Enriching adenosine by thymine-rich DNA oligomers.

Adenosine levels are important in various physiological and pathological activities, but detecting them is difficult because of interference from a complex matrix. This study designed a series of DNA oligomers rich in thymine to enrich adenosine. Their binding affinity (Kd range: 1.25-5.0 mM) to adenosine varied based on the DNA secondary structures, with a clamped hairpin structure showing the highest binding affinity. Compared to other designs, this clamped DNA hairpin underwent the least conformational change during adenosine binding. These DNAs also suppressed the precipitation of supersaturated adenine. Taken together, these results suggest that thymine-rich DNAs could be used to enrich and separate adenosine.

Solution-Phase Synthesis of DNA Amphiphiles for DNA Micellar Assembly.

Hydrophobic moieties of amphiphilic DNAs can help DNAs penetrate cell membranes, but the conjugation of hydrophobic moieties to DNAs in solution phase remains challenging. Herein we report a solution-phase synthesis method to conjugate hydrophobic molecules to DNAs. This method is simple and efficient. The resulted amphiphilic DNAs can spontaneously assemble into micelles, which may serve as nanocarriers for cellular delivery of nucleic acids and water-insoluble drugs.

Phylogeography of the endangered "eyed" turtles (genus Sacalia) and the discovery of a lineage derived from natural interspecific hybridization.

The herpetofauna of the Indomalayan bioregion of Asia suffers from severe habitat loss, unsustainable harvesting, and lack of research and conservation. Here, we investigated the range-wide phylogeography of the endangered "eyed" turtles (genus Sacalia, including the Beale's Eyed Turtle S. bealei and the Four-eyed Turtle S. quadriocellata) and discovered a natural interspecific hybrid turtle population in China. Based on phylogeny of the mitochondrial Cytochrome b gene of 101 samples in this study and public data, three major clades and six subclades were identified: S. bealei (SBE) in eastern-southern China, east S. quadriocellata in South China (northern east [SQUen] and southern east [SQUes] subclades), and west S. quadriocellata mainly in Vietnam (northern west [SQUwn], central west [SQUwc], and southern west [SQUws] subclades). We sequenced 16 nuclear DNA loci of 87 samples from SBE, SQUen, SQUes, and SQUwn subclades. Population genetic clustering analysis suggested a structure similar to the mitochondrial phylogeny, where most samples were classified into four genetic clusters corresponding to the four mtDNA subclades. However, a proportion of samples carrying SQUen mtDNA haplotypes formed an additional distinct cluster SHY. Those samples are found in the contact zone of the two species bearing mosaic and intermediate morphological characteristics. We detected an admixed ancestry in SHY from SBE and SQUen that conformed to an intrapopulation breeding scenario for at least hundreds of generations after the initial hybrid event, leading to a conclusion that SHY is a distinct and near-panmictic population derived from natural interspecific hybridization. In addition, SQUes (Hainan Island endemic) is of special concern due to significant isolation and low genetic diversity. We suggest that seven evolutionarily significant units should be recognized to facilitate appropriate conservation actions. These findings also highlight the urgent need for further herpetological research and conservation in this region.

Rapid authenticity testing of artificially bred green turtles (Chelonia mydas) using microsatellite and mitochondrial DNA markers.

Sea turtles are threatened by climate change and human activity, and their global populations continue to decline sharply. The Chinese government encourages artificial breeding of sea turtles to reduce the use of wild populations. However, artificial breeding of sea turtles is still fairly difficult, and some facilities may illegally purchase wild turtle eggs and then sell incubated turtles by marketing them as artificially bred turtles, which adds another threat to an already endangered species. Therefore, it is necessary to find a reliable method to distinguish the authenticity of artificially bred individuals. In this study, we investigated a turtle farm in southern China, that contained more than 400 green turtles, which were claimed to have been bred in captivity. Parentage testing of turtles from this farm was successfully conducted using two nuclear microsatellites combined with a mitochondrial D-loop DNA marker. Genetic matching of all 19 adults and randomly selected 16 juvenile turtles revealed that none of the juvenile turtles had a matching parent combination among the adult turtles. Therefore, we speculated that the green turtles in this farm were from the wild and that their origin of birth was mainly the Sulu Sea. The methods and molecular markers used in this study could be a reference for rapid authenticity testing of green turtles in future forensic enforcement and population management.

Discovery of first active breeding den of Chinese mountain cat ( Felis bieti).

In mid-September 2018, during a field survey in Chiat'ung, Sanjiangyuan (Three-River-Source) Region, Tibetan Plateau, China, we discovered the first active breeding den of the Chinese mountain cat (Felis bieti), inhabited by one adult female and two kittens. Based on fieldwork over the following months, five breeding dens were discovered, and 33 sightings were recorded. In addition, at least five individuals were confirmed to inhabit this overlooked region, and much previously unknown information concerning this cat species and its ecology was revealed for the first time.

Characterization and comparison of mitogenomes of three 'eyed' turtles Sacalia spp.

In this paper, we characterized the mitogenomes of three 'eyed' turtles, the Beal's Eyed turtle (Sacalia bealei), the Four Eye-spotted turtle (S. quadriocellata), and the Hainan Four Eye-spotted turtle which was a unique lineage in the four-eyed turtle and considered as an independent species S. insulensis recently. The full lengths of the S. bealei and S. quadriocellata mitogenomes are 16,564 bp, and 16,555 bp, respectively, while the length of partial mitochondrial genome of S. insulensis is 16,433 bp without tailed part of D-loop. All the genes exhibit the typical mitochondrial gene arrangement and transcribing directions of turtles. Phylogenetic analysis indicating that a deep divergence about 7.8% p distance was found between S. bealei and (S. quadriocellata + S. insulensis), and the divergence (2.8% in patristic distance) between S. quadriocellata and S. insulensis is comparable with other closely related species in turtles.

Adhesive properties of YapV and paralogous autotransporter proteins of Yersinia pestis.

Yersinia pestis is the causative agent of plague. This bacterium evolved from an ancestral enteroinvasive Yersinia pseudotuberculosis strain by gene loss and acquisition of new genes, allowing it to use fleas as transmission vectors. Infection frequently leads to a rapidly lethal outcome in humans, a variety of rodents, and cats. This study focuses on the Y. pestis KIM yapV gene and its product, recognized as an autotransporter protein by its typical sequence, outer membrane localization, and amino-terminal surface exposure. Comparison of Yersinia genomes revealed that DNA encoding YapV or each of three individual paralogous proteins (YapK, YapJ, and YapX) was present as a gene or pseudogene in a strain-specific manner and only in Y. pestis and Y. pseudotuberculosis. YapV acted as an adhesin for alveolar epithelial cells and specific extracellular matrix (ECM) proteins, as shown with recombinant Escherichia coli, Y. pestis, or purified passenger domains. Like YapV, YapK and YapJ demonstrated adhesive properties, suggesting that their previously related in vivo activity is due to their capacity to modulate binding properties of Y. pestis in its hosts, in conjunction with other adhesins. A differential host-specific type of binding to ECM proteins by YapV, YapK, and YapJ suggested that these proteins participate in broadening the host range of Y. pestis. A phylogenic tree including 36 Y. pestis strains highlighted an association between the gene profile for the four paralogous proteins and the geographic location of the corresponding isolated strains, suggesting an evolutionary adaption of Y. pestis to specific local animal hosts or reservoirs.

Osteogenic differentiation of amniotic epithelial cells: synergism of pulsed electromagnetic field and biochemical stimuli.

BACKGROUND: Pulsed electromagnetic field (PEMF) is a non-invasive physical therapy used in the treatment of fracture nonunion or delayed healing. PEMF can facilitate the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Amniotic epithelial cells (AECs) have been proposed as a potential source of stem cells for cell therapy. However, whether PEMF could modulate the osteogenic differentiation of AECs is unknown. In the present study, the effects of PEMF on the osteogenic differentiation of AECs were investigated. METHODS: AECs were isolated from amniotic membrane of human placenta by trypsin digestion and were induced by PEMF and/or osteo-induction medium. After 21 days we used real time RT-PCR and immunocytochemistry to study the expression of osteoblast markers. The signal transduction of osteogenesis was further investigated. RESULTS: The PEMF stimulation, or osteo-induction medium alone could induce osteogenic differentiation of AECs, as shown by expression of osteoblast specific genes and proteins including alkaline phosphatase and osteocalcin. Furthermore, a combination of PEMF and osteo-induction medium had synergy effects on osteogenic differentiation. In our study, the gene expression of BMP-2, Runx2, ß-catenin, Nrf2, Keap1 and integrinß1 were up-regulated in the osteogenic differentiation of AECs induced by PEMF and/or osteo-induction medium. CONCLUSIONS: Combined application of PEMF and osteo-induction medium is synergistic for the osteogenic differentiation of AECs. It might be a novel approach in the bone regenerative medicine.

Antigen-specific H1N1 influenza antibody responses in acute respiratory tract infections and their relation to influenza infection and disease course.

BACKGROUND: Early antibody responses to influenza infection are important in both clearance of virus and fighting the disease. Acute influenza antibody titers directed toward H1-antigens and their relation to infection type and patient outcomes have not been well investigated. OBJECTIVE: Using hemagglutination inhibition (HI) assays, we aimed to characterize the H1-specific antibody titers in patients with influenza infection or another respiratory infection before and after the H1N1-pandemic influenza outbreak. Among patients with acute influenza infection we related duration of illness, severity of symptoms, and need for hospitalization to antibody titers. METHODS: There were 134 adult patients (average age 34.7) who presented to an urban academic emergency department (ED) from October through March during the 2008-2011 influenza seasons with symptoms of fever and a cough. Nasal aspirates were tested by viral culture, and peripheral blood serum was run in seven H1-subtype HI assays. RESULTS: Acutely infected influenza patients had markedly lower antibody titers for six of the seven pseudotype viruses. For the average over the seven titers (log units, base 2) their mean was 7.24 (95% CI 6.88, 7.61) compared with 8.60 (95% CI 8.27, 8.92) among patients who had a non-influenza respiratory illness, p<0.0001. Among patients with seasonal influenza infection, titers of some antibodies correlated with severity of symptoms and with total duration of illness (p<0.02). CONCLUSION: In patients with acute respiratory infections, lower concentrations of H1-influenza-specific antibodies were associated with influenza infection. Among influenza-infected patients, higher antibody titers were present in patients with a longer duration of illness and with higher severity-of-symptom scores.

Cross reactivity of serum antibody responses elicited by DNA vaccines expressing HA antigens from H1N1 subtype influenza vaccines in the past 30 years.

In the past three decades, ten H1 subtype influenza vaccines have been recommended for global seasonal flu vaccination. Some of them were used only for one year before being replaced by another H1 flu vaccine while others may be used for up to seven years. While the selection of a new seasonal flu vaccine was based on the escape of a new emerging virus that was not effectively protected by the existing flu formulation, there is limited information on the magnitude and breadth of cross reactivity among H1 subtype virus circulation over a long period. In the current study, HA-expressing DNA vaccines were constructed to express individual HA antigens from H1 subtype vaccines used in the past 30 y. Rabbits naïve to HA antibody responses were immunized with these HA DNA vaccines and the cross reactivity of these sera against HA antigen and related H1 viruses in the same period was studied. Our data indicate that the level of cross reactivity was different for different viral isolates and the key mutations responsible for the cross reactivity may involve only a limited number of residues. Our results provide useful information for the development of improved seasonal vaccines than can achieve broad protection against viruses within the same H1 subtype.

[Static magnetic fields and its biomedical effects].

Nowadays, health care products based on static magnetic fields (SMF) and merchandise of magnetic therapy are popular around the world. But the biomedical effects of SMF to animals or human beings remain a widely concerned controversy. In this paper, the recent researches in China and abroad about the biomedical effects of SMF were reviewed in three levels: the cellular, animal and human levels. Nevertheless, these data were not consistent with each other and even some contradicts others' researches. So, it is necessary to do more and further studies on SMF dosing regiman, sham control magnetic device and blinding procedures to obtain the optimal magnetic intensity, the desired therapeutic effects in practical cases and prepare for applying the SMF in biomedical fields more effectively in the future.

[Investigation on disturbing of adipocytic differentiation of rBMSCs by loaded titanium particles].

Our previous investigations demonstrated that varying sizes of loaded titanium particles could inhibit proliferaion, adhension and osteoblastic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). The present study aims to validate the hypothesis that particled-shaped wear debris from prosthetic implants influence the adipocytic differentiation of rBMSCs. The effects of different sizes of loaded titanium particles (6.9 microm, 2.7 microm and 0.9 microm) on the adipocytic differentiation of rBMSCs were studied by observing lipid droplet formation, assaying the expression of lipoprotein lipase (LPL) mRNA by RT-PCR and Triglycerides (TG) secretion. The loaded titanium particles were found to influence adipogenesis of rBMSCs, but had different effects, depending upon particle size, concentration and loading time duration. 2.7 microm and 0.9 microm titanium particles promoted lipid droplet formation, LPL mRNA expression and TG secretion, while at a higher concentration of titanium particles and a longer loading duration, 6.9 microm titanium particles gradually inhibited adipogenic differentiation of rBMSCs. Three sizes of loading titanium particles obviously disturbed the adipocytic differentiation capability of rBMSCs: the smaller particles promoted but the larger inhibited the adipogenic differentiation of rBMSCs.

Relationship between concentration difference of different density lipoproteins and shear stress in atherosclerosis.

Previous research has observed concentration polarization in LDL and HDL in the arterial system. However, there is no report that links this concentration polarization to the development of vascular atherosclerosis (AS). Therefore, the purpose of this study is to establish the relationship between concentration difference of LDL and HDL and shear stress using a carotid bifurcation vascular model. PTFE was employed to create the carotid bifurcation model. Endothelial cells were coated on the inner wall of the graft. In a recirculation system, HDL and LDL concentration were measured under two different ICA flow velocities at 5 different locations within our model. We report the following: (1) LDL and HDL concentration difference was observed in both high flow and low flow environments; (2) the degree of LDL and HDL concentration polarization varied depending of high flow and low flow environment; (3) absolute values of concentration difference between LDL and HDL at the inner wall surface decreased with the increase in shear stress when shear stress was more than 1.5 Pa. This variation trend would be more pronounced if shear stress were less than 0.5 Pa. Our study suggests that under the action of shear stress, concentration differences of LDL or HDL create a disturbance in the balance of atherogenic factors and anti-As factors, resulting in the occurrence of AS.

AP-1 and NF-κB transcriptionally regulate interleukin-8 in EA.Hy926 cells under shear stress.

Cardiovascular and cerebrovascular diseases remain the leading cause of death in the world. AS (atherosclerosis) is not only an inflammatory disease in which chemokines play the main role but also a disorder that is related to blood SS (shear stress). We have investigated the action of IL-8 (interleukin-8) mRNA expression in human endothelial cells line-EA.Hy926 under SS at different intensities and duration. Expression increases with time in an intensity dependent manner. With regard to the transcriptional mechanism involved, transient transfection of the human wild-type IL-8 promoter (-162/+44)/luciferase reporter plasmid, or site mutation of one of the binding sites [AP-1 (activator protein 1) or NF-κB (nuclear factor κB)] in the IL-8 promoter region was investigated. Both AP-1 and NF-κB were essential for SS-activated transcription, with the cells responding to NF-κB activation within minutes. After stimulated at low SS (4.20 dyne/cm2) for 30 min, the P65 subunit was translocated from the cytoplasm to nucleus for at least 60 min, while the cytoplasmic level of IκB (inhibitory κB) gradually decreased. The combined activation of NF-κB and AP-1 are the upstream regulators of low SS-induced IL-8 production in EA.Hy926 cells, which subsequently trigger an inflammatory reaction in endothelium.

[Influence of titanium particles loading on releases of cytokines of osteoblasts].

Aseptic loosening is mainly mediated by bone resorption cytokines in the surrounding area of orthopaedic implants. Our previous investigation demonstrtated that different-sized titanium particles loading can inbibit the osteoblastic differentiation and mineraliztion. In order to investigate the hypothesis that particulate wear debris derived from prosthetic biomaterials affects the release of bone resorption related cytokines, we studied the influence of different-sized titanium particles loading on the osteoblastic cytokines by assaying the secretion of IL-6, IL-10 with use of ABC-quantitative sandwich enzyme-linked immunosorbent assay, and on the expression of osteoclast differentiation factors (ODF) by RT-PCR. The results showed that the 0.9 microm titanium particles promoted osteoblasts producing bone resorption cytokines (IL-6, ODF), and simultaneously secreted bone absorption restraining factor (IL-10) quickly and transitorily. In comparison, the 2.7 microm and 6.9 microm titanium particles,especially the latter primarily promoted osteoblasts secreting bone absorption promoting factors powerfully and slowly. The results suggested that there was a biphasic response appearing in titanium particles loaded-osteoblastic cultures, the level of which varied according to the different size and the loading time of titanium particles. This in vitro experimental result showed that attentaion to the inhibition of bone resorption cytokines stimulated by wear debris and to the screen potential favourable biomaterials for implants must be taken.

[Stimulative and regulative functions of osteoblasts loaded under the titanium particles on osteoclasts].

Our previous studies on the function of the osteoblasts (OBs) have shown that worn titanium particles decrease osteoblast function and promot secretion of bone resorption cytokines of OBs surrounding the synovium-like interface membrane of loosening implants. The current study was aimed to test the hypothesis that osteoclasts (OCs) bone absorption function is induced by conditioned media (CM) prepared from OBs loaded in the presence or absence of titanium particles (with three mean diameters 6.9 microm, 2.7 microm, and 0.9 microm, respectively). The effects of CM on OCs function were examined using a combination of the morphological characteristics tests, i.e., TRAP dyeing, scanning electron microscopy, F-actin immunofluorescence protocol for confocal microscopy, bone resorption lacunae assay, osteoclastic calcium tracking, with biochemical evaluation, i.e., C-terminal cross-linked telopeptides of type I collagen evaluated with ABC-ELISA method. The results showed that CM from 0.9 microm titanium particles could induce osteoclastic differentiation and formation, could partially influence the survival of the OCs; while CM of 2.7 microm and 6.9 microm titanium particles, especially the latter, could obviously augmented osteoclastic activity, survival, or differentiation. The stimulation of osteoclast function may be due to a parallel increase in the intracellular free calcium concentration. The present study provides strong support for the hypothesis that osteoclastic activity, survival, or differentiation are very important in the development of aseptic loosening. The development of therapeutic interventions to reduce osteoclastic function and optimization of biomaterials may be useful approaches for improving the performance of orthopaedic implants.

[Potential of cell transplantation as an alternative to liver transplantation].

Human hepatocyte transplantation to treat liver-based metabolic deficiencies and acute liver failure has shown promising early improvement in liver function; however, long-term success has not been achieved. Stem cell transplantation to restore liver function as an alternative to whole liver transplantation has not been successful in humans. As alternative sources of cells for human hepatocyte transplantation, stem cells are under investigation. The liver extracellular matrix presents an ideal scaffold for stem cell differentiation into hepatocytes, as well as cell transplantation. The innovative technique of the decellularized liver matrix presents great potential as the scaffold for hepatocyte maturation and transplantation, and allows the development of engineered recellularized liver graft for transplantation.

Significant evidence of association between polymorphisms in ZNF533, environmental factors, and nonsyndromic orofacial clefts in the Western Han Chinese population.

The etiology of nonsyndromic orofacial clefts (NSOC) has been considered "complex" or "multifactorial." Etiologic heterogeneity induces disparities in the results among different populations. The zinc finger protein 533 (ZNF533) and several environmental factors have been revealed to be associated with NSOC in several populations. We investigated three single-nucleotide polymorphisms (SNPs) and 10 environmental factors in 211 case-parent trios and 188 control individuals in the Western Han Chinese population to confirm the relationship between ZNF533, environmental factors, and the etiology of NSOC in the Western Han Chinese population. The transmission disequilibrium test, case-control analysis, multiple logistic regression, log-linear model, and conditional logistic regression were tested to confirm the contribution of the ZNF533 gene and environmental factors to the etiology of NSOC. Strong statistically significant evidence of association was found between the rs6757845 and rs1139 markers and NSOC. The haplotype G-G for rs6757845-rs1139 showed significant overtransmission among cleft lip with or without cleft palate (CL/P) trios and among cleft palate only trios. Additional 11 and 5 haplotypes were significantly overtransmitted and undertransmitted among CL/P and among cleft palate only trios, respectively. Maternal disease, use of medication, and passive smoking during the first trimester of pregnancy may increase the risk of NSOC. Maternal folic acid supplementation during the first trimester of pregnancy showed a protective effect on the etiology of NSOC. Genotype-environment interaction test showed a significant evidence of interaction effects between the genotypes at rs6757845 and maternal passive smoking during the first trimester among CL/P trios. These results confirm the effects of the ZNF533 gene and environmental factors on the etiology of NSOC.

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration.

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.