5th Passage Research Articles

The effect of the source of microorganisms on adaptation of hydrolytic consortia dedicated to anaerobic digestion of maize silage

The main aim of this study was to evaluate the effect of the source of microorganisms on the selection of hydrolytic consortia dedicated to anaerobic digestion of maize silage. The selection process was investigated based on the analysis of changes in the hydrolytic activity and the diversity of microbial communities derived from (i) a hydrolyzer of a commercial agricultural biogas plant, (ii) cattle slurry and (iii) raw sewage sludge, during a series of 10 passages. Following the selection process, the adapted consortia were thoroughly analyzed for their ability to utilize maize silage and augmentation of anaerobic digestion communities.The results of selection of the consortia showed that every subsequent passage of each consortium leads to their adaptation to degradation of maize silage, which was manifested by the increased hydrolytic activity of the adapted consortia. Biodiversity analysis (based on the 16S rDNA amplicon sequencing) confirmed the changes microbial community of each consortium, and showed that after the last (10th) passage all microbial communities were dominated by the representatives of Lactobacillaceae, Prevotellaceae, Veillonellaceae.The results of the functional analyses showed that the adapted consortia improved the efficiency of maize silage degradation, as indicated by the increase in the concentration of glucose and volatile fatty acids (VFAs), as well as the soluble chemical oxygen demand (sCOD). Moreover, bioaugmentation of anaerobic digestion communities by the adapted hydrolytic consortia increased biogas yield by 10–29%, depending on the origin of the community. The obtained results also indicate that substrate input (not community origin) was the driving force responsible for the changes in the community structure of hydrolytic consortia dedicated to anaerobic digestion.

English

Bovine herpesvirus-1 (BHV-1) causes Infectious bovine rhinotracheitis/Pustular vulvovaginitis in cattle. Glycoprotein D (gD) of BHV-1 represents a major component of the viral envelope and is a dominant immunogen. gD encoding gene was expressed in baculovirus-insect cell system. Viral genomic DNA extracted from BHV-1 grown on Madin-Darby Bovine Kidney (MDBK) cell monolayer was used as a template for PCR amplification of gD gene (1255 bp). Gel purified gD gene was used for directional cloning into pENTR/SD/D Directional TOPO vector to produce entry clone. Recombinant plasmids were screened by PCR and restriction enzyme (RE) digestion for gD gene insert.  Endotoxin free purified plasmids were then subjected to LR recombination reaction with baculovirus linear DNA. LR recombination mix was transfected into Sf-9 cells and observed for appearance of cytopathic effects (CPE). Recombinant virus was serially passaged for 3 more generations and the 4th passage viral stock was used to infect fresh Sf-9 cells for gene expression study. Recombinant gD protein was immunoprecipitated and when subjected to SDS-PAGE and western blot analysis protein band of ~70 kDa was detected consistently. The recombinant gD protein was further weakly confirmed by dot-ELISA indicating its limited potential as a coating antigen in gD-based diagnostic ELISA. Key words: Bovine herpesvirus-1 (BHV-1), Madin-Darby Bovine Kidney (MDBK) cells, Sf-9 cells, pENTR TOPO vector, Baculovirus mediated gene expression, glycoprotein D, eukaryotic expression,

Novel Approach for Isolation and Identification of Porcine Epidemic Diarrhea Virus (PEDV) Strain NJ Using Porcine Intestinal Epithelial Cells.

Porcine epidemic diarrhea virus (PEDV), which is the causative agent of porcine epidemic diarrhea in China and other countries, is responsible for serious economic losses in the pork industry. Inactivated PEDV vaccine plays a key role in controlling the prevalence of PEDV. However, consistently low viral titers are obtained during the propagation of PEDV in vitro; this represents a challenge to molecular analyses of the virus and vaccine development. In this study, we successfully isolated a PEDV isolate (strain NJ) from clinical samples collected during a recent outbreak of diarrhea in piglets in China, using porcine intestinal epithelial cells (IEC). We found that the isolate was better adapted to growth in IECs than in Vero cells, and the titer of the IEC cultures was 104.5 TCID50/0.1 mL at passage 45. Mutations in the S protein increased with the viral passage and the mutations tended towards attenuation. Viral challenge showed that the survival of IEC-adapted cultures was higher at the 45th passage than at the 5th passage. The use of IECs to isolate and propagate PEDV provides an effective approach for laboratory-based diagnosis of PEDV, as well as studies of the epidemiological characteristics and molecular biology of this virus.

Production of Podophyllotoxin by Plant Tissue Cultures of Juniperus virginiana

Plant tissue cultures are a potential source of secondary metabolites. However, their production, when compared with intact plants, is usually lower. Phenylalanine, a biogenetic precursor of podophyllotoxin, was used to stimulate podophyllotoxin production in callus and suspension cultures of Juniperus virginiana L. The best phenylalanine effect on podophyllotoxin production was manifested in three-years-old callus cultures after a 21-days application of a 10 mmol/L concentration. A podophyllotoxin content of 0.15 mg/g DW was determined, which was about 400% higher in comparison with the control. The maximum content (0.48 mg/g DW) in newly derived suspension cultures (the 4' passage) was induced by 14-days application of a I mmol/L concentration; this was about 243% higher than the control. In one-year-old suspension cultures the highest podophyllotoxin content (0.56 mg/g DW) was recorded also after 14-days application of a I mmol/L concentration; this was about 211% higher than in the control cultures.

Genetic stability of human endometrial mesenchymal stem cells assessed with morphological and molecular karyotyping

The aim of this study was to monitor the genetic stability of endometrial mesenchymal stem cells (eMSCs) by G-banding and molecular karyotyping. We evaluated the sensitivity of each method to assess the genetic stability of eMSCs. G-banding karyotyping performed on passages 6 and 15 showed that more than 80% cells had normal karyotype. Random karyotypic changes were found in a small part of the cell population: aneuploidy, isochromosomes, chromosome breakages, interchromosomal association. Molecular karyotyping carried out on the 6th and 14th passages revealed genomic stability, except for in the case of chromosomes 7 and 14. Microduplications 7q36.3 (62 kb) and 14q11.2 (165kb) were found in these chromosomes. We interpreted these aberrations as being derived from the donor of these cells. The morphological and molecular karyotyping complemented each other. Using these methods, we can analyze karyotypic stability at different levels of the genomic organization.

Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes.

Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

Effects of severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts

Objective: To simulate the environmental factors during the process of formation and evolution of hypertrophic scar, so as to explore the effects of moderate and severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts (HSFs). Methods: Human HSFs were routinely cultured. Cells of the 3rd to the 6th passage were divided into 10.0% oxygen+ 10.0% fetal calf serum (FCS), 5.0% oxygen+ 5.0% FCS, and 0.5% oxygen+ 0.5% FCS groups according to the random number table. After being cultured with DMEM nutrient solution with no FCS for 24 h, the cells were cultured with the corresponding volume fraction of oxygen and FCS. Cell proliferation activity was determined with methyl-thiazole-tetrazolium assay (denoted as actual cell number). Content of total collagen was detected with Sirius red staining method (denoted as absorbance value). Protein expression levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), B-cell lymphoma 2 (Bcl-2), and P53 were determined with Western blotting (denoted as ratio of gray value). Cell apoptosis rate was detected by in situ end labeling method. The sample numbers of each group in the above experiments were all 3. Data were processed with Kruskal-Wallis test and Dunnett test. Results: (1) Compared with 11 000±1 306 in 10.0% oxygen+ 10.0% FCS group, the cell proliferation activity was higher in 5.0% oxygen+ 5.0% FCS group (13 290±1 500, P<0.05), but lower in 0.5% oxygen+ 0.5% FCS group (6 999±765, P<0.05). (2) Compared with 0.039 6±0.004 2 in 10.0% oxygen+ 10.0% FCS group, the content of total collagen of cells was higher in 5.0% oxygen+ 5.0% FCS group (0.051 6±0.005 1, P<0.05), but lower in 0.5% oxygen+ 0.5% FCS group (0.015 6±0.002 4, P<0.05). (3) Compared with those in 10.0% oxygen+ 10.0% FCS group, the protein expression levels of HIF-1α, VEGF, TGF-β1, and Bcl-2 were increased (with P values below 0.05), with no obvious difference in protein expression level of P53 in 5.0% oxygen+ 5.0% FCS group (P>0.05), whereas the protein expression levels of HIF-1α, VEGF, TGF-β1, and Bcl-2 were decreased (with P values below 0.05), while the protein expression level of P53 was increased in 0.5% oxygen+ 0.5% FCS group (P<0.05). (4) Compared with (1.2±0.9)% in 10.0% oxygen+ 10.0% FCS group, the cell apoptosis rate in 5.0% oxygen+ 5.0% FCS group showed no significant difference [(2.6±0.9)%, P>0.05], while it was significantly increased in 0.5% oxygen+ 0.5% FCS group [(13.3±4.1)%, P<0.05]. Conclusions: Severe hypoxia and low concentration of serum protein can inhibit proliferation activity and production of total collagen of human HSFs and induce their apoptosis.

First detection of canine parvovirus type 2b from diarrheic dogs in Himachal Pradesh.

Aim:The present study was conducted to detect the presence of canine parvovirus (CPV) among diarrheic dogs in Himachal Pradesh and to identify the most prevalent antigenic variant of CPV based on molecular typing and sequence analysis of VP2 gene.Materials and Methods:A total of 102 fecal samples were collected from clinical cases of diarrhea or hemorrhagic gastroenteritis from CPV vaccinated or non-vaccinated dogs. Samples were tested using CPV-specific polymerase chain reaction (PCR) targeting VP2 gene, multiplex PCR for detection of CPV-2a and CPV-2b antigenic variants, and a PCR for the detection of CPV-2c. CPV-2b isolate was cultured on Madin-Darby canine kidney (MDCK) cell lines and sequenced using VP2 structural protein gene. Multiple alignment and phylogenetic analysis was done using ClustalW and MEGA6 and inferred using the Neighbor-Joining method.Results:No sample was found positive for the original CPV strain usually present in the vaccine. However, about 50% (52 out of 102) of the samples were found to be positive with CPV-2ab PCR assay that detects newer variants of CPV circulating in the field. In addition, multiplex PCR assay that identifies both CPV-2ab and CPV-2b revealed that CPV-2b was the major antigenic variant present in the affected dogs. A PCR positive isolate of CPV-2b was adapted to grow in MDCK cells and produced characteristic cytopathic effect after 5th passage. Multiple sequence alignment of VP2 structural gene of CPV-2b isolate (Accession number HG004610) used in the study was found to be similar to other sequenced isolates in NCBI sequence database and showed 98-99% homology.Conclusion:This study reports the first detection of CPV-2b in dogs with hemorrhagic gastroenteritis in Himachal Pradesh and absence of other antigenic types of CPV. Further, CPV-specific PCR assay can be used for rapid confirmation of circulating virus strains under field conditions.

Baby Grivet Monkey Kidney (BGM) a new cell line for adaptation of fowl pox virus

The need to economic, sensitive and easily maintained cell cultures use for massive production of fowl pox vaccine wasnecessary. We adapted Fowl pox virus (FPV) Baudette strain on Primary chicken embryo fibroblast (CEF), Baby GrivetMonkey Kidney cell line (BGM) and African green monkey kidney (Vero) cell lines. The highest virus titers were 106.5after the 9th passage in CEF and 106.2 after the 12th passage on BGM while Vero cells were abortive to the virus. BGMcells were selected for study cytopathic effect (CPE) and growth kinetic as it easily maintained than primary CEF cells.The characteristic CPE were rounding till 6th passage with intracytoplasmic inclusion by the 7th passage and syncytiumformation appeared from the 9th passage till the 13th passage. The virus was highly cell associated for the first 84h postinoculation(PI) with a titer of 104.5 then a maximum titer was 106.2 in cell free portion after 120h PI. In conclusion, BGMwere considered a new susceptible cell for growth of FPV with best harvesting time 120h PI to obtain a maximum titerfor subsequent vaccine production

Microsaccades during reading

Background. Extensive research has focused on the control of saccades during reading. However, very little is known in this context about microsaccades (saccades< 0.5 deg), i.e., whether they occur and how they shift the gaze. Using new methods for precisely localizing the line of sight, we recently showed that in high acuity tasks microsaccades precisely position the preferred retinal locus, the tiny region of maximum acuity within the foveola (Ko et al, 2010; Poletti et al, 2013). These findings raise the possibility that microsaccades may also play a role in reading. Methods: Observers (N=10) were instructed to read 10th grade passages of text and answer questions regarding their content. Eye movements were recorded by means of a high resolution DPI eyetracker. Gaze-contingent calibrations were used to accurately localize the preferred retinal locus. Results: Microsaccades occur during reading: the average rate across all subjects was 18 microsaccades per minute. Interestingly, their characteristics differed from those measured when fixating on a marker. During fixation, microsaccades had approximately equal probability to shift the gaze to the left or to the right, and a considerable number (~32%) possessed vertical components. In contrast, during reading, most microsaccades (61%) directed the gaze leftwards and only 16% vertically. Therefore, microsaccades primarily relocated the gaze backward on the text, most of them just 2-3 letters back from the end of long words. Further analyses showed that this pattern of microsaccades differed significantly from what would be obtained by chance. Conclusion: Our results show that microsaccades occur and are controlled during reading. It is well known that the perceptual span normally extends asymmetrically toward the reading direction. Microsaccade backward shifts could compensate for this asymmetry. Meeting abstract presented at VSS 2016

Establishment and characterization of a clear cell carcinoma cell line, designated NOCC, derived from human ovary.

A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70-74 at the 30th passage. NOCC cells secreted 750.5ng/ml of VEGF over 3days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction.

In vitro assessment of growth kinetics and differentiation potential of bone marrow derived mesenchymal and fetal fibroblast cells of caprine

The present study assessed comparative growth kinetics and differentiation potential of mesenchymal and fetal fibroblast cells of goats in vitro. The mesenchymal cells were isolated from bone marrow and fibroblasts were obtained from skin of 3–4 months old fetus. During culture, more than 99% viability of both cell groups indicated optimum culture conditions for in vitro experiments. The population doubling time (PDT) of BM-MSCs and FFCs were similar at third (18.25±0.05 and 18.41±0.07) and seventh (19.61±0.06 and 19.68±0.12) passages. But at 12th passage, FFCs cells had a significantly higher PDT (35.77±0.68 vs 32.063±1.0) as compared to BM-MSCs. Both groups of cells showed a characteristic sigmoid curve on plotting cell concentration verses days of culture. The stem cell like property was exhibited by BM-MSCs by differentiating into osteoblast, chondrocytes and adipocytes under specific media, which were further confirmed by specific staining of cells. Also, the BM-MSCs showed colonogenic property by forming different number of clones at third (60.5±0.59), seventh (56.6±0.5) and 12th (33.4±0.45) passages. The fetal fibroblast cells did not exhibit colonogenic and differentiation potentials. This study concluded that although BM-MSCs and FFCs shared similar morphology and growth kinetics during initial passages but FFCs does not exhibit the stem cell like properties in terms of differentiation potency.

Characterization of Recent Isolates of Newcastle Disease virus

Fifty five tissue samples were collected aseptically from 5 flocks suspected to be naturally infected with NDV in EL- Ismalia, Al-sharkia, El- Gharbia, kafr-Elshiekh & El-Geza and subjected to trials of virus isolation. Nine field samples from 2 flocks (Kafr-Elshiekh and Giza) were positive for virus isolation. Inoculation of SPF chicken eggs induced death of the embryo within 48-72 hours post inoculation. HA test applied on the obtained allantoic fluids showed positive results suggesting that inoculated samples contained NDV. It was noticed that highest infectivity titer of the virus was 9.5 log10 EID50/ml by the 5th passage in SPF chicken eggs. Cell culture passage of the obtained virus isolate from the allantoic fluid through ten consecutive passages in BHK cell line. the CPE developed at the 3rd passage within 24-48 hours post cell infection where polykaryocytosis, cell syncitia, rounding of cells. Tissue cultured fluids (TCF) were found to be direct HA possitive. The virus titer was 3 log10 TCID50 /ml at the 3rd passage and reach to 7 log10 TCID50 /ml by the 6th passage. At the 9th passage, the virus titer was reached to 8.5 log10 TCID50 /ml. Identification of the obtained virus isolate was confirmed by VNT using specific anti-NDV in BHK cell. The isolate of NDV with HA-positive allantoic fluids of El-Giza flockes were virulent NDVand the F gene give expected size of 724 bp. The isolate of this study also compared against the reference and vaccinal strains from gene bank and the results revealed that the isolate NDV/chicken/Egypt/Giza/2015 was velogenic type resembling the genotype VIId strains. F gene analysis of the NDV/chicken/Egypt/Giza/2015 revealed that the most nucleotides identity from gene bank with this isolate were NDV/Chicken/GC/IS/2010/1224 and NDV/buzzard/Israel/714/2011 with a nucleotide identity of 92.7%. It was also closely related to Chicken/China/SDWF07/2011; Chicken/China/Shandong/01/2012 and NDV/Turkey/Israel/111/2011 with a nucleotide identity of 92.5%. Otherwise nucleotides identity between the selected NDV field isolate in this study and NDV/Lasota was 72.4%. Phylogenetic analysis of NDV/chicken/Egypt/Giza/2015 with other reference and vaccinal strains of NDV revealed that this isolate was related to NDV/B7/RLQP/CH/EG/12 and present in the same group with NDV/F388/RLQP/CH/EG/14 and NDV/F460/RLQP/CH/EG/13. Regarding evaluation of the prepared experimental vaccines, it was found that the NDV infected tissue culture fluid, were completely inactivated with 3% BEI at 30 °C for 18 hrs post treatments showing no pathological changes in the ECE and no CPE in the BHK cell culture. Humoral immune response of chickens to the prepared vaccines showed that vaccinated chicks with NDV-Montanide ISA 70 adjuvanted vaccine exhibited antibody titer of 8.3 log2 three weeks post vaccination recording the higher titer of (10.6 log2) at the 6th week post vaccination with and 3rd week post challenge test. The high antibodies values were persist till the 12th weeks post NDV-Montanide ISA 70 vaccination followed by gradual decrease till the end of the experiment (24th weeks). On the other hand, vaccination of chicks with inactivated NDV isolate without adjuvant failed to induce protective HI antibodies (3 log2) all over the experiment. Challenge test showed that chickens vaccinated with ISA-70 adjuvant vaccine showed no sign of disease and no mortalities while the antigen without adjuvant could not provide any protective efficiency to SPF chickens.

Swine small intestine submucosa combined with bone marrow mesenchymal stem cells for repair of Achilles tendon defect in rats: a histological and biomechanical study

Objective To explore the effect of repairing rat Achilles tendon defect with swine small intestine submucosa (SIS) combined with rat bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were isolated from Wister fetal bone marrow aspirate and expanded to 5th passage. These P5 BMSCs were incorporated into swine SIS to form a construct. A tendon defect model was created in 54 Wistar rats by removing 0.8 cm segment of the left Achilles tendon. The rats were randomly divided into 3 groups with 18 each. In group A, the tendon defect was repaired with the SIS/BMSC construct. In group B, the defect was repaired with SIS only. In group C, the removed tendon segment was sutured back as an autologous graft. Biomechanical tests and histological examinations were carried out 3, 6 and 9 weeks postoperatively. Results Masson trichrome staining showed gradual increase of type Ⅰ collagen and gradual decrease of type Ⅲ collagen in both group A and B. At 9 weeks postoperatively, type Ⅰ collagen content in group A was significantly higher than in group B (P>0.05), while the content of type Ⅲ collagen was lower in group A than in group B (P>0.05). The content of type Ⅰ and Ⅲ collagen in group A was comparable to that in group C (P>0.05). Biomechanical tests revealed gradual increase in tensile strength of the repair in all 3 groups. At 9 weeks postoperatively the tensile strength in group A was (52.17±2.43) N, being significantly higher than the tensile strength in group B (46.75±2.89) N (P 0.05). Conclusion Swine SIS can be used as a scaffold for xenogenic tendon graft. Combining SIS with host BMSCs can accelerate the engrafting process. Key words: Tissue engineering; Transplantation; Small intestinal submucous; Bone mesenchymal stem cells

REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence.

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30th passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

Cytogenetic analysis of rat pancreatic cell cultures at early passages

Cell culture obtained from the pancreas can serve as a source of physiologically competent substitute for primary islets of Langerhans in the treatment of diabetes. However, it is possible to obtain the required number of cells only at long-term cultivation in vitro. Therefore, it is necessary to investigate the risks of neoplastic transformation of cells in vitro before transplantation.Materials and methods. Cell culture was obtained by explant method from pancreas of 12-day-old rats. Cell cultures of the first to sixth passages were used for the cytogenetic analysis. In this study the number of cells with altered karyotype, cells with micronuclei, binucleated cells and the cells in a state of apoptosis were considered, mitotic index was calculated.Results. Aneuploid cells were noted at all passages in an amount of 2.2 % (1st) to 16.6 % (4th). Polyploidy manifested in a population of cells from the second (1.1 %) to the sixth passage (4.4 %) with a maximum at passage four (7.8 %). A significant increase in their number was observed since the second passage (0.3 %). We have seen a significant increase in the number of binucleated cells from the first (0.1 %) to the sixth passage (0.8 %). During the study there was a decrease in mitotic index from the first (2.7 %), to the third passage (1.5 %) and its gradual increase in fourth (1.7 %) and sixth (2.0 %) passages. In addition, there was discovered a small percentage of cells in apoptosis, their number gradually increased to the 4th passage (0.5 %). The 5th-6th passages showed decrease in the number of apoptotic cells to 0.1 %.Conclusion. There have been revealed changes in the rat pancreatic cells culture as aneuploidies, polyploidies and micronuclei, the intensity of which varied depending on the passage. However, karyotype variability of mentioned cell did not exceed the level of spontaneous mutations characteristic of mammalians.

Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion.

Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV) and foot-and-mouth disease virus (FMDV) mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE) of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP). PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture) transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV) and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus) in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of viruses, but could also help in establishing better guidelines for trading animals that could transmit further infections and epidemics in disease free nations.

EFFECT OF HUMAN PLACENTAL DECIDUA BASALIS DERIVED MESENCHYMAL STEM CELLS IN REPAIR OF NUDE MICE SKIN WOUND

To investigate the effectiveness of human placental decidua basalis derived mesenchymal stem cells (PDB-MSCs) in repairing full-thickness skin defect of nude mice. Human placenta samples were obtained from healthy donor mothers with written informed consent. PDB-MSCs were isolated through enzymic digestion and density gradient centrifugation; the 4th passage cells were identified by cellular morphology, cell adipogenic and osteogenic differentiation, and phenotype evaluation. Forty-two 4-5-week-old BALB/c female nude mice were randomly divided into experimental group (n=21) and control group (n=21). The 4th passage PDB-MSCs solution (200 μL, 5×106/mL) was injected into the mice of experimental group via caudal vein; the mice of control group were given equal volume of PBS. The full-thickness skin defect model of 1.5 cm×1.5 cm in size was made after 3 days. The wound healing was observed generally at 1, 2, 4, 7, 14, 18, 21, 25, and 30 days after operation, and the wound healing rate was calculated after wound decrustation. HE staining was used to observe the wound repair at 1, 7, 14, 21, and 31 days; immunofluorescent staining was used for cellular localization at 7, 14, and 31 days after operation. Cells isolated from human placenta were MSCs which had multipotential differentiation ability and expressed MSCs phenotype. Animals survived to the end of the experiment. The general observation showed that the experimental group had a faster skin repairing speed than the control group; the time for decrustation was 12-14 days in experimental group and was 14-17 days after operation in the control group. The wound healing rate of experimental group was significantly higher than that of control group at 14, 18, and 21 days (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018), but no significance was found at 25 and 30 days (t=1.565, P=0.193; t=1.000, P=0.423). HE staining showed lower inflammatory reaction, and better regeneration of the whole skin and glands with time in the experimental group. The immunofluorescent staining was positive in skin defect area of experimental group at different time points which displayed that human PDB-MSCs existed. Through enzymic digestion and density gradient centrifugation, PDB-MSCs can be obtained. Pre-stored PDB-MSCs can mobilize to the defect area and participate in repair of nude mice skin.

Regulation of the Two-Component Regulator CpxR on Aminoglycosides and β-lactams Resistance in Salmonella enterica serovar Typhimurium.

The two-component signal transduction system CpxAR is especially widespread in Gram-negative bacteria. It has been reported that CpxAR contributes to the multidrug resistance (MDR) in Escherichia coli. CpxR is a response regulator in the two-component CpxAR system. The aim of this study was to explore the role of cpxR in the MDR of S. enterica serovar Typhimurium. The minimal inhibitory concentrations (MICs) of various antibiotics commonly used in veterinary medicine for strains JS (a multidrug-susceptible standard strain of S. enterica serovar Typhimurium), JSΔcpxR, JSΔcpxR/pcpxR, JSΔcpxR/pcpxR*, JSΔcpxRΔacrB, JSΔcpxRΔacrB/pcpxR, JSΔcpxRΔacrB/pcpxR*, 9 S. enterica serovar Typhimurium isolates (SH1–9), and SH1–9ΔcpxR were determined by the 2-fold broth microdilution method. The relative mRNA expression levels of ompF, ompC, ompW, ompD, tolC, acrB, acrD, acrF, mdtA, marA, and soxS in strains JS, JSΔcpxR, and JSΔcpxR/pcpxR were detected by real-time PCR. The results showed 2- to 4-fold decreases in the MICs of amikacin (AMK), gentamycin (GEN), apramycin (APR), neomycin (NEO), ceftriaxone (CRO), ceftiofur (CEF), and cefquinome (CEQ) for strain JSΔcpxR, as compared to those for the parental strain JS. Likewise, SH1–9ΔcpxR were found to have 2- to 8-fold reduction in resistance to the above antibiotics, except for NEO, as compared to their parental strains SH1–9. Furthermore, 2- to 4-fold further decreases in the MICs of AMK, GEN, APR, and CEF for strain JSΔcpxRΔacrB were observed, as compared to those for strain JSΔacrB. In addition, CpxR overproduction in strain JSΔcpxR led to significant decreases in the mRNA expression levels of ompF, ompC, ompW, ompD, tolC, acrB, marA, and soxS, and significant increases in those of stm3031 and stm1530. Notably, after all strains were induced simultaneously by GEN to the 15th passage at subinhibitory concentrations, strain JSΔcpxR/pcpxR showed significant increases in mRNA expression levels of the efflux pump acrD and mdtA genes, as compared to strain JSΔcpxR. Our results indicate that the two-component regulator CpxR contributes to resistance of S. enterica serovar Typhimurium to aminoglycosides and β-lactams by influencing the expression level of the MDR-related genes.

Establishment, characterization and cryopreservation of Fars native goat fetal fibroblast cell lines

Abstract Objective To biologically develop and evaluate the caprine fetal fibroblast cell cultures before and after freezing. Methods Goat fetuses (ages 51, 53 and 55 d) were collected from slaughterhouse. Their skin was cut into small pieces (1 mm 3 ) and cultured in DMEM and FBS. When reaching 80%–90% confluence, cells were passaged. Cells of the 8th passage were cultured in 24-well plates (1.5 × 10 5 cells/well) for 9 d and three wells were counted every day. The average cell counts at each time point were plotted against day number and the population doubling time (PDT) was determined. Then, 42 vials of cells (2 × 10 6 cells/mL) were frozen. Samples were thawed and cultured after 1 month. Cell viability and PDT were evaluated after thawing. Results After eight passages, the goat fetal fibroblast cells had a latent phase of about 48 h and after an exponential phase, cells entered the plateau phase on day 5. Before freezing, PDT was about 22 h and after thawing it was about 28 h. Conclusions The goat fetal fibroblast cell culture can be established using the adherent culture method and can be cryopreserved, too. After thawing, growth and viability indices of these cells were acceptable.