Cloning and expression analysis of piRNA-like RNAs: adult testis-specific small RNAs in chicken

Many small RNAs have been cloned from animal gonads, for example, endogenous small interfering RNAs (endo-siRNAs) were found in oocytes and piwi-interacting RNAs (piRNAs) were found in testis. Gallus gallus (chicken) is an important model organism, but few small RNAs have been identified from its gonads. In this study, we isolated and cloned 156 small RNAs from adult chicken testes. Since there is a reasonably even distribution from 22 to 33 nt, these small RNAs are slightly longer than miRNAs and endo-siRNAs. Genome mapping indicated that these small RNAs were derived from intergenic regions, exons, introns, and repetitive elements including chicken repeat 1, long terminal repeats, and simple repeats. Since they are similar with piRNAs, we named them piRNA-like RNAs (pil-RNAs). Northern blotting of 16 selected sequences showed that nine are specifically expressed in the adult testis. The vast majority of these pil-RNAs are poorly conserved between species, suggesting that they are unique to the adult chicken testis. Further analysis of the cloned pil-RNAs will improve our understanding of the function of small RNAs in animal gonad development.     

The significant increase of FcγRIIIA (CD16), a sensitive marker, in patients with coronary heart disease

Our previous studies suggest that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is closely correlated to coronary heart disease (CHD). However, whether or not deregulated FcγRIIIA expression is involved in the development of CHD remains largely unclear. Herein, we investigated the FcγRIIIA mRNA expression in the leukocytes, the serum protein level of soluble CD16 (sCD16) and membrane CD16 on monocytes from 100 diagnosed CHD patients and 40 healthy individuals. Our results demonstrated that there was a significant increase of FcγRIIIA at the mRNA level in leukocytes, and at the protein level for both sCD16 in sera and membrane CD16 on monocytes from CHD patients compared to the healthy control. Similarly to the soluble CD14 (sCD14), the level of macrophage colony stimulating factor (M-CSF) in sera was also higher in CHD patients than that in the control individuals. Furthermore, the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), in sera and the mean fluorescent intensity of intercellular adhesion molecule 1 (ICAM-1, CD54) on CD14(+) CD16(+) monocytes were increased in CHD patients. Overall, these data demonstrated that FcγRIIIA (CD16) is involved in the pathogenesis of CHD by activating monocytes and stimulating inflammation. The significant increase of CD14(+) CD16(+) monocytes in CHD patients therefore suggested that the increase of the FcγRIIIA level might be a sensitive marker for the CHD diagnosis.     

Covariation in plant functional traits and soil fertility within two species-rich forests

The distribution of plant species along environmental gradients is expected to be predictable based on organismal function. Plant functional trait research has shown that trait values generally vary predictably along broad-scale climatic and soil gradients. This work has also demonstrated that at any one point along these gradients there is a large amount of interspecific trait variation. The present research proposes that this variation may be explained by the local-scale sorting of traits along soil fertility and acidity axes. Specifically, we predicted that trait values associated with high resource acquisition and growth rates would be found on soils that are more fertile and less acidic. We tested the expected relationships at the species-level and quadrat-level (20 × 20 m) using two large forest plots in Panama and China that contain over 450 species combined. Predicted relationships between leaf area and wood density and soil fertility were supported in some instances, but the majority of the predicted relationships were rejected. Alternative resource axes, such as light gradients, therefore likely play a larger role in determining the interspecific variability in plant functional traits in the two forests studied.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morphology always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermediate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence comparison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) intergenic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we examined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species. __________ Translated from Acta Botanica Yunnanica, 2007, 29 (1): 91–97 [译自:云南植物研究]     

Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.     

Preoperative education with image illustrations enhances the effect of tetracaine mucilage in alleviating postoperative catheter-related bladder discomfort: a prospective,randomized, controlled study

Background

Catheter-related bladder discomfort (CRBD), secondary to catheterization of urinary bladder is distressing. The aim of this study was to assess the efficacy of preoperative education on CRBD with image illustration for alleviating CRBD.

Methods

Sixty adult male patients, undergoing elective colonal and rectal surgery, were randomized to receive tetracaine mucilage instilled into the urethra and applied to the catheter (tetracain group), or receive tetracaine mucilage in combination with image illustration on CRBD (image group) before urethral catheterization. The incidence and severity of CRBD were assessed at 0.5, 1, 2, and 6?h after patients’ extubation. The severity of postoperative pain, incidence of postoperative agitation and other adverse events were also recorded.

Results

Patients in image group reported remarkably less CRBD than those in tetracaine group at 0.5,1, 2 and 6?h after extubation (20, 20, 6.7 and 6.7% v.s. 60, 73.3, 53.3 and 53.3%, respectively, P<0.01). Severe CRBD was not reported in either group. However, the incidence of moderate CRBD was significantly lower in image group, with 6.7% at 1?h and thereafter none occurred, compared to 6.7% at 0.5?h, and increasing to 20% at 1?h, 2?h and 6?h in tetracaine group, respectively. Moreover, patients in image group suffered less moderate to severe postoperative pain than that of tetracaine group (13.3% v.s. 40.0% at 1?h, P?=?0.039, 33.3% v.s. 60% at 2?h and 6?h, P?=?0.038).

Conclusions

Preoperative education on uretheral catheterization via image illustrations could enhance the effect of tetracaine mucilage in reducing both the incidence and severity of CRBD.

Trial registration

The trial was registered at www,clinicaltrials.gov with registration number NCT03199105 (retrospectively registered). Date of trial registration which is “June 26, 2017”.

     

Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

Hepatocellular carcinoma is the most frequent primary liver cancer worldwide. The use of antioxidants as cancer prevention and treatment agents has become a focus of research in recent years due to their limited adverse effects. Alpha lipoic acid (ɑ‐LA) is synthesized in the liver and is considered a naturally occurring antioxidant. In this study, a total of 4446 differentially expressed genes (2097 down‐regulated and 2349 up‐regulated) were identified via RNA‐Seq in HepG2 cells after exposure to α‐LA for 24 hrs. Moreover, GO and KEGG pathway analyses showed that cancer‐relevant cell membrane proteins were significantly affected. An interaction network analysis predicted that Grb2 might mediate the key target pathways activated by exposure to ɑ‐LA. Verification of the RNA‐Seq and iTRAQ results confirmed that Grb2 mediated the ɑ‐LA‐induced inhibition of cell proliferation in vitro. Furthermore, the analysis of human hepatocellular carcinoma specimens obtained from the GEO database showed that the expression of EGFR and Met correlated with that of Grb2. These findings provide a novel mechanism through which ɑ‐LA regulates cell proliferation via the down‐regulation of growth factor‐stimulated Grb2 signalling.