Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.     

Detection of pathogenic bacteria in food samples using highly-dispersed carbon particles

There is an unmet need for detection methods that can rapidly and sensitively detect food borne pathogens. A flow through immunoassay system utilizing highly dispersed carbon particles and an amperometric technique has been developed and optimized. A sandwich immunoassay format was utilized in which pathogenic cells were captured by antibodies immobilized onto activated carbon particles, and labeled with horseradish peroxidase (HRP) conjugated antibodies. Flow of the peroxidase substrates resulted in an amperometric signal that is proportional to the number of captured cells. Factors influencing the analytical performance of the system, such as the quantity of carbon particles and concentrations of capture antibody, enzyme labeled antibody, and enzyme substrates, were investigated and optimized. Detection and quantification of Escherichia coli, Listeria monocytogenes and Campylobacter jejuni were demonstrated with low detection limits of 50, 10, and 50 cells/ml, respectively, and an overall assay time of 30 min. Milk and chicken extract samples were spiked with various concentrations of these pathogens and were used to challenge the system. The system design is flexible enough to allow its application to the detection of viruses and proteins.     

The cardiac biomarkers troponin I and CK-MB in nonpregnant and pregnant goats, goats with normal birth, goats with prolonged birth, and goats with pregnancy toxemia

This study was designed to establish the reference range for the cardiac biomarkers cardiac troponin I (cTnI) and creatine kinase myocardial band (CK-MB) in nonpregnant and pregnant goats, goats with normal birth, goats with prolonged birth associated with dystocia, and goats with pregnancy toxemia. Fifty-seven does, categorized into three groups (G1 to G3), were used. These groups were comprised of 20 healthy does (G1), 19 does with prolonged birth (G2), and 18 does with pregnancy toxemia (G3). Six blood samples (T0 to T5) were collected from G1. The first blood sample (T0) was taken before insemination, the second (T1) at the first trimester, the third (T2) at the second trimester, the fourth (T3) at the last trimester, the fifth (T4) within 12 h of parturition, and the sixth blood sample (T5) was taken 10 days after parturition. A sample of blood was obtained from G2 and G3 upon admission to the hospital. At T0 to T3, no cTnI was detected in any of the 20 does in G1. At parturition (T4), seven of the 20 goats (35%) exhibited slightly elevated cTnI concentrations (range, 0.01 to 0.04 ng/mL). Ten days after parturition (T5), cTnI was not detected in any of the 20 goats. In 10 of the 19 goats (53%) with prolonged birth (G2), the cTnI was significantly elevated to a mean value of 0.094 ± 0.155 ng/mL, with a maximum value of 0.61 ng/mL. In 16 of the 18 goats (89%) with pregnancy toxemia (G3), the cTnI was significantly elevated to a value of 0.852 ± 1.472 ng/mL, with a maximum value of 5.219 ng/mL. Comparing the values of CK-MB in G1 (T0 to T5), G2 and G3 revealed nonsignificant differences. Only a slight elevation in the CK-MB levels in goats with prolonged birth (G2) was noted. We concluded that in healthy does, the cardiac biomarker cTnI is not elevated during normal pregnancy. The serum cTnI concentration may be elevated in a number of goats at normal vaginal or cesarean delivery. Finally, cTnI is significantly elevated in does with pregnancy toxemia and could be used as a prognostic indicator in such cases. The cardiac biomarker CK-MB is not a good indicator of parturition stress in does. Serum cTnI is elevated in goats with pregnancy toxemia, indicating some degree of cardiac dysfunction.     

Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Introduction

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.

Methods

Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.

Results

Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).

Conclusions

This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.     

Analysis of IL28B variants in an Egyptian population defines the 20 kilobases minimal region involved in spontaneous clearance of hepatitis C virus

Spontaneous clearance of hepatitis C virus (HCV) occurs in ~30% of acute infections. Host genetics play a major role in HCV clearance, with a strong effect of single nucleotide polymorphisms (SNPs) of the IL28B gene already found in different populations, mostly infected with viral genotypes 1 and 3. Egypt has the highest prevalence of HCV infection in the world, which is mostly due to viral genotype 4. We investigated the role of several IL28B SNPs in HCV spontaneous clearance in an Egyptian population. We selected nine SNPs within the IL28B genomic region covering the linkage disequilibrium (LD) block known to be associated with HCV clearance in European populations. These SNPs were genotyped in 261 HCV-infected Egyptian subjects (130 with spontaneous clearance and 131 with chronic infection). The most associated SNPs were rs12979860 (P = 1.6 × 10(-7)) and the non-synonymous IL28B SNP, rs8103142 (P = 1.6 × 10(-7)). Interestingly, three SNPs at the two bounds of the region were monomorphic, reducing the size of the LD block in which the causal variants are potentially located to ～20 kilobases. HCV clearance in Egypt was associated with a region of IL28B smaller than that identified in European populations, and involved the non-synonymous IL28B SNP, rs8103142.     

Evaluation of cDNA Libraries from Different Developmental Stages of Schistosoma mansoni for Production of Expressed Sequence Tags (ESTs)

A comparative study of the gene expression profile in differentdevelopmental stages of Schistosoma mansoni has been initiatedbased on the expressed sequence tag(EST) approach. A total of1401 ESTs were generated from seven different cDNA librariesconstructed from four distinct stages of the parasite life cycle.The libraries were first evaluated for their quality for a large-scalecDNA sequencing program. Most of them were shown to have lessthan 20% useless clones and more than 50% new genes. The redundancyof each library was also analyzed, showing that one adult wormcDNA library was composed of a small number of highly frequentgenes. When comparing ESTs from distinct libraries, we coulddetect that most genes were present only in a single library,but others were expressed in more than one developmental stageand may represent housekeeping genes in the parasite. When consideringonly once the genes present in more than one library, a totalof 466 unique genes were obtained, corresponding to 427 newS. mansoni genes. From the total of unique genes, 20.2% wereidentified based on homology with genes from other organisms,8.3% matched S. mansoni characterized genes and 71.5% representunknown genes.