Charged Nylon Membrane Research Articles

Fabrication of Robust Forward Osmosis Membrane by Assembling FeOxCly Nanoparticles

AbstractOwing to its inherent advantages like low‐energy consumption, low‐fouling propensity, and high recovering ability over high‐pressure‐driven membrane processes, the future of membrane technology is shifting toward forward osmosis (FO). However, the utility of FO in challenging areas is hindered by the lack of suitable FO membranes. Here, the development of innovative FO membranes by depositing weakly magnetic and positively charged FeOxCly nanoparticles on a negatively charged nylon membrane (FeOx‐Nyl) is reported. Remarkably, the FeOx‐Nyl membrane possesses outstanding stability in an aqueous medium and survived in both acidic (pH = 3.3) and basic (pH = 12.2) solutions. Upon varying the loading amount of FeOxCly, the water flux of the FeOx‐Nyl membrane varies from 27.8 to 61.3 LMH (from 5 to 30 mg). Due to its hydrophilic nature, the FeOx‐Nyl membrane exhibits excellent flux recovery rates (FRR), up to ≈70%. The outstanding robustness and high flux of FeOx‐Nyl are exploited in challenging applications like recovering reactive chemical wastes and dehydration of acetic acid.

Dot and Slot Hybridization of Purified RNA.

Dot blotting and slot blotting are techniques for immobilizing several preparations of nucleic acids on the same solid support, usually a charged nylon membrane. The concentrations of the target sequence of interest can be estimated by hybridizing the immobilized samples to an appropriate probe. The amounts of target sequence are estimated by comparing the intensity of signals emitted by dots containing the test samples with standards containing known concentrations of the target sequence. This protocol describes the blotting and subsequent hybridization of RNA that has been purified from cells or tissues.

Telomere length in dromedary camels (Camelus dromedarius) produced by somatic cell nuclear transfer (SCNT) and their age-matched naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Design and Development of Reverse Slot Blot for the Simultaneous Detection of Rare and Regional Specific Mutations in the Beta Globin Gene in Khuzestan Province of Iran.

Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin (HBB) gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the HBB gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire HBB gene using labelled 5'-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.

Performance and cost-efficiency of eDNA and eRNA capture methodologies: experimental assessment using cultured microalgae

Following the recent leap in biotechnologies and particularly in high-throughput sequencing techniques, environmental DNA and RNA (eDNA and eRNA) are increasingly being used for biodiversity assessments and monitoring of complex ecosystems – lakes, streams and coastal waters. Growing interest and affordability of eDNA/eRNA based tools have led to the emergence of manifold protocols for capturing genetic material from variable biological matrices, e.g. soil, water, feces, biofilms, etc. The variability in eDNA and eRNA material (ranging from free-floating molecules to cellular complexes to intact organisms) and its patchy distribution in the environment may substantially affect effective capture from the environment. Therefore, it is extremely challenging for stakeholders to standardize or choose optimal protocols, impeding incorporation of eDNA/eRNA methods into routine monitoring and surveillance programs. Although there is still no consensus on the standardized workflow for processing eDNA/eRNA samples, the common practice for water samples is to concentrate nucleic acids (NA) via filtration, ranging from 0.22 to 20-micron pore size. Although using the smallest pore is assumed efficient for NA capture from a wide range of material (including sub-cellular particles), a trade-off between detection of the meaningful molecular signal and time(cost)-efficiency is needed. Using finer pore membranes increases the likelihood of clogging and prohibits processing larger volumes of water, thus reducing the chances of detecting rare biodiversity. Moreover, large sample volumes may be accompanied by increased concentrations of inhibitory substances (e.g. humic compounds), suppressing target molecular signal. Comparative studies involving formal cost-efficiency assessments are lacking, restricting informed decision-making around the optimized sampling approach for addressing a particular research or surveillance question. Identifying the optimal combination of time effort and signal detection efficiency is particularly crucial for targeted surveillance, e.g. detection and monitoring of nuisance organisms, endangered and indicator taxa or other species of particular economic or cultural importance. Although it has been previously shown that larger pore filters can be as efficient for species detection from waterborne eDNA, more data are needed on the amount and type of NA material capture and loss. Here, we present a comparison study using an easily cultured microalgal species (Alexandrium pacificum) as a proxy to test the effectiveness of different filter membranes (cellulose acetate membranes of 5 μm, 1.2 μm or 0.45 μm pore size, and positively charged nylon membrane with 1.2 μm pore size) in the context of targeted species detection. We performed an efficiency analysis to identify the method that delivered the optimal use of resources. A tiered experimental design was applied to: i) assess the impact of membranes on capturing various fractions of target eDNA/eRNA (intact cells, partially lysed cells, naked NAs) spiked into pre-filtered and ambient environmental seawater and ii) establish efficiency and utility of different membranes in terms of optimizing the performance – maximized output (capture of target eDNA and eRNA) balanced against minimized time and cost input. The results showed no statistically significant difference between membranes for capturing DNA signal from intact and partially lysed cell treatments. However, positively charged nylon membranes were more efficient in capturing naked NAs, as well as RNA from partially lysed cells. In terms of time effort and volume processed, higher efficacy was reported for the larger pore size cellulose membranes. However positively charged nylon consistently performed better for capturing RNA signal across treatments.

14 Telomere length in cloned camels produced by somatic cell nuclear transfer is not different from that in their naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, and their age-matched naturally produced counterparts by terminal restriction fragment (TRF) analysis. Genomic DNA was extracted from venous blood collected from 6 cloned animals (aged 3, 12, and 24 months), and their age-matched counterparts by a conventional phenol/chloroform protocol. Denatured and neutralized DNA was blotted onto a positively charged nylon membrane and fixed by baking at 80°C for 3h. After washing in a prewarmed digoxigenin (DIG) easy hybridization solution at 42°C for 1h, DNA hybridization was carried out using a telomere (TTAGGG)n-specific, DIG-labelled hybridization probe (Roche Diagnostics, Germany) at 42°C for 4h. Stringent washes were carried out at the same temperature, followed by chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. Molecular weights of the unknown TRF bands on the gel were calculated using known molecular weight marker provided by Telo TAGGG Telomere Length Assay Kit (Roche Diagnostics). A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated 3 times, and the data, presented as mean±s.e.m., were analysed using a 2-sample t-test (Minitab statistical software). No difference (P&amp;gt;0.05) was found in the mean telomere length of cloned camels compared with their naturally produced age-matched counterparts (21.7±0.3 vs. 22.1±0.3; 21.9±0.3 vs. 22.1±0.3; 22.2±0.5 vs. 22.0±0.1; 20.5±0.5 vs. 22.5±0.7; 20.1±0.1 vs. 22.5±0.7; 21.7±1.1 vs. 22.6±0.2), respectively. In conclusion, this is the first study where telomere length has been reported in naturally produced and cloned dromedary camels produced by SCNT. We found that telomere lengths in cloned camels were similar to those of their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Assessment of ISO Method 15216 to Quantify Hepatitis E Virus in Bottled Water.

Hepatitis E virus (HEV) is one of the causative agents of water-borne human viral hepatitis and considered in Europe an emerging zoonotic pathogen. Analysis of bottled water through a standard method validated for HEV can contribute towards the risk management of this hazard. Putting some recent reports by the European Food Safety Authority in place, this study aimed to assess the performance of the concentration and extraction procedures described in ISO 15216-1:2017 for norovirus and hepatitis A virus on HEV detection. Following the ISO recommendation, the bottled water samples were spiked using serially diluted HEV fecal suspensions together with mengovirus as process control and concentrated by filtration via positively charged nylon membranes. In order to extract viral RNA from the resulting concentrates, two different methods were compared in this study: The one recommended in the ISO norm, NucliSens® MiniMag® system (NS), and an alternative commercially available kit NucleoSpin®RNA virus kit (MN). Finally, three reverse transcription quantitative PCR (RT-qPCR) assays were used to quantify HEV titers. The evaluated procedures resulted in average HEV recoveries of 14.08 ± 4.90% and 3.58 ± 0.30% for the MN and NS methods, respectively. The limit of detection (LoD95%) was 1.25 × 104 IU/L for both extraction methods combined with the three RT-qPCR assays tested, with the exception of NS extraction coupled with RT-qPCR1 that showed a LoD95% of 4.26 × 103 IU/L. The method characteristics generated in this study support the limited suitability of the ISO 15216-1:2017 concentration procedure coupled with the evaluated RT-qPCR assays for detecting HEV in bottled water.

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis invitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

Tissue-Print and Squash Capture Real-Time RT-PCR Method for Direct Detection of Citrus tristeza virus (CTV) in Plant or Vector Tissues.

Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.

Molecular probes for identification of UDP-glucuronosyltransferase 1 gene polymorphisms for Gilbert's syndrome diagnosis

The T-3279G mutation in Phenobarbital Responsive Enhancer Modulein (PBREM), TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of the A(TA)6TAA, and G211A mutation in coding exon 1, particularly in Asians, of human UGT1A1 (UDP-glucoronosyltransferase) gene are the three common polymorphisms of Gilbert’s syndrome. This article employed dot blot hybridization to detect the T-3279G, A(TA)6/7TAA and G211A mutation of UGT1A1 gene in 15 patients. In the dot blot hybridization assay, PCR products were denatured and spotted on positively charged nylon membranes instead of acrylamide-modified slides. Then three pairs of probes were labeled by digoxigenin and hybridized with the spotted array. After hybridization, the signal was visualized using TMB color development solution and all possible polymorphisms of the T-3279G, A(TA)6/7TAA and G211A in 15 Chinese patients with hyperbilirubinemia were successfully identified. The results of 15 samples by dotblot hybridization were consistent with the sequencing results. The data showed that dot blot hybridization assay is a quick and low cost method to detect UGT1A1 gene polymorphisms and helpful to diagnosis of Gilbert’s syndrome.

Visual detection of cyanide ions by membrane-based nanozyme assay

In this paper, we report a simple one-step synthesis of well-dispersed amorphous cobalt hydroxide/oxide-modified graphene oxide (CoOxH-GO) possessing peroxidase-like catalytic activity, and its application for the detection of H2O2, glucose, and CN– ions. CoOxH is formed and deposited in situ on the GO surface through the reaction between GO (size ~ 240nm) and Co2+ in basic solution at room temperature. We investigated the enzyme-mimicking activity of the CoOxH-GO nanohybrid in detail via the H2O2-mediated oxidation of Amplex Red (AR) to form fluorescent resorufin. The peroxidase-like activity of CoOxH-GO is utilized herein for the quantitation of H2O2 in a wide concentration range, from 100nM to 100μM. When coupled with glucose oxidase (GOD), the AR/CoOxH-GO system can determine glucose level in blood samples. Interestingly, cyanide ions (CN–) significantly inhibit the catalytic activity of the CoOxH-GO nanohybrid, which allows for the construction of a probe for the detection of CN– in water samples and laboratory wastes. We fabricated a membrane-based CoOxH-GO probe for the visual detection of CN– by preparing a thin film of CoOxH-GO on a positively charged and porous nylon membrane (N+M). The CoOxH-GO/N+M operates on the principle that CN– inhibits the catalytic activity of CoOxH-GO towards the H2O2-mediated oxidation of AR to form reddish resorufin on the membrane. The intensity of the red color of the membrane decreases with increasing CN– concentration, which can be easily observed with the naked eye at the nanomolar level. This cost-effective sensing system allows for the rapid and simple determination of the concentrations of CN− in complicated wastewater samples.

Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae.

Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms.

Northern blots for small RNAs and microRNAs.

This protocol describes the detection of small RNAs (~10-200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a (32)P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea-polyacrylamide gel electrophoresis.

Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization

Harmful algal blooms (HABs) caused by microscopic algae present a threat to human health, ecosystem, fishery, tourism, and aquaculture worldwide. HAB warning and monitoring projects require a simple and rapid method for accurate parallel identification of causative algae. This study presents a useful method for simultaneous detection of harmful algae by multiple PCR coupled with reverse dot blot hybridization (MPCRDBH). A variety of probes, including positive, negative, and specific, were first developed by sequencing and consequent sequence analysis of large subunit rDNA D1–D2 from target species and used for specificity test by blot hybridization. The MPCRDBH assay mainly included five steps: (1) microalgal DNA isolation; (2) amplification and labeling of target DNA by multiple PCR; (3) probe tailing and fixation onto positively charged nylon membrane; (4) reverse dot blot hybridization; and (5) hybridization signal recognition by naked eyes. The reverse dot blot hybridization conditions were optimized, and the appropriate parameters were as follows: ultraviolet cross-linking time, 0.5min; probe density, 2μM; Dig-labeled PCR product density, 200ng; hybridization time and temperature, 2h and 42°C; and washing time and temperature, 2×5min and 47°C. Sensitivity tests showed that MPCRDBH demonstrated a detection limit of 0.6 cell. MPCRDBH recovered all target species and was not affected by background DNA. MPCRDBH also demonstrated a stable detection performance for fixative (acidic Lugol's solution)-preserved samples over 30d using simulated field samples. MPCRDBH applicability was assessed and proven effective for parallel detection of target microalgae in the field samples. The developed MPCRDBH exhibited a simple membrane-based DNA array preparation and hybridization signal recognition compared with other current DNA arrays. The assay presented in this study is specific and sensitive for parallel detection of microalgae, with stable performance. Therefore, this assay is promising for field monitoring of natural samples.

Extensive Genetic Diversity among Clinical Isolates of Mycobacterium tuberculosis in Central Province of Iran.

Human tuberculosis caused by Mycobacterium tuberculosis (Mtb) remains a significant disease in many countries. According to Iran's borders with Afghanistan and Pakistan, which are among the 22 high burden countries around the world, this study was conducted to analyze the current molecular epidemiology of tuberculosis and survey genetic diversity of Mtb strains in Markazi Province in center of Iran. In this experimental study, 75 sputum specimens and one gastric lavage from all smear-positive TB patients admitted to the public hospitals across the Markazi Province were cultured on specific mycobacterial culture media. Genomic DNA was digested by PvuII and transferred to positively charged nylon membrane by southern blotting method and hybridization by PGRS and DR probes. Genotyping of the isolates by PGRS-RFLP and DR-RFLP displayed a wide range of genetic diversity as 25 and 26 genotypes were identified, respectively. Generally speaking, despite the relatively limited number of isolates in the study, high age of patients and also large heterogeneity found in the setting are both in opposition to active circulation of Mtb strains between patients under study either Iranian or Afghan nationals. Thus, it seems that reactivation of latent infection has had the main role in the spread of tuberculosis.

Parallel detection of harmful algae using reverse transcription polymerase chain reaction labeling coupled with membrane-based DNA array

Harmful algal blooms (HABs) are a global problem, which can cause economic loss to aquaculture industry's and pose a potential threat to human health. More attention must be made on the development of effective detection methods for the causative microalgae. The traditional microscopic examination has many disadvantages, such as low efficiency, inaccuracy, and requires specialized skill in identification and especially is incompetent for parallel analysis of several morphologically similar microalgae to species level at one time. This study aimed at exploring the feasibility of using membrane-based DNA array for parallel detection of several microalgae by selecting five microaglae, including Heterosigma akashiwo, Chaetoceros debilis, Skeletonema costatum, Prorocentrum donghaiense, and Nitzschia closterium as test species. Five species-specific (taxonomic) probes were designed from variable regions of the large subunit ribosomal DNA (LSU rDNA) by visualizing the alignment of LSU rDNA of related species. The specificity of the probes was confirmed by dot blot hybridization. The membrane-based DNA array was prepared by spotting the tailed taxonomic probes onto positively charged nylon membrane. Digoxigenin (Dig) labeling of target molecules was performed by multiple PCR/RT-PCR using RNA/DNA mixture of five microalgae as template. The Dig-labeled amplification products were hybridized with the membrane-based DNA array to produce visible hybridization signal indicating the presence of target algae. Detection sensitivity comparison showed that RT-PCR labeling (RPL) coupled with hybridization was tenfold more sensitive than DNA-PCR-labeling-coupled with hybridization. Finally, the effectiveness of RPL coupled with membrane-based DNA array was validated by testing with simulated and natural water samples, respectively. All of these results indicated that RPL coupled with membrane-based DNA array is specific, simple, and sensitive for parallel detection of microalgae which shows promise for monitoring natural samples in the future.

Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.

Selection of an apt support for the immobilization of microbes for the development of a BOD biosensor

The present study entails the screening of different supports for the immobilization of a bacterial consortium for the development of a BOD biosensor. The supports used were PVA (polyvinyl alcohol) + nylon cloth, agarose, nitrocellulose and a nylon membrane. The stability of the prepared membranes was checked at regular time intervals in terms of their response with a reference standard, i.e. glucose–glutamic acid, while stored at different temperatures as well as at different pH values. It was observed that microbes immobilized on the nylon membrane exhibited a maximum stability compared to those immobilized on the other membranes. The study revealed that pH 7.0 was best suited for the storage of membranes harboring immobilized bacteria, preferably at 4 °C, with an observed stability of more than 400 days. Studies also showed that leaching was negligible in the case of a charged nylon membrane. The selected support material, i.e. the immobilized microbial nylon membrane, maintained its stability and activity after an intermittent use for 400 cycles.

Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.