Investigation and analysis of a human orf outbreak among people living on the same farm

Human orf is a viral zoonotic infection caused by Parapoxvirus. The skin lesions of human orf can be misdiagnosed as cutaneous anthrax leading to overtreatment and also fear. This study was conducted to analyze an outbreak which led to deaths among kids and lambs in the same flock, and skin lesions in some persons who were living on the same farm that were initially diagnosed as cutaneous anthrax by a practitioner. Eight patients with skin lesions and eleven persons who had no skin lesion were considered as patients and control groups, respectively. The cultures obtained from the lesions of all patients were negative for Bacillus anthracis. The diagnosis of skin lesions was done by clinical findings, histopathological examination and PCR as human orf. To be under 20 years of age, direct contact with the animals, and contact with flayed skin of sick animals were the risk factors for human orf (Odds Ratio 7.5; 95% Confidence Interval 1.02-54.54, OR 12.25; 95% CI:1.3-100.9, OR 16.67; 95% CI:1.65-148.20, respectively). Orf should be kept in mind in the differential diagnosis of skin lesions resembling anthrax. For control and prevention of orf, transmission routes should be known; good hand hygiene and other personal protective measures have to be implemented.     

Cryopreservation of spin-dried mammalian cells

This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2)) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.     

High-throughput single cell arrays as a novel tool in biopreservation

Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes.Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields.For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology.     

Mathematical modeling of the indole-3-butyric acid applications on rooting of northern highbush blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.) softwood-cuttings

The objective of the present study is to develop a mathematical model to predict the effect of indole-3-butyric acid (IBA) on mean rooting (%) and mean root growth of northern highbush blueberry cultivars (Vaccinium corymbosum L.). The best estimating equations for the rooting (%) and root growth are formulized as: RG = (5.672183) + [0.002851 × (IBA)] − [2.0E⁻⁶ × (IBA)2] + (−0.27211 × Cv.) and R = (82.00649) + [0.030801 × (IBA)] − [2,4E⁻⁵ × (IBA)2] − [2.36218 × (Cv.)] where RG is root growth, R is rooting, IBA is indole-3-butyric acid (ppm) and Cv. is cultivar. Cultivars are Ivanhoe [1], Jersey [2], Rekord [3], Northland [4], Berkeley [5] and Bluejay [6]. The numbers given in square brackets represent the blueberry cultivars for the equations. Multiple regression analysis was carried out until the least sum of squares (R2) was obtained. R ² value 0.90 for rooting and 0.95 for root growth. Standard errors were found to be significant at the p < 0.001 level. The actual rooting differed to the blueberry cultivars and it was between 57.76 and 83.23% while estimated rooting percentage calculated by the produced mathematical model was between 59.04 and 83.80%.     

Comparison and Optimisation of Transfection of Human Dental Follicle Cells,a Novel Source of Stem Cells,with Different Chemical Methods and Electro-poration

Introduction Human dental follicle cells (HDFCs) derived from human impacted third molars (wisdom teeth) have been shown to be a significant source of adult stem cells. Generation of mesenchymal stem cell-like cells from dental follicles causes minimal surgical stress. In vitro and in vivo reports showed that HDFCs can be utilized in gene and cell therapy applications which make them an attractive alternative source for different gene-cell therapy applications. However, there are currently no systematic comparative studies on transfection potential of HDFC cells using different chemical and electro-poration techniques. Methods Stem cells from impacted third tooth molars were isolated, and analyzed for expression of surface markers. Transfection efficiencies of four commercially available transfection reagents (Transfast, Escort V, Superfect and FuGene HD) and electro-poration on isolated stem cells were compared. Results Isolated HDFCs were stained positive for CD105, CD90, CD73, CD166, and negative for CD34, CD45, and CD133. Among the chemical transfection reagents used in this study, FuGene HD was the most efficient in transfecting HDFCs, even in the presence of 10% serum. Conclusion Electro-poration of HDFCs yield relatively high transfection rates and cell viability when compared to chemical transfection techniques. Our observations might be useful for developing gene and cell therapy applications using dental follicle stem cells.     

Direct electrochemical genosensing for multiple point mutation detection of Mycobacterium tuberculosis during the development of rifampin resistance

We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples.     

Laboratory evaluation of Turkish entomopathogenic nematodes for suppression of the chestnut pests,Curculio elephas (Coleoptera: Curculionidae) and Cydia splendana (Lepidoptera: Tortricidae)

The lepidopteran, Cydia splendana, and the coleopteran, Curculio elephas, are the most serious pests of chestnut fruit in Turkey. We evaluated the biological control potential of three Turkish entomopathogenic nematode species, Steinernema feltiae, S. weiseri and Heterorhabditis bacteriophora, against the last instar larvae of C. splendana and C. elephas, both of which occur in the soil from fall (October–November) until mid-summer (August). The optimal temperature for infection, time to death of the hosts, and reproductive potential of the nematodes were determined at 10, 15, 20 and 25°C for both pest species. Cydia splendana was more susceptible to nematode infection than C. elephas. Temperature had a significant effect on the infectivity and development of entomopathogenic nematodes. The cold-adapted S. weiseri and S. feltiae were the most virulent species at 10 and 15°C, whereas the warm-adapted H. bacteriophora was the most effective at 20 and 25°C. In soil pot experiments conducted at 15°C, S. weiseri was the most virulent species against C. elephas and C. splendana. However, our data show that C. elephas larvae had a lower and C. splendana larvae had a higher susceptibility to the nematode species tested. Accordingly, we recommend that future efforts of using entomopathogenic nematodes, especially S. weiseri, be directed against C. splendana and that there be a continued effort to find more virulent nematode isolates against larvae of C. elephas.     

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.