Furthermore, control samples, not exposed to labelled insulin, did not give PLX4032 a positive reaction when developed with DAB. The initial binding experiments used a concentration of insulin that was much higher than the physiological concentration, but in-line with what previous workers had used (Christopher & Sundermann, 1996; Souza & López, 2004). These experiments were repeated with insulin-binding positive bacteria using insulin at a normal physiological concentration.

The insulin-binding assay was repeated on B. multivorans and A salmonicida using 80 pM of insulin peroxidase at different exposure times 2, 5, 10, 20, 40 and 80 min. These experiments showed that A salmonicida produced a positive reaction after 5 min, and this grew stronger with time up to 80 min. However, the B. multivorans showed no reaction at exposure times of 2, 5 and 10 min, and the first positive reaction was seen at 20 min and grew stronger at 40 and 80 min. Also included is a microscopic image of cells of A. salmonicida CM30 showing binding of FITC-labelled insulin (Fig. 1b). Variation in the intensity of staining of individual cells may be attributable to the method of fixation, different planes of focus and/or the possibility that some labelled insulin may have entered

cells. Both wild-type A. salmonicida and B. multivorans showed significant insulin binding at all the time points tested; however, the amount of insulin binding to the fish pathogen A. salmonicida was about 105 ng per 109 cells after 15 min incubation time,

which was much higher compared JQ1 research buy to 28.3 and 21.1 ng per 109 cells binding to B. multiv-orans and the A. salmonicida A-layer mutant, respectively (Fig. 2). Furthermore, wild-type A. salmonicida and B. multivorans showed significant binding relatively early (after 1 min) compared to the mutant A. salmonicida MT004, which showed significant FITC-insulin binding only after 10 min. Cyclooxygenase (COX) The amount of nonspecific insulin that bound to the P. aeruginosa and Escherchia coli was about 0.08 and 0.03 ng per 109 cells, respectively. Insulin binding to wild-type A. salmonicida increased steadily with time; however, B. multivorans showed no significant increase in insulin binding up to 5 min (13.1 ng per 109 cells) but produced strong binding of 19.1 and 23.8 ng per 109 cells after 10 and 15 min, respectively. Whereas the mutant A. salmonicida MT004 showed significant binding of 15.5 and 21.1 ng per 109 cells only after 10 and 15 min, respectively, with no significant binding at earlier times. Various protocols were applied during this work to separate bacterial proteins on different gels using native, SDS-PAGE (Laemmli, 1970), blue native (BN-PAGE; Nijtmans et al., 2002) and agarose gel electrophoresis (Henderson et al., 2000) and both Burkholderia and A. salmonicida samples initially showed no IBP bands on Western ligand blotting.

“
“Listeria monocytogenes is a food-borne pathogen that can survive

under a wide range of environmental and energy stress conditions. The general stress response controlled by σB largely contributes to stress resistance in L. monocytogenes. Moreover, the bacterial cell wall is the first defense PLX4032 molecular weight against cellular stress and as such is the target of numerous antibiotics. We therefore hypothesize that σB contributes to monitoring the integrity of cell walls. We evaluated σB activity in wild type and ΔsigB mutant L. monocytogenes containing reporter fusions (σB-dependent opuCA promoter and a lacZ reporter gene) during the early exponential growth phase by measuring the specific activity of β-galactosidase after vancomycin (2 μg mL−1 final concentration) stress. σB activity is significantly induced only in the wild-type strain by addition of vancomycin. In

addition, we identified σB-dependent vancomycin-inducible proteins using LC-ESI-MS/MS analysis. Two independent proteomic analyses confirmed the minimum twofold upregulation of 18 vancomycin-inducible σB-dependent stress response proteins in the wild-type strain compared with the ΔsigB mutant. The functions PD0332991 purchase of these proteins are associated with cell wall biogenesis, intracellular transport, general stress response, cell metabolism and virulence. These results suggest that the σB protein may contribute to the monitoring of cell wall integrity. Listeria monocytogenes is a widely distributed intracellular pathogen that causes listeriosis, a serious illness from which children, pregnant

women and immunocompromised individuals are at risk. Infection is most often caused by the ingestion of contaminated foods, such as those most frequently associated with raw milk, soft cheeses, raw vegetables and refrigerated ready-to-eat products (Farber & Peterkin, 1991). Listeria monocytogenes has the ability to grow in very diverse environments; it can survive in a wide temperature range (−1 to 45 °C), a broad pH range (4.5–9.0) and in high salt concentrations (10% NaCl) (Cole et al., 1990; Sleator et al., 2001). These unique resistance properties are related to the general stress response, which is controlled by the Resveratrol alternative sigma factor σB (Wiedmann et al., 1998; O’Byrne & Karatzas, 2008). Listeria monocytogenesσB was found to play a role in the general stress response. σB-null mutants demonstrate increased sensitivity under salt, acid, cold, heat, ethanol and oxidative stress, as well as carbon starvation (Becker et al., 1998, 2000; Wiedmann et al., 1998). About 150 σB-dependent genes are known to be expressed under stress conditions, where they contribute to stress resistance in L. monocytogenes (Raengpradub et al., 2008).

, 2007). Selfing occurs under conditions favourable for sexual development, with closed fruiting bodies (cleistothecia), containing ascospores, being formed by all fertile strains (see Todd et al., 2007). Previously, we found that pex mutants, impaired in PTS1 protein import, were affected in sexual development, producing low numbers of small cleistothecia in selfings or homozygous crosses (Hynes et al., 2008). However, it was clear that meiosis was not blocked. We have find more now deleted the gene encoding Pex2 in order to test whether meiotic commitment is dependent on the RING-finger complex in A. nidulans. Unlike P. anserina, meiosis is not

affected, indicating a fundamental difference between these species. The media and conditions for growth of A. nidulans and standard genetic manipulations were as described previously (Todd et al., 2007). DNA from transformants was analysed by Southern blotting to confirm predicted integration events. Standard methods for DNA manipulations, nucleic acid Compound Library blotting and hybridization have been described (Hynes et al., 2006). Unless otherwise indicated, strains contained the veA1 mutation.

This mutation results in increased conidiation and reduced sexual reproduction relative to veA+ strains (Kim et al., 2002). The isolation of the pexC∷bar and pexC∷bar; ve+ strains has been described (Hynes et al., 2008). A single gene encoding the homologue of Pex2 was identified as AN4056.3 in the A. nidulans genome database (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). A 2.7-kb fragment corresponding to coordinates −598 to +2098 (relative

to the predicted translation start) was amplified by the PCR using the primers 5′-AACATCCCCGCAAGATACAG-3′ and 5′-ATGAGTTCGAGAAGCGTCGT-3′ and inserted into EcoRV cut pBluescript SK+ to generate the plasmid pFK7442. A 2.1-kb XhoI–E coICRI fragment containing the Aspergillus fumigatus riboB gene (Nayak et al., 2006) was inserted between the XhoI and StuI sites of the insert of pFK7442 to generate pFK7447, thereby replacing sequences of AN4056 (+209 to +1177) corresponding to amino acids 71–379 (Fig. 1a). A SSR128129E linear PCR fragment generated with the above primers was used to transform strain TNO2A21 (genotype pyroA4 nkuAΔ; veA1 riboB2) selecting for riboflavin prototrophy using standard methods (Nayak et al., 2006). The recipient strain contained the nkuAΔ to promote homologous integration events. blastp searches of the predicted proteins from the A. nidulans genome sequence with the P. anserina Pex2 sequence revealed a single homologue (AN4056.3) in agreement with the analysis of Kiel et al. (2006). The A. nidulans gene contains only one intron, while the predicted pex2 genes of other Aspergillus spp. contain two introns (Kiel et al., 2006). AN4056 was designated pexB in accordance with the standard nomenclature for A. nidulans.

In-hospital costs (for both in-patient and day-care admissions) were based on the DRG system in use in Italy since 1994, in which disease groups Fluorouracil cell line are defined according to the hospital discharge form data. Costs

of out-patient consultations and examinations (laboratory and clinical imaging) were calculated based on the official standard costs assigned by the Italian Ministry of Health. According to an agreement between the Italian Ministry of Health and all pharmaceutical companies, hospitals pay half price for antiretroviral drugs, instead of the full cost paid by the public in Italy. All costs in this analysis were annualized and expressed in nominal terms for the year in which they were incurred. Costs incurred by patients (e.g. costs of travel to hospital services or costs of additional services incurred by staying at home), intangible costs (e.g. stress and anxiety) and indirect costs to society (e.g. loss of productivity) were Bleomycin datasheet not estimated, as they did not affect the comparison of medical sector burden between chronic diseases, which was the main objective of the study. For the

same reasons, the cost analysis did not take into consideration the inflation rate, which the Italian National Institute of Statistics confirmed to be 2.1% on average at the time of our study. The criteria for the identification of HIV-infected persons were as follows: a diagnosis of HIV infection based on serological testing; For an HIV-infected person to be considered as having a concomitant chronic disease, they had to satisfy at least one of the above criteria for that chronic disease. HIV-infected persons who were at any time on antiretroviral treatment during the year were classified as ‘on antiretroviral treatment’. A verification procedure to assess the sensitivity of the BLHA database for detecting HIV-infected patients was performed through cross-checking of patients registered in the databases O-methylated flavonoid of the following institutions operating

in the Province: the Institute of Infectious and Tropical Diseases, the Clinic for Sexually Transmitted Diseases, Methadone Dispensing Units, and Primary Health Care Services for HIV Patients. Death certificates were also reviewed. Cross-checking verified that the BLHA database missed only 4% of patients registered in the other available databases. Starting from 2004, all newly identified cases were considered as ‘incident’ cases, by which we mean ‘newly diagnosed’ rather than ‘newly infected’. To calculate a denominator for the annual prevalence and incidence, we used an estimate of the mid-year average number of people who received services from the Brescia Local Health Authority during a calendar year.

Since raltegravir is generally well tolerated and has now been approved by the US Food and Drug Administration for treatment-naive patients, it represents a potential alternative to protease inhibitors for use in expanded regimens. However, there are only limited data on its safety in healthy uninfected individuals. The choice of regimen should take into consideration the most common ART regimens being used in the country where the trainee is rotating. Furthermore, if the source

is found to be HIV positive with a history of ART, further guidance will be required to assess documented or suspected viral resistance and adjust the regimen accordingly as the patient might have a drug-resistant virus strain. In more complex cases such as those involving pregnancy, breast-feeding, or exposure to a source patient with documented poor ART adherence, an infectious disease specialist should be consulted Protein Tyrosine Kinase inhibitor to help decide the most appropriate regimen. However, in the absence of immediate access to a specialist or an alternative PEP regimen, the standard PEP

protocol should CDK inhibitor be followed until the specialist makes alternative recommendations or access to more appropriate ART becomes possible. With the rapid rise of interest in global health and increasing numbers of health care trainees participating in international electives, the medical community has an obligation to develop provisions to adequately support and protect them. Medical trainees are at considerable risk for contracting HIV, and in the event of an occupationally acquired infection, the consequences can be devastating for both the trainee and their home institution. As an infected health care professional, these trainees

may potentially face difficulties securing health insurance, possible problems resulting in loss of income, and as their illness progresses, long-term disability and premature death. Given their tenuous status, students may not be eligible for workers’ compensation and private insurance, leaving them vulnerable to considerable financial difficulties with a debilitating illness. As most students did not receive compensation Fossariinae for their contributions, they do not fall under the purview of workers’ compensation laws, unless the law specifies the coverage of apprentices. Trainees, left with no other options, may be compelled to pursue legal action, leaving medical schools and teaching hospitals at risk for civil litigation.22 Ultimately, academic institutions have a commitment to educate, guide, and protect their students and residents. Thus, pre- and postdeparture travel clinic visits, immunizations, PEP starter pack, and 24-hour access to a home-based clinician with appropriate expertise should be made available by the institutions themselves.

The 143Cys mutant, however, still maintains some activity and indicates that the role of the –S-S- bond is not similar to the ferredoxin:thioredoxin reductase system. The disulphide bond appears to have a structural role, ensuring close proximity of PQQ to cytochrome c. Substitution of one or both of the Cys with Ser residues would increase flexibility of the enzyme leading

to a conformational change with a negative 5-FU mw impact on the electron flow. Homology structure prediction indicates that mutation to either one or both Cys residues would result in a conformation change, notably, protein homology structure of the 143CysSer mutant (Fig. 5b) with Chimera software (Pettersen et al., 2004) predicted three major deviations from wild-type LH structure (Fig. 5a) in terms of α-helices Galunisertib mw and four differences in β-pleated sheets. The predicted tertiary structure of the 124,143CysSer mutant (Fig. 5c) appeared to deviate even more from the wild type with six changes in α-helices and nine differences in β-pleated sheets. More importantly, the N-terminal and cytochrome c domain linker appears

to be significantly affected. These mutations appear to have resulted in the enlargement of the molecule with a possible significant effect on the active site. Thus, the loss of disulphide bond alters the structure dramatically and probably affects the enzyme activity because of changes to the cytochrome c domain. In conclusion, SPTBN5 LH is in possession of a disulphide bond formed between spatially distal residues 124Cys and 143Cys. Although this bond is not undergoing cycles of reduction and oxidation during catalytic breakdown of the substrate, its formation is crucial for enzyme activity as it ensures structural rigidity and correct protein conformation. This work was privately funded and supported by IBERS, Aberystwyth University. We would like to acknowledge Dr Ian Mercer and

Dr Maurice Bosch for proof reading the drafts. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco (supported by NIH P41 RR001081). “
“Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with ‘Archaeoglobus lithotrophicus’ being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin–Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in ‘A.

In another classical conditioning MEG study with two different CS tones, Kluge et al. (2011) reported stronger tangential field gradients at left and right central sensor clusters at mid-latency (85–115 ms) and late (180–270 ms) intervals for CS+ (with omitted electric shock) compared to CS− tones during conditioning. When comparing all CS+ (also CS+ with US presentation) with CS− tones they found relatively enhanced gradient

fields for CS− processing in these sensor groups in an early interval between 30 and 50 ms. A following phase with contingency reversal eliminated the CS+/CS− differentiation at early and mid-latency intervals but reversed effects at the late interval. The authors interpret the effects at mid-latency and late intervals as enhanced processing of the pain-signalling CS+ in the auditory cortex. However, as electric shocks (UCS) were presented at 100, 175 and 250 ms after CS onset and source analysis was not performed, it remains unclear whether these click here effects reflect preferential auditory CS+ processing, a somatosensory CR (see above) or a mixture of both. Effects in the early interval were interpreted

as reflecting preferential auditory sensory processing of the safety signalling Selleck PR-171 CS− tones. A post hoc analysis of N1m interval identified by Kluge et al. (2011; 85–115 ms) revealed very similar results as our 100–150 ms interval analysis i.e. relatively enhanced CS− processing at the left temporoparietal junction and left occipitocerebral junction. An Cobimetinib nmr analysis of their P2m (180–270 ms) interval in fact showed indications for enhanced CS+ processing but at bihemispheric somatosensory and right hemispheric parietal but not auditory sensory regions. Again, the N1m and P2m CS+ processing identified by Kluge and co-workers may at least partly reflect a conditioned response. The late effect might, however, additionally represent some form of conscious CS+ processing depending on contingency awareness which might have contributed to the unique reversal effects in this late component. Please note that, in the above classical conditioning studies, CS stimuli have been paired several hundred times with or without US and most subjects should have

been at least partly aware of the reinforcement plan, whereas absence of contingency awareness is one important factor within this MultiCS conditioning study. Importantly, the only interval which should not be superposed by a conditioned response (20–50 ms) revealed enhanced processing for safety signalling CS− identical to our study. The length of the CS stimuli (200 ms in Moses et al., 2010; and 250 ms in Kluge et al., 2011) is another important difference between these previous classical and our MultiCS conditioning studies (20 ms CS length). Although a differentiation of CS+ and CS− tones should be available immediately after CS onset, as our results would suggest, it remains unclear how differential processing of the subsequent parts of the auditory CS superimpose (e.

Samples were pelleted and resuspended in Laemmli buffer containing 5% 2-mercaptoethanol and stored at −20 °C. Proteins were separated on 4–20% Tris–HCl SDS-PAGE TGX gels in running

buffer (25 mM Tris base, 192 mM glycine, 10% SDS). Frozen lysates were boiled for 5 min and held on ice for 5 min before use. The RC DC Protein Assay was performed to equalize the amount of total protein loaded in each lane. All protein supplies were obtained from Bio-Rad unless otherwise stated. Proteins were transferred to an Immun-Blot PVDF membrane using a Trans-Blot apparatus. The membrane was blocked overnight at 4 °C in 0.05% Tween 20 in Tris-buffered saline (TBS) containing 5% nonfat dry milk on a Belly Dancer. Primary antibodies used at 1 : 10 000 dilutions were either an antipeptide PD-0332991 solubility dmso antibodies directed against amino acids 5–19 of UmuDAb or polyclonal antibody prepared by GenScript by injection of purified UmuDAb ABT 199 (produced by GenScript) into rabbits and purified by protein A chromatography. Goat anti-rabbit HRP-conjugated secondary antibody was used at a dilution of 1 : 32 000. All antibody incubations were carried out for 1 h in 0.05% TBS Tween 20

in 2.5% milk on a Belly Dancer. Precision StrepTactin-HRP Conjugate was added with the secondary antibody to visualize the protein size marker (Precision Plus Protein WesternC Standards). The membrane was washed five times (10 min each) with 0.01% TBS Tween 20 after each antibody incubation. SuperSignal West Pico chemiluminescent substrate (Pierce) was used to visualize

proteins after exposure to X-ray film. UmuDAb expression and cleavage was investigated after transforming E. coli AB1157 wild-type and mutant cells with plasmids bearing various umuDAb alleles. This allowed us to test the effects of recA and umuD mutations on UmuDAb cleavage in a context of the otherwise intact and well-studied DNA damage response of E. coli. Escherichia coli cells were exposed to DNA-damaging agents, and immunoblot analyses of cell lysates were performed with anti-UmuDAb peptide or polyclonal antibodies. To test whether the umuDAb ORF truly encoded an extra-large UmuDAb protein, plasmid pJH1, which contains 2.2 kbp of DNA from ADP1, including umuDAb in its native chromosomal context, was used as a UmuDAb expression source. This approach was feasible Cytidine deaminase because Acinetobacter promoters are typically highly expressed in E. coli (Shanley et al., 1986). Lysates from E. coli wild-type and ΔumuD cells, carrying pJH1 but not treated with MMC, expressed a c. 24-kDa protein (Fig. 2), consistent with the predicted molecular weight of 23.4 kDa, and demonstrating that the protein encoded by umuDAb was indeed larger than the 15-kDa UmuD (Kitagawa et al., 1985). This protein was not expressed in cells containing only the pUC19 vector of pJH1. This UmuDAb expression in uninduced E. coli may be due to the lack of an E.

Wildtype DJ-1 scavenges H2O2 by cysteine oxidation in response to oxidative stress, and thus confers neuroprotection. Activation of the transcription factor NF-E2-related factor-2 (Nrf2) has also been shown to be important for protection against oxidative stress in many models of neurodegenerative diseases. Previous data indicate that DJ-1 affects the transcriptional functions and stability of Nrf2. However, this observation has not been confirmed. In the current study, the role of DJ-1 in the regulation CX5461 of Nrf2 is examined in primary cultured neurons,

astrocytes and in vivo. The prototypical Nrf2 activator tBHQ protected primary cortical neurons derived from DJ-1-knockout (KO) as well as DJ-1 wildtype mice by activation of Nrf2-ARE pathway. Nrf2 nuclear translocation, robust increases in canonical Nrf2-driven genes and proteins, and dramatic activation of the ARE reporter gene, hPAP, were observed after tBHQ treatment. These results were further confirmed by siRNA-mediated DJ-1 knockdown in primary cortical astrocytes from ARE-hPAP mice and tBHQ administration into the striatum of mouse brain. In addition, overexpression of Nrf2 with adenovirus preferentially in astrocytes from DJ-1-KO mice enhanced survival

of neurons under oxidative insults. These findings indicate that activation of the Nrf2–ARE pathway is independent of DJ-1, and Nrf2 activation is a potential therapeutic target to prevent neurodegeneration in sporadic and DJ-1 familial Parkinson’s disease. “
“Neuronal firing sequences that occur during behavioral tasks are precisely Alectinib supplier reactivated in the neocortex and the hippocampus during rest and sleep. These precise firing sequences are likely to reflect latent memory traces, and their reactivation

is believed to be essential for memory consolidation and working memory maintenance. However, how the organized repeating patterns emerge through the click here coordinated interplay of distinct types of neurons remains unclear. In this study, we monitored ongoing spatiotemporal firing patterns using a multi-neuron calcium imaging technique and examined how the activity of individual neurons is associated with repeated ensembles in hippocampal slice cultures. To determine the cell types of the imaged neurons, we applied an optical synapse mapping method that identifies network connectivity among dozens of neurons. We observed that inhibitory interneurons exhibited an increase in their firing rates prior to the onset of repeating sequences, while the overall activity level of excitatory neurons remained unchanged. A specific repeating sequence emerged preferentially after the firing of a specific interneuron that was located close to the neuron first activated in the sequence. The times of repeating sequences could be more precisely predicted based on the activity patterns of inhibitory cells than excitatory cells.

p.m. Actinobacillus pleuropneumoniae isolates were either obtained from existing collections maintained in our

University, or kindly provided by Dr Huanchen Chen (Huazhong Agricultural University, Wuhan, China) and Dr Youxiang Diao (Shandong Agricultural University, Tai’an, China). The chromosomal DNA from A. pleuropneumoniae was extracted using the AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen) according to the manufacturer’s instructions. RDA was performed using a previously check details described method (Lisitsyn & Wigler, 1993). The following adapters and primers were used for RDA (listed in Table 2): R-Bgl 12/R-Bgl 24, J-Bgl 12/J-Bgl 24, and N-Bgl 12/N-Bgl 24. Briefly, the DNA fragments were digested with Sau3AI (TaKaRa), the R-Bgl 12/R-Bgl 24 adapters were ligated to the digested DNA to be used as the tester. The first differential product (DP1) was obtained by performing hybridization (20 h at 67 °C) with a driver : tester ratio

of 100 : 1, and this product was amplified by PCR with an R-Bgl 24 primer. The second (DP2) and third (DP3) differential products were generated by ligating the N-Bgl and J-Bgl adapters to the tester in the second and third rounds of subtractive hybridization, with driver : tester ratios of 400 : 1 and 8000 : 1, respectively. The differential DNA fragments for CVCC259 were obtained using CVCC259 as the tester and CVCC261 as the driver; this combination was designated as ‘a.’ Similarly, the differential DNA fragments for CVCC261 were obtained using CVCC261 as the tester and CVCC259 as the driver; this combination was designated as ‘b. The DP3 differential products were 5-FU manufacturer purified using the

Qiaquick PCR purification kit (Qiagen) and ligated into the pGEM-T vector (Promega). The RDA library was constructed by transforming the ligation mixture into competent Escherichia coli DH5α cells (TaKaRa). The inserts were sequenced by the BGI-GBI Biotech Company (Beijing, China). The blastn program was used to locate the sequence similarity nearly in the GenBank database. The differential nature of the DNA sequences was confirmed using a novel application of the reverse Southern hybridization procedure (Lancashire et al., 2007). The differential DNA fragments were successively spotted onto a nylon membrane. The membrane was baked at 120 °C for 30 min. The probes were prepared using 6 μg of the Sau3AI-digested genomic DNA obtained from the CVCC259 and CVCC261 strains and separately labeled using digoxigenin (DIG)-High Prime (Roche). Nonradioactive labeling, hybridization, and detection were performed using the DIG-High Prime DNA Labeling and Detection Starter Kit (Roche) according to the manufacturer’s instructions. Because all the amplified differential sequences contained the J-Bgl 24 primer, the J-Bgl 24 primer was considered as the negative control. To further characterize the differential DNA sequences, we designed specific primers using the primer 5.0 software (listed in Table 2).