A simple and efficient method for isolating polymorphic microsatellites from cDNA

<p>Abstract</p> <p>Background</p> <p>Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.</p> <p>Results</p> <p>The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.</p> <p>Conclusion</p> <p>Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.</p

Acoustic black holes from supercurrent tunneling

We present a version of acoustic black holes by using the principle of the Josephson effect. We find that in the case two superconductors $A$ and $B$ are separated by an insulating barrier, an acoustic black hole may be created in the middle region between the two superconductors. We discuss in detail how to describe an acoustic black hole in the Josephson junction and write the metric in the langauge of the superconducting electronics. Our final results infer that for big enough tunneling current and thickness of the junction, experimental verification of the Hawking temperature could be possible.Comment: 15pages,1 figure, to appear in IJMP

Effects of two Lactobacillus strains on lipid metabolism and intestinal microflora in rats fed a high-cholesterol diet

<p>Abstract</p> <p>Background</p> <p>The hypocholesterolemic effects of lactic acid bacteria (LAB) have now become an area of great interest and controversy for many scientists. In this study, we evaluated the effects of <it>Lactobacillus plantarum </it>9-41-A and <it>Lactobacillus fermentum </it>M1-16 on body weight, lipid metabolism and intestinal microflora of rats fed a high-cholesterol diet.</p> <p>Methods</p> <p>Forty rats were assigned to four groups and fed either a normal or a high-cholesterol diet. The LAB-treated groups received the high-cholesterol diet supplemented with <it>Lactobacillus plantarum </it>9-41-A or <it>Lactobacillus fermentum </it>M1-16. The rats were sacrificed after a 6-week feeding period. Body weights, visceral organ and fat pad weights, serum and liver cholesterol and lipid levels, and fecal cholesterol and bile acid concentrations were measured. Liver lipid deposition and adipocyte size were evaluated histologically.</p> <p>Results</p> <p>Compared with rats fed a high-cholesterol diet but without LAB supplementation, serum total cholesterol, low-density lipoprotein cholesterol and triglycerides levels were significantly decreased in LAB-treated rats (p < 0.05), with no significant change in high-density lipoprotein cholesterol levels. Hepatic cholesterol and triglyceride levels and liver lipid deposition were significantly decreased in the LAB-treated groups (p < 0.05). Accordingly, both fecal cholesterol and bile acids levels were significantly increased after LAB administration (p < 0.05). Intestinal <it>Lactobacillus </it>and <it>Bifidobacterium </it>colonies were increased while <it>Escherichia coli </it>colonies were decreased in the LAB-treated groups. Fecal water content was higher in the LAB-treated groups. Compared with rats fed a high-cholesterol diet, administration of <it>Lactobacillus plantarum </it>9-41-A resulted in decreases in the body weight gain, liver and fat pad weight, and adipocytes size (p < 0.05).</p> <p>Conclusions</p> <p>This study suggests that LAB supplementation has hypocholesterolemic effects in rats fed a high-cholesterol diet. The ability to lower serum cholesterol varies among LAB strains. Our strains might be able to improve the intestinal microbial balance and potentially improve intestinal transit time. Although the mechanism is largely unknown, <it>L. plantarum </it>9-41-A may play a role in fat metabolism.</p

Biofeedback Therapy Combined with Traditional Chinese Medicine Prescription Improves the Symptoms, Surface Myoelectricity, and Anal Canal Pressure of the Patients with Spleen Deficiency Constipation

In order to observe the clinical therapeutic effects of Yiqi Kaimi Prescription and biofeedback therapy on treating constipation with deficiency of spleen qi, the 30 cases in the control group were given oral administration of Yiqi Kaimi Prescription, in combination with anus-lifting exercise; the 30 cases in the treatment group were given biofeedback therapy on the basis of the afore mentioned methods for the control group. The TCM symptom scores and anorectal pressures before and after treatment were observed and evaluated. There were significant differences in TCM symptom scores, anorectal pressure, and clinical recovery rate before and after treatment. In the treatment group, the total recovery rate was 86.66%, while in the control group it was 50%; there were significant differences between the two groups (P<0.01). Yiqi Kaimi Prescription coupled with biofeedback therapy is clinically effective for treating constipation with deficiency of spleen qi, and thus this method is applicable for functional constipation with deficiency of spleen qi

Stage-Specific Expression of TNFα Regulates Bad/Bid-Mediated Apoptosis and RIP1/ROS-Mediated Secondary Necrosis in Birnavirus-Infected Fish Cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0–6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6–12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12–18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses