Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

[Phylogenetic relationships of seabuckthorn based on ITS sequences].

Sequences of the internal transcribed spacer (ITS) was compared in three seabuckthrons (Hippophae rhamnoides subsp. sinensis, H. tibetana and H. neurocarpa) distributed in Qinghai Province, then the systematic positions of 15 seabuckthron samples were analyzed with Elaeagnaceae angustifolia being outgroup. The results showed that ITS sequences in three seabuckthron species varied in length from 600 to 605 bp. ITS-1, 5.8S and ITS-2 varied from 201 to 203 bp, 166 to 167 bp and 232 to 236 bp, respectively. The sequence divergence among three seabuckthorn species was also remarkably high; Cluster analysis based on ITS indicated that H. gonicocarpa subsp. litangensis and H. gonicocarpa subsp. goniocarpa were distinct and could be classified as H. gonicocarpa and H. litangensis. H. gyantsensis had the closest genetic relationship with H. salicifolia and the distant relationship with H. rhamnoides subsp. sinensis. The positions of nine subspecies of H. rhamnoides based on morphological classification were different from the results of ITS.

Cytopathic effect of classical swine fever virus NS3 protein on PK-15 cells.

In order to further research the relationship between classical swine fever virus' (CSFV) NS3 protein and the cytopathic effect (CPE) in cells infected with the CSFV, and to reveal the effect of protein NS3 on the host cells, the NS3 of CSFV Shimen strain amplified by RT-PCR was subcloned into the pEGFP-C1, named pEGFP-C1-NS3. The insert position, the size and the reading frame were correct for restriction enzyme digestion and sequence analysis. The pEGFP-C1-NS3 and pEGFP-C1 were transfected into PK-15 cells by liposome, and positive cell clones were gained by G418. The NS3-EGFP fusion protein expressed in pEGFP-C1-NS3 cells was observed by inverted fluorescence microscopy and identified by Western blot. The CPE appeared in positive pEGFP-C1-NS3 cells 72 h after passaging, apoptosis detection was also performed on positive pEGFP-C1-NS3 cells and pEGFP-C1 cells 72 h after passaging by TUNEL assay. The apoptosis rates in the positive pEGFP-C1-NS3 and pEGFP-C1 cells were 43.4 and 13.1%, respectively (p < 0.05). The results suggest that the CPE in positive pEGFP-C1-NS3 cells was induced by apoptosis and there is a relationship between the expression of NS3 and apoptosis.