma (HCC). We aimed to compare the survival of patients with HCC, with a potential live donor (pLDLT) at listing vs. no potential donor (pDDLT), on an intention-to-treat basis.

Memories for daily events require that individuals integrate initial fragile traces of events over time. Recent evidence suggests that reward anticipation enhances memory performance and amplifies frontal theta activity for remembered items vs. forgotten items. However, little is known about how incidental rewards after item presentation retrospectively modulate memory and the neural basis of this processing. Here, we used EEG combined with an incidental memory task to study how incidental reward association biased the post-encoding process. In the anticipatory stage, participants saw photos in win, loss and neutral contexts. Each photo was presented in a color frame that indicated the incentive condition (win vs. loss vs. neutral) and participants were asked to make a binary choice to predict whether the photo was associated with the left/right button. Feedback was presented to indicate arbitrary correctness and monetary outcomes. Recognition memory was tested after a short delay. During the encoding phase, left central-parietal theta power predicted subsequent memory performance in the win context. The post-encoding theta power at right central-frontal and central-parietal sites predicted later memory performance only in the win context. The size of frontal post-encoding related theta activity in the win context was correlated with the discriminate accuracy of the test stimulus. Our results suggest that post-encoding theta activity is closely linked to reward-based associative learning, providing evidence of a potential post-encoding mechanism of information binding.

S-Nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) are known to produce nitric oxide (NO) through thermal, photolytic, and metal ion-promoted pathways, which has led to their increasing use as exogenous sources of therapeutic NO. Despite the burgeoning NO release applications for RSNOs, their susceptibility to metal-promoted decomposition has rarely been examined in a uniform manner through the specific measurement of NO release. In this study, the ability of various transition and post-transition metal ions to promote NO release from GSNO was surveyed by chemiluminescence-based NO detection. Substantial NO formation (>10-fold increase relative to GSNO baseline) was detected after the addition of Cu2+, Au3+, Pd2+, Pt2+, and V3+. Modest increases were observed in the cases of Co2+, Hf4+, Fe2+, Fe3+, Mn2+, Hg2+, Ni2+, Ag+, Sn2+, and Zr4+, while no effect was evident for Al3+, Cr3+, Pb2+, Sc3+, and Zn2+. It was further observed that In+ compounds initiate the apparent NO-forming decomposition of GSNO, while In0 and In3+ are inactive, indicating that In+ exerts a previously unknown effect on GSNO.

The goal of current investigations was to reveal the molecular mechanism triggered through feeding a diet with high-concentrate to dairy cows for subacute ruminal acidosis (SARA) induction and to examine the oxidative stress parameters in their mammary epithelial tissue. In an eighteen-weeks feeding trial, 12 Holstein Friesian cows with a standard weight of 455 ± 28 kg were evenly divided into two groups and given either a low-concentrate (LC, forage to concentrate ratio = 6:4) or a high-concentrate (HC, forage to concentrate ratio = 4:6) diet. A remarkable reduction in ruminal pH also increased ruminal lipopolysaccharide (LPS) concentration that was observed in the high-concentrate group of cows at 4 h post-feeding in the morning. Moreover, reduced milk yield was observed in the HC group. The relative mRNA abundance of glutathione peroxidase (GPX) 1 and 3 and superoxide dismutase (SOD) 1 and 2 were down-regulated in high-concentrate fed animals than in the LC, while mRNA was expressed with no change in the of SOD3 among groups. In addition, genes responsible for oxidative stress e.g., ERK, JNK, and p38 were also showed dramatically high mRNA intensity in HC group. The protein concentration of ERK, pERK, pJNK, with pp38, were up-regulated significantly as JNK & p38 showed no big difference. While Nrf2 and pNrf2 were down-regulated considerably in HC group. The total antioxidant capacity (T-AOC) was significantly decreased but of Malondialdehyde (MDA) concentration was raised in HC group than in LC. We thus proposed that higher levels of endogenous LPS may affect the Mitogen-activated protein kinases (MAPK) and nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response.

Whole-genome duplications are an important source of evolutionary novelties that change the mode and tempo at which genetic elements evolve within a genome. The Cucurbita genus experienced a whole-genome duplication around 30 million years ago, although the evolutionary dynamics of the coding and noncoding genes in this genus have not yet been scrutinized. Here, we analyzed the genomes of four Cucurbita species, including a newly assembled genome of Cucurbita argyrosperma, and compared the gene contents of these species with those of five other members of the Cucurbitaceae family to assess the evolutionary dynamics of protein-coding and long intergenic noncoding RNA (lincRNA) genes after the genome duplication. We report that Cucurbita genomes have a higher protein-coding gene birth-death rate compared with the genomes of the other members of the Cucurbitaceae family. C. argyrosperma gene families associated with pollination and transmembrane transport had significantly faster evolutionary rates. lincRNA families showed high levels of gene turnover throughout the phylogeny, and 67.7% of the lincRNA families in Cucurbita showed evidence of birth from the neofunctionalization of previously existing protein-coding genes. Collectively, our results suggest that the whole-genome duplication in Cucurbita resulted in faster rates of gene family evolution through the neofunctionalization of duplicated genes.

Social cognitive ability is a significant determinant of functional outcome, and deficits in social cognition are a disabling symptom of psychotic disorders. The neurobiological underpinnings of social cognition are not well understood, hampering our ability to ameliorate these deficits.

The wings of Ptiliidae, the coleopteran family containing the smallest free-living insects, are analyzed in detail for the first time. A reconstruction of the evolutionary sequence of changes associated with miniaturization is proposed. The wings of several species are described using light microscopy and scanning electron microscopy. The morphology and scaling are analyzed in comparison with larger representatives of related groups. The wings of all studied ptiliids show some degree of ptiloptery (feather-like shape, typical for extremely small insects). In larger ptiliids the wing contains at least five veins, has a wide blade, and bears a marginal fringe of 200-300 setae; in the smallest species it has three veins or fewer, a narrow blade, and about 40 setae along the margin. The setae are brush-like; peculiar outgrowths, denser towards the apex, increase the effective diameter of the setae. Morphometric analysis shows that the geometry of the wings and their elements strongly differs from those of other staphyliniform beetles, suggesting that the aerodynamics of the feather-like wings may also differ distinctly from the usual pattern.

Oxidized low-density lipoprotein (ox-LDL) can impair endothelial function and lead to the atherosclerosis development. Protein S-nitrosylation is sensitive to cellular redox state and acts as a crucial regulator and executor of nitric oxide (NO) signaling pathways. Aberrant S-nitrosylation contributes to the pathogenesis of cardiovascular and cerebrovascular diseases. However, the effect of ox-LDL on S-nitrosylation and its significance for endothelial dysfunction have not been studied at proteome level. Herein, the combined quantitative analysis of proteome and S-nitrosoproteome was performed using an integrated biotin switch and iTRAQ labeling approach in EA.hy926 cell line derived from human umbilical vein endothelial cell (HUVEC) treated with ox-LDL. A total of 2204 S-nitrosylated (SNO) peptides of 1318 SNO-proteins were quantified. Notably, 352 SNO-peptides of 262 SNO-proteins were significantly regulated after excluding S-nitrosylation changes caused by protein expression alterations. Many of them belonged to mRNA splicing, ribosomal structure and translational regulatory proteins, covering the entire translation process. The results indicated that S-nitrosylation of the splicing and translational machinery in vascular endothelial cells was susceptible to ox-LDL. Abnormal protein S-nitrosylation may be one pivotal mechanism underlying endothelial dysfunction induced by ox-LDL. This study potentially enriches the present understanding of pro-atherogenic effect of ox-LDL from the perspective of S-nitrosylation. SIGNIFICANCE: The role of ox-LDL in endothelial dysfunction and atherosclerosis development has been recognized from the aspect of impaired NO production. However, its effect on S-nitrosylation, which is directly related to NO signaling pathway, still remains largely unexplored. Our work initially provided a systematic characterization of S-nitrosoproteome in ox-LDL-treated endothelial cells after ruling out the changes of S-nitrosylation modification caused by protein expression alone. MS-based approach coupled with iTRAQ technique indicated 262 SNO-proteins were significantly regulated. Functional enrichment and interaction network analysis revealed that proteins involved in mRNA splicing and translational machinery were susceptible to abnormal S-nitrosylation under ox-LDL treatment. This achievement suggested one potential mechanism underlying endothelial dysfunction induced by ox-LDL from the perspective of S-nitrosoproteome.