OBJECTIVES: To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval （PMI）. METHODS: Twelve individuals with known PMI （range from 4.3 to 22.5 h） were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. RESULTS: 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β-actin was 24.6%, while GAPDH was 41.0%. CONCLUSIONS 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β-actin correlates well with PMI, which can be used as an additional index for early PMI estimation.
Actins/analysis* ; Autopsy ; Brain/metabolism* ; Humans ; MicroRNAs/analysis* ; Models, Theoretical ; Postmortem Changes ; RNA Stability ; RNA, Ribosomal, 18S/analysis* ; RNA, Ribosomal, 5S/analysis* ; RNA, Small Nuclear/analysis* ; Real-Time Polymerase Chain Reaction ; Software

Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry.
Base Sequence ; Cloning, Molecular ; Cold Temperature ; Escherichia coli ; metabolism ; Lipase ; genetics ; metabolism ; Open Reading Frames ; Penicillium ; enzymology ; genetics ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S ; genetics ; Recombinant Proteins ; genetics ; metabolism

In order to transform main active ingredient of volatile oil, endophytic fungi were screened from the root of Atractylodes lancea. Transformation method was used in vitro. The changes of volatile oil were traced by gas chromatography. One endophytic fungus (strain ALG-13) which could uitilize volatile oil selectively was screened. Single factor experiment were conducted for exploring the effects of various factors that including kinds of carbon source, speed, liquid volume, pH and concentration of plant tissue on degradation by this strain. Subsequently, the main affecting factors carbon source, speed, pH and liquid volume were optimized using orthogonal array design. Results showed that endophytic fungus ALG-13 selectively used the volatile oil, change the relative percentage of the main components of volatile oil, Atractylon and Atractydin were increased, While, beta-eudesmol and Atractylol decreased. After selectively degradation by fungus, volatile oil components percentage were closer to the geo-herbs. Strain ALG-13 was identified as Bionectria ochroleuca according to its morphological characteristics and systematic analysis of ITS sequence. The optimal conditions were as follows: sucrose used as carbon source, rotating speed was 200 r x min(-1), initial pH for medium was 4.5, 50 mL liquid was added in 250 mL flask. The endophytic fungus ALG-13 could degrade the volatile oil selectively, which was benefit for forming geoherbs A. lancea volatile oil composition.
Atractylodes ; chemistry ; microbiology ; Fungi ; classification ; genetics ; isolation & purification ; metabolism ; Oils, Volatile ; chemistry ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; genetics

OBJECTIVETo screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.

METHODEndophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.

RESULTFungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.

CONCLUSIONEndophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Acetylcholinesterase ; metabolism ; Acremonium ; genetics ; metabolism ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Chromatography, Thin Layer ; DNA, Fungal ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; Diazonium Compounds ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Huperzia ; microbiology ; Hydrolysis ; Naphthaleneacetic Acids ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; classification ; genetics ; RNA, Ribosomal, 5.8S ; classification ; genetics ; Sequence Analysis, DNA

OBJECTIVE: To explore the stability of internal controls in human cardiac muscle by real-time RT-PCR during early postmortem interval (PMI) in order to find the most stable marker. METHODS: Ten individuals with similar environmental conditions (the average store temperature: 25 degrees C) and different PMI ranging from 4.3 to 22.3 h were selected. Total RNA was extracted from each sample and six commonly internal controls were used including beta-actin, GAPDH, B2M, U6, 18S rRNA and HSA-miR-1, and the expression was detected in cardiac muscle by real-time RT-PCR. The expression stability of internal controls was evaluated using genormPLUS software during early PMI. The internal control with the most stability was selected. The relationship between the most stable marker and its expression level affected by some other parameters such as age, gender and cause of death was also analyzed. RESULTS: The U6 showed the most stable expression during early PMI in cardiac muscle, and its expression level was not affected by those parameters including age, gender and cause of death (P > 0.05). CONCLUSION U6 may be a valuable internal control for the study of relationship between PMI determination and degradation of nucleic acid in human cardiac muscle by real-time RT-PCR.
Actins/metabolism* ; Adolescent ; Adult ; Cause of Death ; Evaluation Studies as Topic ; Female ; Forensic Pathology/methods* ; Humans ; Male ; MicroRNAs/metabolism* ; Middle Aged ; Myocardium/metabolism* ; Postmortem Changes ; RNA/metabolism* ; RNA Stability ; RNA, Ribosomal, 18S/metabolism* ; RNA, Small Nuclear/metabolism* ; Real-Time Polymerase Chain Reaction/methods* ; Time Factors ; Young Adult

OBJECTIVE: To investigate the degradation changes of beta-actin mRNA and 18S rRNA in different time points and temperature after death, and to explore the relationship between the changes and postmortem interval (PMI) in the brain of mice. METHODS: Twenty-four health adult C57BL/6 mice were randomly divided into two groups (12 each group). They were sacrificed by cervical dislocation and placed in chamber with two different temperature (4 degrees C and 37 degrees C, humidity was 80%). The mice brains were sampled at 6 different time points(immediately, 0.5h, 2h, 6h, 24h, 48h), and total brain RNA were extracted. Ct value of each sample was obtained using RT-PCR and real-time PCR technology, and beta-actin mRNA and 18S rRNA content ratio was calculated. The correlation between the content ratio and PMI was expressed using statistical regression analysis. RESULTS: At 37 degrees C, RNA degradation rate was faster than 4 degrees C, which showed that there was correlation between temperature and RNA degradation. Comparing with the stability of beta-actin mRNA, 18S rRNA was more stable. CONCLUSION The study on degradation of beta-actin mRNA and 18S rRNA in mice brain using real time PCR technology could provide a new theoretical basis for estimation of PMI and would be supplementary to the traditional methods.
Actins/metabolism* ; Animals ; Brain/metabolism* ; Female ; Forensic Medicine/methods* ; Male ; Mice ; Mice, Inbred C57BL ; Postmortem Changes ; RNA Stability ; RNA, Messenger/metabolism* ; RNA, Ribosomal, 18S/metabolism* ; Random Allocation ; Real-Time Polymerase Chain Reaction/methods* ; Regression Analysis ; Temperature ; Time Factors

With the development of molecular biology, the evidences of genetics has been used widely in forensic sciences. DNA technology has played an important role in individual identification and paternity testing, RNA technology is showing more and more wide application in prospect. This article reviews the application and progress of RNA in forensic science including estimation of postmortem interval, bloodstain age, wound age, as well as determination of cause of death and the source of body fluids.
Actins/metabolism* ; Animals ; Blood Stains ; Body Fluids/metabolism* ; Cause of Death ; Forensic Medicine/methods* ; Gene Expression ; Genetic Markers/genetics* ; Humans ; MicroRNAs/genetics* ; Polymerase Chain Reaction/methods* ; Postmortem Changes ; RNA/genetics* ; RNA Stability ; RNA, Messenger/metabolism* ; RNA, Ribosomal, 18S/genetics*

OBJECTIVE: To investigate the expression of 18S rRNA and beta-actin mRNA in bloodstain between 8 and 15 days after death and extrapolate the time of bloodstain formation. METHODS: RNA in dried bloodstain at different times was extracted, then quantified for 18S rRNA and beta-actin mRNA by real-time RT-PCR. The bloodstain formation time was deduced based on the changes of the ratio of 18S rRNA to beta-actin mRNA at different time points. RESULTS The ratio of 18S rRNA to beta-actin mRNA increased gradually with time, indicating that rRNA and mRNA degraded in different rate with time. CONCLUSION; The ratio of 18S rRNA to beta-actin mRNA could be used for estimating the time of bloodstain formation in some period.
Actins/metabolism* ; Blood Stains ; DNA, Complementary/genetics* ; Forensic Medicine/methods* ; Humans ; Postmortem Changes ; RNA, Messenger/metabolism* ; RNA, Ribosomal, 18S/metabolism* ; Real-Time Polymerase Chain Reaction ; Time Factors

OBJECTIVE: To explore the relationship between the time-dependent level changes of microRNA and 18S rRNA and the different postmortem interval (PMI) in rat cardiac muscle. METHODS: SD rats were sacrificed by cervical dislocation and placed at ambient temperature 25 degrees C with a humidity of 50%. Total RNA was extracted from the rat cardiac muscle at different time points after death. The levels of miR-1-2 and 18S rRNA were examined using real-time PCR in rat cardiac muscle. The results were expressed by cycle threshold (Ct) value to explore relationship between PMI and Ct value, and the regression functions were established to estimate PMI. RESULTS: The miR-1-2 level in rat myocardial tissue showed no significant changes within 120 h after death, and then began to decline. The 18S rRNA level increased gradually within 96 h after death, and then declined slowly. The nonlinear relationships were established between Ct value (18S rRNA), deltaCt value (difference between 18S rRNA and miR-1-2) and PMI. The R2 of conics fitting were 0.9487 and 0.8072, respectively. CONCLUSION Ct value of 18S rRNA and deltaCt value present a good correlation with PMI, and can be markers for estimating early PMI.
Animals ; Forensic Pathology ; Male ; MicroRNAs/metabolism* ; Myocardium/pathology* ; Postmortem Changes ; RNA Stability ; RNA, Messenger/metabolism* ; RNA, Ribosomal, 18S/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction/methods* ; Time Factors

The endosymbionts of 4 strains of Acanthamoeba (KA/E9, KA/E21, KA/E22, and KA/E23) isolated from the infected corneas of Korean patients were characterized via orcein stain, transmission electron microscopic examination, and 16S rDNA sequence analysis. Double membrane-bound, rod-shaped endosymbionts were distributed randomly throughout both the trophozoites and cysts of each of Acanthamoeba isolates. The endosymbionts of KA/E9, KA/E22, and KA/E23 were surrounded by electron-translucent areas. No lacunae-like structures were observed in the endosymbionts of KA/E21, the bacterial cell walls of which were studded with host ribosomes. Comparative analyses of the 16S rDNA sequences showed that the endosymbionts of KA/E9, KA/E22 and KA/E23 were closely related to Caedibacter caryophilus, whereas the KA/E21 endosymbiont was assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. In the 4 strains of Acanthamoeba, the hosts of the endosymbionts were identified as belonging to the Acanthamoeba castellanii complex, which corresponds to the T4 genotype. Acanthamoeba KA/E21 evidenced characteristics almost identical to those of KA/E6, with the exception of the existence of endosymbionts. The discovery of these endosymbionts from Acanthamoeba may prove essential to future studies focusing on interactions between the endosymbionts and the amoebic hosts.
Acanthamoeba/genetics/isolation & purification/*microbiology ; Acanthamoeba Keratitis/*microbiology/*parasitology ; Animals ; Bacteria/*genetics/isolation & purification ; Base Sequence ; Cornea/microbiology/*parasitology ; DNA, Mitochondrial/genetics ; Humans ; Korea ; Microscopy, Electron, Transmission/methods ; Oxazines/metabolism ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 18S/genetics ; Symbiosis