Growth Kinetics Assay Research Articles

Impact of acquisition of 16S rRNA methylase RmtB on the fitness of Escherichia coli.

The aim of this study was to elucidate the biological phenotypes of 16S rRNA methylase RmtB in Escherichia coli and the impact of RmtB acquisition on the fitness of the target bacterium. An rmtB in-frame deletion mutant in E. coli was constructed using a suicide vector (pDMS197)-based double crossover allelic exchange, and its corresponding complemented strain was established. Combined studies of microdilution susceptibility testing, conjugation experiments, growth kinetics assays, competitive experiments, biofilm formation tests and motility assays were performed to study the rmtB-mediated fitness among the prototype E. coli strain, its isogenic mutant and the corresponding complemented strain. The minimum inhibitory concentrations (MICs) of 4,6-disubstituted 2-deoxystreptamines for the rmtB wild-type strain, its isogenic mutant and the complemented strain were ≥1024, ≤2 and ≥1024mg/L, respectively. Both the growth rates and the competitive abilities of the wild-type and complemented strains were relatively inferior to the ΔrmtB mutant. There was no significant difference in biofilm formation and motility among the three strains. In conclusion, the data presented here suggest that acquisition of the 16S rRNA methylase gene rmtB in E. coli can exact a fitness cost on the bacteria, subsequently reducing the growth rate slightly and decreasing the competitive capacity of the bacterium, whereas it does not affect biofilm formation or motility.

A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro.

Several studies have demonstrated the rabies virus to be a perfect potential vaccine vector to insert foreign genes into the target genome. For this study, a green fluorescent protein (GFP) gene was cloned into the rabies virus (RABV) genome between the N and P gene. CT dinucleotide was inserted as intergenic region. The recombinant high egg passage Flury strain (HEP-Flury) of RABV, carrying GFP (rHEP-NP-GFP), was generated in BHK-21 cells using reverse genetics. According to the viral growth kinetics assay, the addition of GFP between N and P gene has little effect on the viral growth compared to the parental strain HEP-Flury. Quantitative real-time PCR (qPCR) indicated that rHEP-NP-GFP showed different viral gene transcription, especially for G gene, compared to HEP-Flury. The same is true for one other recombinant RABV carrying GFP between G and L gene in NA cells. In addition, parent HEP-Flury showed more expression of innate immune-related molecules in NA cells. Compared to HEP-Flury, Western blotting (WB) indicated that insertion of a foreign gene following N gene enhanced the expression of M and G proteins. According to the qPCR and WB, GFP expression levels of rHEP-NP-GFP were significantly higher than rHEP-GFP. This study indicates HEP-Flury as valid vector to express exogenous genes between N and P.

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

Genetic divergence of Chikungunya virus plaque variants from the Comoros Island (2005).

Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.

Abstract 4468: Anticancer activity of novel cucurbitacin analogue in pancreatic cancer

Abstract Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2015-4468

Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

Genetically modified mesenchymal stem cells have been used in attempts to increase the expression of interleukin-1 receptor antagonist (IL-1Ra); however, the attempts thus far have been unsuccessful. The aim of the present study was to investigate whether the lentivirus transduced IL-1Ra gene was able to be stably expressed in murine bone marrow-derived mesenchymal stem cells (mBMSCs) in vitro. In the present study, third generation lentiviral (Lv) vectors transducing the IL-1Ra/green fluorescent protein (copGFP) gene were constructed and transfected into mBMSCs to establish the Lv.IL-1Ra.copGFP/mBMSCs, which were evaluated using fluorescence microscopy, flow cytometry, cell viability analysis using a cell counting kit-8 kit, Trypan blue staining and an MTT growth kinetics assay. The expression of IL-1Ra was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that the Lv.IL-1Ra/copGFP vector was successfully constructed. The mBMSCs exhibited a short proliferation life, however they had good growth kinetics at an early stage and improved viability following efficient transduction of the IL-1Ra gene. IL-1Ra was overexpressed following transfection of mBMSCs. In conclusion, lentiviral vector transduced mBMSCs were able to efficiently express exogenous Il-1Ra under certain conditions and had a marked capacity for proliferation.

Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007–2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community.

Abstract 2714: A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in United States. Tremendous efforts have witnessed the failure of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), extends patient survival by only a few weeks. Numerous studies implicate the need for the drugs that can target multiple signaling events in pancreatic cancer. Recently, cucurbitacins, tetracyclic triterpenoid compund/molecules, belonging to a family of Cucurbitaceae have shown promising anti-cancer activities such as antiproliferation, cell cycle arrest, and apoptosis induction. Here, we report the anticancer activity of a novel analogue of cucurbitacin (Cuc D) that is able to target important signaling proteins involved in pancreatic cancer cell proliferation, invasion and metastasis. Methods: Herein, we investigated the use of Cuc D for the treatment of pancreatic cancer using a panel of pancreatic cancer cells. The effect of Cuc D on the growth of pancreatic cancer cells was determined by CellTiter 96® AQueous One Solution cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96h. The clonogenic potential of cancer cells was also studied using the colony formation assay. microRNA-21 expression levels were investigated through Real-time PCR. Immunoblotting techniques were performed to determine the effects of Cuc D at the molecular level. Results: Our results indicate the anticancer effects of Cuc D in pancreatic cancer cells. Cuc D induces concentration dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell lines at nano molar concentrations. It also inhibits colony formation and metastasis potential of pancreatic cancer cells in dose and time dependent manner. Importantly, Cuc D targets and inhibits proteins, MUC13, Bcl2 and survivin (inhibitor of apoptosis protein family member: IAP) that are involved in proliferation, metastasis and tumor progression as seen through Western blotting. Additionally, the treatment of Cuc D induces the phosphorylation of p53 (p-p53; ser-15) in pancreatic cancer cells. We also observed that Cuc D effectively inhibits miR-21 levels in pancreatic cancer, HPAF-II cells as seen through Real time PCR analysis. miR-21 is an oncogenic miRNA that is overexpressed in pancreatic cancer. Conclusion: Our findings demonstrate that Cuc D exerts multi-focal action in pancreatic cancer cells targeting multiple signaling events. Overall, this study suggests that Cuc D can be a potential and promising therapeutic modality for pancreatic cancer treatment. Citation Format: Mohammed Sikander, Mohd Saif Zaman, Neeraj Chauhan, Murali M. Yallapu, Sheema Khan, Fathi T. Halaweish, Bhavin Chauhan, Meena Jaggi, Subhash C. Chauhan. A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2014-2714

Isolation, characterization and antimicrobial evaluation of a novel compound N-octacosan 7β ol, from Fumaria parviflora Lam

BackgroundFumaria parviflora Lam. (Fumaraceae) is widely used in traditional as well as folkloric system of medicine from ancient. It is commonly known as ‘Pitpapra’ or ‘Shahtrah’ in Indian traditional system of medicine and used for treating numerous ailments like diarrhea, fever, influenza, blood purifier and other complications. The object of the present study was to evaluate the Antileishmanial, antibacterial, antifungal and cytotoxic potential of isolated compound.MethodsMethanolic extract of whole plant of Fumaria parviflora was dried under reduced pressure to obtain a dark brown residue which was adsorbed on silica gel column grade (60–120 mesh) to obtain a slurry and chromatographed over silica gel loaded column in petroleum ether – chloroform (3:1, 1:1 and 1:3 v/v). The in vitro antileishmanial evaluation of isolated compound against Leishmania donovani promastigotes was investigated by growth kinetics assay, reversibility assay, analysis of cellular morphology, adverse toxicity and determination of 50% growth inhibitory concentration (GI50). Disc diffusion and broth micro dilution methods were used to study the antibacterial (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium) and antifungal (Candida albicans and Aspergillus niger) potential in vitro.ResultsStructure elucidation by spectral data analysis revealed a novel compound, n-octacosan-7β-ol (OC), yield (0.471%), having significant antimicrobial activity against Leishmania donovani promastigotes, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Aspergillus niger in vitro with GI50 = 5.35, MIC 250, MIC 250 and MFC 500 and MIC 250 μg ml-1 respectively. The isolated compound did not show adverse effect against mammalian macrophages.ConclusionsThe available evidence of compound suggested that it may be used as antimicrobial agent in future and may provide new platform for drug discovery programmes for leishmaniasis.

Characterization of human vaginal mucosa cells for autologous in vitro cultured vaginal tissue transplantation in patients with MRKH syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) is a rare syndrome characterized by congenital aplasia of the uterus and vagina. The most common procedure used for surgical reconstruction of the neovagina is the McIndoe vaginoplasty, which consists in creation of a vaginal canal covered with a full-thickness skin graft. Here we characterized the autologous in vitro cultured vaginal tissue proposed as alternative material in our developed modified McIndoe vaginoplasty in order to underlie its importance in autologous total vaginal replacement. To this aim human vaginal mucosa cells (HVMs) were isolated from vaginal mucosa of patients affected by MRKH syndrome and characterized with respect to growth kinetics, morphology, PAS staining, and expression of specific epithelial markers by immunofluorescence, Western blot, and qRT-PCR analyses. The presence of specific epithelial markers along with the morphology and the presence of mucified cells demonstrated the epithelial nature of HMVs, important for an efficient epithelialization of the neovagina walls and for creating a functional vaginal cavity. Moreover, these cells presented characteristics of effective proliferation as demonstrated by growth kinetics assay. Therefore, the autologous in vitro cultured vaginal tissue might represent a highly promising and valid material for McIndoe vaginoplasty.

Mesenchymal stromal cells promote tumor progression in fibrosarcoma and gastric cancer cells.

BackgroundExtensive evidence has accumulated regarding the role of mesenchymal stromal cells (MSCs) in tumor progression, but the exact effects and mechanisms underlying this role remain unclear. We investigated the effects of MSC-associated tumor progression in MSC-sarcoma models and a gastric cancer metastatic model.MethodsWe conducted an in vitro growth kinetics assay and an in vivo tumor progression assay for sarcoma cells and gastric cancer cells in the presence or absence of MSCs.ResultsMSC-cocultured human fibrosarcoma cells (HT1080) showed accelerated growth compared with HT1080 alone (79- vs 37-fold change, p<.050). For HT1080, human MSC-coinjected tumors showed significantly greater and highly infiltrative growth compared to those of HT1080 alone (p=.035). For mouse fibrosarcoma cells (WEHI164), mouse MSC-coinjected tumors had greater volume than those of WEHI164 alone (p=.141). For rat sarcoma cells (RR1022), rat MSC-coinjected tumors exhibited greater volume and infiltrative growth than those of RR1022 alone (p=.050). For human gastric cancer cells (5FU), tumors of 5FU alone were compact, nodular in shape, and expansile with good demarcation and no definite lung metastatic nodules, whereas tumors grown in the presence of human MSCs showed highly desmoplastic and infiltrative growth and multiple lung metastasis.ConclusionsWe observed morphological evidence for MSC-associated tumor progression of fibrosarcomas and gastric cancer cells.

Isolation and Identification of Phenol Degrading Bacteria from Mangrove Sediments in the Persian Gulf (Asaluyeh) and their Growth Kinetics Assay

Phenol and phenolic compounds are the most common pollutants result of petrochemical industries. Their carcinogenic and toxic effects have been recorded on human being. Since chemical methods of removing these contaminants are expensive, applying microorganisms is suitable substitution to remove these compounds. The purpose of this research is to isolate and identify of phenol degrading bacteria from mangrove sediments in the Persian Gulf (Asaluyeh) and study of their growth kinetics and degradation level. Nine samples of mangrove sediments were collected. Isolation of bacteria was done by culture in a mineral salt-medium containing 0.5 gL-1 phenol. The growth ability of bacteria was evaluated by bacteria culture in 0.3, 0.5, 0.7, 0.9, 1, 2 and 3 gL-1 phenol concentrations. Gibbs indicator was used in order to measure the amount of phenol removal. All of isolated bacteria showed high ability in phenol removal so that Pseudomonas putida and Acinetobacter sp were able to grow in 0.9 to 2 gL-1 phenol concentrations. The findings of this research indicated that mangrove sediments have numerous phenol degrading bacteria such as; Pseudomonas putida, Acinetobacter sp, Bacillus thuringiensis, Brevibacterium iodinum, and Staphylococcus aureus.

Interaction of silver nanoparticles with Escherichia coli and their cell envelope biomolecules

The antibacterial effect of AgNPs was investigated by determining MIC/MBC and growth kinetics assay. The lowest MIC/MBC was found to be in the range of 11.25-22.5 µg ml(-1) . The growth kinetics curve shows that 25 µg ml(-1) AgNPs strongly inhibits the bacterial growth. Confocal laser scanning electron microscopy (CLSM) shows that as the concentration of NPs increases, reduction in the number of cells was observed and at 50 µg ml(-1) of NPs, 100% death was noticed. Scanning electron microscopy (SEM) shows cells were severely damaged with pits, multiple depressions, and indentation on cell surface and original rod shape has swollen into bigger size. High resolution-transmission electron microscopic (HR-TEM) micrograph shows that cells were severely ruptured. The damaged cells showed either localized or complete separation of the cell membrane. The NPs that anchor onto cell surface and penetrating the cells may cause membrane damage, which could result in cell lysis. The interaction of AgNPs to membrane biomolecules; lipopolysaccharide (LPS) and L-α-phosphatidyl-ethanolamine (PE) were investigated by attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy. LPS and PE showed IR spectral changes after AgNPs exposure. The O-antigen part of LPS was responsible for interaction of NPs through hydrogen bonding. The phosphodiester bond of PE was broken by AgNPs, forming phosphate monoesters and resulting in the highly disordered alkyl chain. The AgNPs-induced structural changes in phospholipid may lead to the loss of amphiphilic properties, destruction of the membrane and cell leaking. The biomolecular changes in bacterial cell envelope revealed by ATR-FTIR provide a deeper understanding of cytotoxicity of AgNPs.

THU0066 Phenotypical and Functional Characteristics of in Vitro Expanded Adipose-Derived Mesenchymal Stem Cells from Patients with Systemic Sclerosis

BackgroundAdult stem cells, expecially those of mesodermal origin (MSCs), have received attention as an ideal source of regenerative capable cells because of their multipotentially and ability to replicate. Regenerative cells...

Marek’s disease virus (MDV) ubiquitin-specific protease (USP) performs critical functions beyond its enzymatic activity during virus replication

Marek’s disease virus (MDV) encodes an ubiquitin-specific protease (USP) within its UL36 gene. USP is highly conserved among herpesviruses and was shown to be important for MDV replication and pathogenesis in MDV’s natural host, the chicken. To further investigate the role of MDV USP, several recombinant (r) MDVs were generated and their in vitro phenotypes were evaluated using plaque size and growth kinetics assays. We discovered that the N-terminus of pUL36 is essential for MDV replication and could not be complemented by ectopic expression of MDV USP. In addition, we demonstrated that the region located between the conserved glutamine (Q85) and leucine (L106) residues comprising the active site cysteine (C98) is also essential for MDV replication. Based on the analyses of the rMDVs generated here, we concluded that MDV USP likely contributes to the structure and/or stability of pUL36 and affects replication and oncogenesis of MDV beyond its enzymatic activity.

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells

BackgroundParticular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. MethodsHAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. ResultsCultured HAMCs displayed intermediate epitheloid–fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. ConclusionThese results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy.

Replication of West Nile virus, Rabensburg lineage in mammalian cells is restricted by temperature

BackgroundThe genus Flavivirus currently consists of approximately 80 single-strand positive-sense RNA viruses. These replicate in a range of hosts including myriad vertebrate, insect, and tick species. As a consequence of this broad host range, the majority of flaviviruses can be propagated in most vertebrate and insect cell cultures. This ability to infect arthropods and vertebrates usually is essential for maintenance of these viruses in nature. But recently, there has been the discovery of a number of flaviviruses that infect mosquitoes but not vertebrates. It remains largely unknown why certain flaviviruses infect vertebrates and mosquitoes while others infect mosquitoes or vertebrates exclusively.MethodsHere, we initiated in vitro host range studies of Rabensburg virus (RABV), an intermediate between the mosquito-specific and horizontally transmitted flaviviruses, to provide information on the factor(s) that underlie the varying host range of flaviviruses. RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses because it does not infect mammalian or avian cell cultures, house sparrows, or chickens, but it does share genetic characteristics with the Japanese Encephalitis serogroup of flaviviruses.ResultsIn vitro growth kinetic assays revealed the complete abrogation of RABV growth on Vero and E6 cells incubated at temperatures 35°C and higher, but surprisingly RABV infected, replicated efficiently, and displayed overt cytopathic effects (CPE) on Vero and E6 cell cultures incubated below 35°C. In contrast, RABV was fully viable, replicated efficiently, and displayed overt CPE on C6/36 cells incubated at 28°C or 37°C, thus implicating temperature as an important factor limiting the host range of RABV.ConclusionsThese data are critical for further study to more fully identify the determinants that mediate the evolution of biological transmission among flaviviruses. It also will be useful for studies that look to provide a comprehensive molecular definition of flavivirus-host cell interactions. And it will provide a cadre of information to design wet lab experiments to investigate the genetic changes that facilitate host switching, which may lead to new vertebrate pathogens or transmission pathways.

Protection against Enterovirus 71 with Neutralizing Epitope Incorporation within Adenovirus Type 3 Hexon

Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens.

Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells

Induction of micronucleus formation (cytogenetic damage) in brain cancer cells upon exposure of dielectric barrier discharge plasma has been investigated. We have investigated the influence of exposure and incubation times on T98G brain cancer cells by using growth kinetic, clonogenic, and micronucleus formation assay. We found that micronucleus formation rate directly depends on the plasma exposure time. It is also shown that colony formation capacity of cells has been inhibited by the treatment of plasma at all doses. Cell death and micronucleus formation are shown to be significantly elevated by 120 and 240 s exposure of dielectric barrier discharge plasma.

Enhancement of antibiotic susceptibility ofStenotrophomonas maltophiliausing a polyclonal antibody developed against an ABC multidrug efflux pump

Stenotrophomonas maltophilia is an emerging nosocomial pathogen capable of causing healthcare-associated infections, including pneumonia and bacteremia. Intrinsic resistance in S. maltophilia is exhibited towards many broad-spectrum antibiotics, and treatment recommendations are controversial. One of the major causes of antimicrobial resistance is attributed to a robust array of efflux pumps that extrude drug compounds from the cell. Using checkerboard and growth kinetic assays, we evaluated the in vitro activity of a polyclonal antibody raised against an ATP-binding cassette efflux protein in S. maltophilia. Six clinical strains of S. maltophilia and one type strain were challenged with co-trimoxazole, ticarcillin-clavulanate, and ciprofloxacin, alone and in combination with antibody. One clinical strain was tested by growth curve experiments for each antibiotic-antibody combination. The use of antibody resulted in significantly increased susceptibility in 71.4% (15/21) of treatments tested, with 33.3% displaying synergy and 38.1% an additive effect. In growth kinetic studies, synergy was obtained for each antibiotic-antibody combination. Thus, the use of antibody raised against multidrug efflux pumps for the treatment of multidrug-resistant organisms warrants further investigation. Antibody targeting substrate recognition sites, or other functionally important epitopes, may lead to inhibition of multiple efflux pumps that share the same substrate and is an attractive area that should be explored.