Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression.     

A comparative study of DNA extraction methods to be used in real-time PCR based quantification of ochratoxin A-producing molds in food products

Quantification of ochratoxin A (OTA)-producing molds in foods by real-time quantitative PCR (qPCR) may be affected by the DNA extraction method used. In the present work, 6 different methods for extraction of DNA from ochratoxigenic molds in foods were tested. Several combinations of mechanical and thermal lysis of conidia with commercialized DNA extraction kits and enzymatic treatments or resins were evaluated. DNA recovery and quality of extracted DNA was measured by testing the extracted DNA with a conventional PCR and an SYBR Green qPCR amplifying the β-tubulin gene and the non-ribosomal peptide synthetase gene, otanpsPN. Inhibition of conventional and qPCR was not observed when the DNA-extraction method includes an initial thermal disruption of conidia before use of commercialized extraction kit or resin, enzymatic treatment and/or lysis buffer. Of the six methods tested, the one combining thermal lysis of conidia followed by a short enzymatic treatment and incubation with Chelex-100 resin and final extraction with the EZNA kit was selected, since the extracted DNA showed good amplification by conventional PCR for β-tubulin gene and the highest DNA recoveries when tested by qPCR. The method was subsequently validated in different food products such as ripened foods, nuts, and grapes inoculated with Penicillium and Aspergillus species. With this Chelex100-enzymatic-EZNA method good DNA recoveries ranging from 69 to 99% were obtained for all food matrices and fungal species tested. This fast method is a promising tool to be used as routine analysis in HACCP systems in the food industry for quantifying OTA-producing molds by qPCR.     

Assessment of Fusarium infection in wheat heads using a quantitative polymerase chain reaction (qPCR) assay

The accuracy of a quantitative polymerase chain reaction assay in quantifying the DNA of trichothecene-producing F. culmorum and F. graminearum within harvested wheat grains and head tissue was evaluated in comparison with incidences of infected kernels and deoxynivalenol levels. In a first experiment, six durum and bread wheat varieties were grown in randomized plots for a 2-year period, and inoculated with Fusarium macroconidia at six growth stages between heading and dough ripening, to obtain a wide range of Fusarium head blight incidences. There was a close relationship between fungal DNA and the amount of deoxynivalenol, and this relationship was consistent over Fusarium species, wheat species and varieties, and over a wide range of Fusarium head blight infection. In a second experiment potted wheat plants were grown under environmentally controlled conditions and inoculated with the two Fusarium species at full flowering; head samples were collected before inoculation and after 6 h to 12 days, and processed by the quantitative polymerase chain reaction assay. This assay made it possible to detect the dynamic of fungal invasion in planta after infection had occurred, and to single out the presence of infection before the onset of the disease symptoms: A robust detection of the infection occurred within 18-24 h for F. culmorum, and within 2-9 days for F. graminearum.     

Applicability of quantitative polymerase chain reaction (qPCR) assays for common bean authentication in processed food

Food authentication by quantitative polymerase chain reaction (qPCR) methods assures food quality. The aim was to evaluate three qPCR assays for DNA quantification after heat processing of common bean grains, genus-specific FAS assay for Phaseolus, species-specific LEC assay for common bean (Phaseolus vulgaris) and genetically modified (GM) event-specific FGM assay for Embrapa 5.1 event GM common bean. FAS assay showed high stability among Phaseolus genus samples. Common bean grains were heat-treated in autoclave (at 120 °C for 15–60 min) and target DNA copy number decreased as processing time increased. Even with DNA degradation, qPCR assays were capable to detect low DNA quantity, and the limit of detection was 100 copy number. Mean efficiency value of FGM assay was 92% in the presence of background DNA. Background DNA did not cause any interference, and 0.39% of GM material can be detected. These qPCR assays are able to quantify common bean in processed food.     

Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples

European Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of T_t is needed for quantification purposes.     

Recommendations following a multi-laboratory comparison of microbial source tracking methods

Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero C_t values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions.     

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1–3.3% and 1.9–7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1–2 log₁₀copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.     

Using rapid indicators for Enterococcus to assess the risk of illness after exposure to urban runoff contaminated marine water

Background

Traditional fecal indicator bacteria (FIB) measurement is too slow (>18 h) for timely swimmer warnings.

Objectives

Assess relationship of rapid indicator methods (qPCR) to illness at a marine beach impacted by urban runoff.

Methods

We measured baseline and two-week health in 9525 individuals visiting Doheny Beach 2007-08. Illness rates were compared (swimmers vs. non-swimmers). FIB measured by traditional (Enterococcus spp. by EPA Method 1600 or Enterolert™, fecal coliforms, total coliforms) and three rapid qPCR assays for Enterococcus spp. (Taqman, Scorpion-1, Scorpion-2) were compared to health. Primary bacterial source was a creek flowing untreated into ocean; the creek did not reach the ocean when a sand berm formed. This provided a natural experiment for examining FIB-health relationships under varying conditions.

Results

We observed significant increases in diarrhea (OR 1.90, 95% CI 1.29-2.80 for swallowing water) and other outcomes in swimmers compared to non-swimmers. Exposure (body immersion, head immersion, swallowed water) was associated with increasing risk of gastrointestinal illness (GI). Daily GI incidence patterns were different: swimmers (2-day peak) and non-swimmers (no peak). With berm-open, we observed associations between GI and traditional and rapid methods for Enterococcus; fewer associations occurred when berm status was not considered.

Conclusions

We found increased risk of GI at this urban runoff beach. When FIB source flowed freely (berm-open), several traditional and rapid indicators were related to illness. When FIB source was weak (berm-closed) fewer illness associations were seen. These different relationships under different conditions at a single beach demonstrate the difficulties using these indicators to predict health risk.     

Expression of Lactobacillus pentosus B96 bacteriocin genes under saline stress

This research studied the influence of sodium chloride on bacteriocin activity of table olives’ strain Lactobacillus pentosus B96. The strain was cultured in MRS under different NaCl concentrations (0, 4, 6 and 8%, in w/v). In MRS, maximum bacteriocin activity was achieved 9 h later. A medium containing 4 or 6% NaCl (w/v) increased the total bioactivity of the strain and an 8% NaCl reduced it. Real-time PCR was used to monitor the genetic expression of the bacteriocin genes plnA, plnB, plnC, plnE/F, plnJ, plnK, plnN and plantaricin S. Cultured in MRS, plantaricin S reached its maximum expression during the lag phase while plnE/F expresses during the exponential phase. The presence of sodium chloride in the medium moved the maximum expression of plantaricin S to the stationary phase, independently of the concentration. 4% (w/v) of NaCl didn’t affect the expression pattern of plnE/F while promotes the expression of plnN during both the lag and the exponential phases. More sodium chloride, 6% (w/v) maintained the expression of plnN in the pag phase but not in the exponential and moved plnE/F expression to the stationary phase. Plantaricin S, plnE/F and plnN over-expressed during the stationary phase in the higher sodium chloride concentration assayed, 8% (w/v). The relative expression level of plsA was 1000-fold higher than that of the plnE/F and plnN genes and even the ldhD constitutive gene used. Under our conditions, expression of plnA, plnB, plnC, plnJ and plnK genes was not observed.