DNA Gel Electrophoresis Research Articles

Targeting Photoinduced DNA Destruction by Ru(II) Tetraazaphenanthrene in Live Cells by Signal Peptide.

Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding of the complex within the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nuclear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal control by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.

Synthesis and Biological Evaluation of Bisthiazoles and Polythiazoles.

Small heterocyclic compounds containing nitrogen and sulfur atoms, such as thiazole derivatives, represent a significant class of organic azoles that exhibit promising bioactivities and have a great potential in medicinal and agricultural fields. A convenient and high-yielding synthetic approach for a range of organic molecules is presented. The nuclease-like activities of compounds were studied with the aid of E. coli AB1157 DNA and agarose gel electrophoresis. The antioxidant evaluation of the compounds was carried out with different antioxidant techniques, such as ABTS and NO scavenging efficiency. The antibacterial behavior was evaluated against various bacterial strains, both Gram-positive and -negative, and the minimum inhibitory concentration (MIC) values of these compounds were determined. The antiproliferative activities and IC50 values of the synthesized organic molecules compounds against HEPG-2, MCF-7, and HCT-116 cell lines were evaluated.

Thiazole orange as an everyday replacement for ethidium bromide and costly DNA dyes for electrophoresis.

DNA gel electrophoresis is a standard tool of biochemistry and molecular biology laboratories. The common dye ethidium bromide suffers from toxicity concerns and requires the use of damaging ultraviolet light. We observe that exposing plasmid DNA to a UV transilluminator for only 1 s results in detectable loss of colonies following transformation, suggesting rapid accumulation of DNA damage. SYBR Safe, a commercial product, is marketed as a safe alternative to ethidium bromide and has excellent sensitivity with nondamaging blue light, but suffers from prohibitively high costs. We show that thiazole orange, the parent compound of SYBR Safe, is an excellent, simple, and inexpensive alternative to these dyes. It is excitable with safe blue light or UV light, with DNA detection limits in agarose gels similar to ethidium bromide and SYBR Safe (1-2ng/lane). Thiazole orange safely allows the use of nondamaging blue light at the same cost as ethidium bromide.

Synthesis of Tetra 3,4‐dimethoxyphenethoxy Peripheral Substituted Metallophthalocyanines and Investigation of Some Properties

AbstractThe synthesis and characterization of tetra 3,4‐dimethoxyphenethoxy substituted cobalt, zinc and copper phthalocyanines are reported. These compounds have been characterized by using electronic absorption, nuclear magnetic resonance spectroscopy, infrared spectroscopy, and elemental analysis. The aggregation behaviors were investigated in different concentrations phthalocyanine complexes. The photodegradation stabilities of pthalocyanine complexes 4–6 were determined in THF. The antioxidant activities of scavenging effect on 1,1‐Diphenyl‐2‐picryl‐hydrazyl (DPPH) radicals and ferrous chelating reducing power were also investigated. The antimicrobial effects of compounds were evaluated. In addition, DNA cleavage activities of the ligand and its phthalocyanine complexes were experimented on plasmid DNA (pBR322) using DNA gel electrophoresis and the complexes exhibited promising cleavage activity. Thermal properties of compounds were determined by thermogravimetric analysis(TG) and differential thermal analysis (DTA).

Qualitative and quantitative assessment of DNA quality of frozen beef based on DNA yield, gel electrophoresis and PCR amplification and their correlations to beef quality

Freezing is a practical method for meat preservation but the quality of frozen meat can deteriorate with storage time. This research investigated the effect of frozen storage time (up to 66 months) on changes in DNA yield, purity and integrity in beef, and further analyzed the correlation between beef quality (moisture content, protein content, TVB-N value and pH value) and DNA quality in an attempt to establish a reliable, high-throughput method for meat quality control. Results showed that frozen storage time influenced the yield and integrity of DNA significantly (p < 0.05). The DNA yield decreased as frozen storage time increased due to DNA degradation. The half-life (t1/2 = ln2/0.015) was calculated as 46 months. The DNA quality degraded dramatically with the increased storage time based on gel electrophoresis results. Polymerase chain reaction (PCR) products from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were observed in all frozen beef samples. Using real-time PCR for quantitative assessment of DNA and meat quality revealed that correlations could be established successfully with mathematical models to evaluate frozen beef quality.

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development.

ABSTRACTUbiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.

Estimation of allele status of β-carotene hydroxylase1 in maize (Zea mays L.) inbred lines

Purpose. Assessing the polymorphism of crtRB1-3’TE gene responsible for the high content of β-carotene in maize inbred lines of Research Institute of Agrarian Business breeding.Methods. DNA isolation, PCR, and gel electrophoresis. Results. Presented in the article are the research results on the allele’s status of the crtRB1 gene with a molecular marker to 3’-ending in 106 new promising inbred maize lines. Three alleles of the gene were identified: allele 1 (543 bp), allele 2 (296 bp) and allele 3 (296+875 bp). According to the electrophoretic separation of the amplification products, the allele 3 is represented by amplification products of two sizes (296 bp and 875 bp) as a result of the adherence of the forward and the two reverse primers. Allele 2 and allele 1 are formed as a result of the adherence of the forward and one reverse primer, so they are represented by amplification products of the single size. The frequency of resulting from PCR alleles was as following: 0.19 for allele 1, 0.36 for allele 2, and 0.45 for allele 3. It was found that allele 1 (543 bp) is linked with the high content of β-carotene in maize grain. According to the research results, 20 lines of maize were identified that contained an allele of 543 bp (allele 1), 38 allele of 296 bp (allele 2) and 48 alleles of 296+875 bp (allele 3).Conclusions. The crtRB1 allele status of the crtRB1-3’TE marker was analyzed. 20 lines of maize were identified which had a favorable allele for the increased accumulation of β-carotene in grain: RL25, RL35, RL39, RL46, RL47, RL50, RL56, RL63, RL64, RL67, RL73, RL74, RL77, RL83, RL85, RL110, RL111, RL112, RL116 and RL121. These lines can be used in further selective work on the high content of β‑carotene in a grain of corn.

Cloning, Over-Expression, and Purification of Carbonic Anhydrase from an Extremophilic Bacterium: An Introduction to Advanced Molecular Biology

The photosynthetic bioreactor research program is a training platform appropriate for introducing advanced molecular biology techniques to undergraduate students and advanced high school biology students. For this advanced molecular biology training exercise, the enzyme carbonic anhydrase was cloned, over-expressed, purified, and functionally characterized. Carbonic anhydrases are industrially important enzymes with potential use in carbon sequestration and biofuel production. Alpha and beta carbonic anhydrases from Photobacterium profundum, a psychrophilic, halotolerant bacterium, were characterized in this study. Carbonic anhydrases that can withstand high salinity and are active at low temperatures can be transformed into oleaginous marine microalgae to enhance biofuel production. Our research program started with a three-day boot camp with lectures in relevant topics of molecular biology, microbiology, and research methods. After the boot camp, the lab phase of the project involved training students to perform polymerase chain reaction, DNA gel electrophoresis, DNA ligation, and bacterial transformation. In the final phase of the project, students were trained in recombinant protein over-expression and protein purification techniques. Here we report successful cloning and over-expression by high school students of two novel carbonic anhydrases from a psychrohalophile with application in biofuel production.

Experimental validation of DNA interactions with nanoparticles derived from metal coupled amphiphiles

In the present report, a facile strategy for the synthesis of copper nanoparticles utilizing copper@cetylpyridinium chloride as the metal precursor in combination with vitamin C, was been developed. Synthesized nanoparticles (NPs) were well characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and powder X-ray diffraction (XRD). The as-obtained NPs were used for binding with deoxyribonucleic acid from calf thymus (CT-DNA). Binding potential of synthesized NPs towards DNA was checked by calculating apparent binding constant and various thermodynamic parameters, like ΔG, ΔH, ΔS and number of binding sites from UV-Vis, circular dichroism, and fluorescence spectroscopy. NPs lead to the change in conformation and mobility of the genomic DNA as notify by the circular dichroism and DNA gel electrophoresis. Synergistic effect of synthesized nanoparticles on DNA was also visualized by the tapping mode atomic force microscopy. Research findings of the present work are expected to have an impact on genomic activities.

Fast detection by loop-mediated isothermal amplification (LAMP) of the three begomovirus species infecting tomato in Panama

Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) of genus Begomovirus (family Geminiviridae) are the only three begomovirus species detected infecting tomato (Solanum lycopersicum L.) in Panama. PYMPV, ToLCSiV and TYMoV induce symptoms of stunting, yellowing, curling, distortion of leaves and reduction of fruit size and cause important economic loses. A loop-mediated amplification under isothermal conditions (LAMP) assay was developed for the individual detection of these three begomovirus species by using a set of three primer pairs specific per each one of them. Amplification products were visualized by gel electrophoresis or direct Gel-Red staining of DNA into the reaction tube. PYMPV, ToLCSiV and TYMoV were detected in total DNA extracts obtained from different plant tissues such as leaves, stems, flowers, fruits and roots of infected tomato plants collected in different production regions of Panama. LAMP sensitivity was similar to that of conventional PCR but, the first procedure was faster and cheaper than the last one. Moreover, all three viruses were successfully detected by LAMP and not by conventional PCR from sap extracts obtained from leaf tissues of infected tomato plants which were embedded into 3MM Whatman paper and stored several days, facilitating the samples processing as well as the material movement among different laboratories. Therefore, LAMP is a specific, rapid and cheap procedure to detect all three begomoviruses infecting tomato in Panama and it is suitable for field surveys and sanitation programs.

Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen.In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established.Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.

Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold.

ABSTRACTLarge icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.

Bufalin induced apoptosis in SCC‑4 human tongue cancer cells by decreasing Bcl‑2 and increasing Bax expression via the mitochondria‑dependent pathway.

The aim of the present study was to investigate the cytotoxic effects of bufalin on SCC-4 human tongue cancer cells. Cell morphological changes and viability were examined using phase contrast microscopy and flow cytometry, respectively. The results indicated that bufalin induced morphological changes and reduced total viable cells. Apoptotic cell death was analyzed by DAPI staining and DNA gel electrophoresis; the results revealed that bufalin induced cell apoptosis. Levels of reactive oxygen species (ROS), Ca2+, nitric oxide (NO) and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry, and bufalin was observed to increase Ca2+ and NO production, decrease the ΔΨm and reduce ROS production in SCC-4 cells. In addition, western blotting was performed to detect apoptosis-associated protein expression. The results demonstrated that bufalin reduced the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and increased the expression of the pro-apoptotic protein, Bcl-2-associated X protein. However, bufalin treatment also increased the expression of other apoptosis-associated proteins such as apoptosis-inducing factor and endonuclease G in SCC-4 cells. Based on these findings, bufalin may induce apoptotic cell death via mitochondria-dependent pathways in human tongue cancer SCC-4 cells.

Biosynthesis, characterization of magnetic iron oxide nanoparticles and evaluations of the cytotoxicity and DNA damage of human breast carcinoma cell lines

Magnetic iron oxide nanoparticles (MNPs) were synthesized using Albizia adianthifolia leaf extract as reducing and protecting agent. Colour changing, UV–Vis spectrum, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) confirmed the biosynthesis and characterization of MNPs. The XRD pattern revealed that MNPs are crystalline in nature. FT-IR spectral analysis showed that MNPs was capped with plant constituents. From SEM analysis, the MNPs were generally found to be spherical in shape and the size was ranged 32–100 nm. Free radical scavenging potentials of the MNPs against DPPH were confirmed based on its stable anti-oxidant effects. The synthesized MNPs were used to capture Staphylococcus aureus under the magnetic field effect. Further, it was observed that the MNPs are able to exert cytotoxic effect towards human breast (AMJ-13) and (MCF-7) cancer cells. The anti-proliferative effect of this treatment is due to cell death and inducing apoptosis. Mitochondrial membrane potential, acridine orange-propidium iodide staining assays as well as single cell and DNA gel electrophoresis analyses indicated that MNPs induce cell death only by apoptosis. The findings of present study suggest that the MNPs might be used for medicinal applications particularly for cancer therapeutics.

PREPARATION OF CALCIUM PHOSPHATE NANOPARTICLES AS NON-VIRAL VECTORS FOR GENE DELIVERY SYSTEM

A major aim of gene therapy is the efficient and specific delivery of therapeutic gene into the desired target tissues. Development of reliable vectors is a major challenge in gene therapy. The aim of this study is to develop calcium phosphate nanoparticles as novel non-viral vectors for the gene delivery system. Calcium phosphate nanoparticles were prepared by water-in-oil microemulsion method with a water to surfactant molar ratio, Wo [Formula: see text] 2–10. This paper studies the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 100[Formula: see text]nm in diameter. The structure of DNA-calcium phosphate nanocomplex observed by TEM was displayed as a shell-like structure. This study used pEGFP as a reporter gene. The encapsulating efficiency to encapsulate DNA inside the nanoparticles was greater than 80%. In the MTT test, both calcium phosphate nanoparticles and DNA-calcium phosphate nanocomplex have no negative effect for 293T cells. By gel electrophoresis of free and entrapped pEGFP DNA, the DNA encapsulated inside the nanoparticles was protected from the external DNaseI environment. In vitro transfection studies in 293T cell-line, the DNA-calcium phosphate nanocomplex could be used safely to transfer the encapsulated DNA into the 293T cells and expression green fluorescent protein. The characteristic of DNA-calcium phosphate nanocomplex to deliver DNA belongs to slow release. The property of DNA-calcium phosphate nanocomplex was fit in the requirement of non-viral vectors for the gene delivery system.

First description in Europe of the emergence of Enterococcus faecium ST117 carrying both vanA and vanB genes, isolated in Greece.

An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.

Postoperative Enterococcus faecalis endophthalmitis: virulence factors leading to poor visual outcome.

Retrospective. E. faecalis isolates from nine acute-onset PBE cases were examined using pulsed-field gel electrophoresis of genomic DNA, plasmid isolation and pheromone response experiments. Virulence determinant genes, including cytolysin (cylA), aggregation substance (asa1), enterococcal surface protein (esp), gelatinase (gelE) and adhesin of collagen from enterococci (ace), were examined by polymerase chain reaction. Expression of cytolysin and secreted protease were also tested. Retinal findings observed during vitrectomy surgery and final visual acuities were compared to obtained microbiological data. Poor visual outcome was defined as final visual acuity of less than 0.1 (decimal). Although nine isolates were collected, two were identical. Out of the eight different isolates, three were positive for both expressions of cytolysin and secreted proteases. Plasmid DNAs were isolated from six of eight isolates, while pheromone response was observed in five isolates. The asa1, esp and ace genes were positive in six, five and eight of the isolates, respectively. Patients with retinal destruction exhibited significantly higher prevalence of secreted protease expression versus those without (P<0.05), and retinal destruction was related to poor visual outcome (P<0.05). Our data suggest that enterococcal secreted proteases were correlated to retinal destruction, thereby fostering poor visual outcomes in eyes with enterococcal endophthalmitis.

Amentoflavone enhances sorafenib-induced apoptosis through extrinsic and intrinsic pathways in sorafenib-resistant hepatocellular carcinoma SK-Hep1 cells in vitro.

The present study aimed to evaluate the effects of amentoflavone on sorafenib-induced apoptosis in sorafenib-resistant hepatocellular carcinoma (HCC) cells. The sorafenib-resistant SK-Hep1 (SK-Hep1R) cell line was established for the present study. Initially, the differences in sorafenib-induced cytotoxicity and apoptosis between wild-type SK-Hep1 and SK-Hep1R cells were verified using the MTT assay and flow cytometry. The effects of amentoflavone on sorafenib-induced cytotoxicity and apoptosis were then investigated using MTT, flow cytometry, DNA gel electrophoresis and western blot analysis. The results demonstrated that cell viability of SK-Hep1R cells was increased compared with that of SK-Hep1 cells following treatment with different concentrations of sorafenib for 24 h. Apoptosis of SK-Hep1R cells was lower than that of SK-Hep1 cells following treatment with 20 µM sorafenib for 24 h. Amentoflavone alone did not inhibit cell viability but significantly triggered sorafenib-induced cytotoxicity and apoptosis in SK-Hep1R cells. Amentoflavone not only reversed sorafenib-induced anti-apoptotic protein levels but also enhanced sorafenib-induced pro-apoptotic protein expression in SK-Hep1R cells. In conclusion, amentoflavone may be used as a sorafenib sensitizer to enhance sorafenib-induced cytotoxicity and trigger sorafenib-induced apoptosis through extrinsic and intrinsic pathways in SK-Hep1R cells.

UBR5 promotes cell proliferation and inhibits apoptosis in colon cancer by destablizing P21.

UBR5 is recently recognized as a key player in a large number of prevalent cancers. In this study, we sought to explore the connection of UBR5 expression with cell proliferation, apoptosis, as well as the regulation mechanism in colon cancer cell line. SiUBR5 or oeUBR5 were separately applied to interfere the expression of UBR5. Western blot, DNA gel electrophoresis and qPCR were performed to detect the expression of UBR5 at mRNA and protein level. Then MTT and flow cytometry were used to explore the proliferation and apoptosis in a colon cancer cell line in vitro. Finally, we explored the interaction and correlation of UBR5 and P21 in the colon cancer regulation. We found that UBR5 was highly expressed in colon cancer not only at mRNA level but also at protein level. Moreover, UBR5 can promote the growth of colon cancer cells, and inhibit apoptosis. The mechanism exploration proved that UBR5 can degrade P21 via ubiquitination. All these findings suggest that UBR5 may be involved in progression of colon cancer and could be a new therapeutic target for this disease.

Effect of Bartonella spp. on the Freezing Tolerance in Female Ixodes scapularis

The aim of the current study was to determine the effect of Bartonella spp. on the freezing tolerance of engorged and non-engorged female Ixodes scapularis (Say). Bartonella is a genus of pathogenic vector-transmitted gram-negative bacteria, implicated in zoonotic disease transmission. I. scapularis ticks collected throughout Nova Scotia, Canada underwent freezing tolerance testing. DNA extraction, PCR and gel electrophoresis were then conducted to determine the presence and prevalence of Bartonella spp. in the experimental population. The experimental results demonstrated that the changes in the freezing tolerance as a result of the presence of Bartonella spp. were not statistically significant. The infection rate observed in the experimental population was 58.61%; this was significantly higher comparable to the infection rate found in earlier research. It was concluded that Bartonella spp. had no altering physiological effect on Ixodes scapularis.