Therefore, HDAC inhibitors might reactivate the expression of plasticity-related genes in the adult cortex by enhancing CREB-mediated gene transcription. This possibility is also supported by the observation that MD in adult mice triggers selleck screening library a labile form of plasticity that can be rendered persistent by the expression of a constitutively active CREB mutant (Pham et al., 2004). Which genes are crucially involved in mediating the action of HDAC inhibitors on visual cortical plasticity, or in other models of brain plasticity, is still poorly known. Further analyses are required to unravel the final effectors of the epigenetic treatment on visual

cortical plasticity (Borrelli et al., 2008; Fagiolini et al., 2009). In addition learn more to the manipulation of epigenetic mechanisms, other factors are able to promote a recovery from amblyopia in adult rodents. Environmental enrichment (Sale et al., 2007) and dark rearing (He et al., 2007), coupled with RS or binocular vision, allow the recovery of a long-term deprived eye to normal levels of acuity and ocular dominance. Both protocols lead to a reduction in GABAergic inhibition and

probably result in a return to an SP-like balance between excitation and inhibition (Spolidoro et al., 2009). Influencing specific molecular and cellular components was also found to promote recovery from amblyopia in adulthood. Visual cortical plasticity is inhibited by the aggregation of extracellular matrix molecules such as chondroitin sulphate proteoglycans (CSPGs). Enzymatic digestion of CSPGs in combination with RS has been shown to restore visual cortical plasticity in adult rats (Pizzorusso et al., 2002) and to reverse the effects of long-term MD on visual acuity and ocular dominance (Pizzorusso et al., 2006). Also, chronic administration of fluoxetine (which increases extracellular serotonin levels), coupled with RS, allows visual acuity and ocular dominance

recovery from long-term MD (Maya Vetencourt Sorafenib et al., 2008); again, a possible mediator of the effect is the lowering of the inhibitory tone (Spolidoro et al., 2009). Intriguingly, environmental enrichment induces histone acetylation, and fluoxetine causes alterations in gene expression overlapping with those induced by HDAC inhibitor treatment (Fischer et al., 2007; Covington et al., 2009); it is therefore possible that epigenetic mechanisms could represent a common endpoint of other treatments enhancing plasticity in the adult visual cortex (Pizzorusso et al., 2007). In summary, our study demonstrates that targeting HDACs can be an effective pharmacological strategy to promote experience-dependent plasticity in the adult visual cortex and recovery from amblyopia.

The use of animal manure as crop fertilizer contributes to the sustainable

recycling MDX-1106 of essential nutrients and organic matter required to maintain good soil quality. However, care must be taken to avoid soil and plant contamination with human pathogenic bacteria present in untreated animal manure as well as dissemination of the bacteria. A large part of the outbreaks caused by pathogenic bacteria is related to the consumption of raw produce contaminated with human pathogens such as Salmonella spp. (Semenov, 2008). Salmonella spp. are more persistent in soil compared with other bacterial pathogens (Guan & Holley, 2003), displaying long periods of survival (Zibilske & Weaver, 1978) and only slightly reduced cell numbers over time (Guo et al., 2002a). Salmonella has been detected in fecal cultures from the majority

of dairies (Kirk, 2003), posing a significant risk of further pathogen dissemination to soil and fresh plant produce through the application of untreated cattle manure to agricultural fields. In several cases, cows carried Salmonella asymptomatically, i.e. they did not have clear symptoms that humans infected with Salmonella show (Semenov, 2008). Salmonella cells present in cattle manure have been shown to survive for at least 60 days at 4 and 20 °C (Himathongkham et al., 1999), but were not detectable after 19 days at 37 °C. Upon application of contaminated manure to soil, Salmonella was shown to survive for up to 300 days, with higher initial bacterial inoculation doses normally resulting in extended survival periods of Salmonella in the soil (Jones, 1986; Baloda et al., 2001; Islam et www.selleckchem.com/products/AP24534.html al., 2004). Whether Salmonella can disseminate to plant roots depends on factors such as the site of colonization (Doyle & Erickson, 2008), i.e. whether bacteria colonize the root surface or exhibit endophytic colonization of roots and aboveground plant tissues. For example, Salmonella enterica has been shown to penetrate epidermal cell walls of barley

roots (Kutter et al., 2006) and has been detected in sterilized leaf samples from crops grown in soil contaminated Farnesyltransferase with Salmonella (Franz et al., 2007). The entry sites of the pathogens are believed to be around cracks (Wachtel et al., 2002) and lateral root junctions (Cooley et al., 2003; Dong et al., 2003; Warriner et al., 2003), which display increased exudation of nutrients (Jablasone et al., 2005). Internalized pathogens may move systemically through plants (Guo et al., 2002b), but contamination of edible plant parts has been also reported to occur via movement along the plant surface (Cooley et al., 2003). Bacteria that manage to reach leaf surfaces must contend with harsh conditions (i.e. lack of nutrients and sunlight), and the persistence of S. enterica is 30–40-fold lower in the phyllosphere compared with in the rhizosphere (Cooley et al., 2003).

Control larvae were injected with 0.9% NaCl. To determine whether DHA confers a protective effect to Burkholderia-infected larvae, a single dose of DHA (50 mM) was administrated 6 h postinfection. To determine intracellular bacterial numbers, hemolymph was obtained from three infected

larvae by puncturing the larval abdomen with a sterile needle. The out-flowing hemolymph was immediately transferred into a sterile Eppendorf tube containing a few crystals of phenylthiourea to prevent melanization. Then, hemolymph was serially diluted in 0.9% selleck kinase inhibitor NaCl and plated on LB agar. Colonies were counted after incubation at 37 °C for 24 h. Three larvae per treatment for each time point (10 and 21 h postinfection) were cryopreserved, sliced and homogenized in 1 mL of Trizol reagent (Sigma–Aldrich). Whole animal RNA was extracted according to the manufacturer’s protocol. RNA was treated with Turbo DNase (Ambio, Applied Biosystems) according to manufacturer’s recommendations and quantified spectrophotometrically (NanoDrop ND-1000). Quantitative real-time reverse transcription PCR (RT–PCR) was performed Belnacasan mouse with the 7500 real-time PCR system (Applied Biosystems), according to the protocols of the manufacturer. Briefly, cDNA was synthesized from 200 ng of total RNA using Taqman kit (Roche, Applied Biosystems) and then analyzed with Power SYBR Green

master mix (Applied Biosystems), using primers to amplify the genes encoding antimicrobial peptides: gallerimycin (Altincicek & Vilcinskas, 2006), galliomycin (Wojda et al., 2009), inducible metalloproteinase inhibitor (IMPI) (Altincicek & Vilcinskas, 2006), lysozyme (Altincicek & Vilcinskas, 2006) and the housekeeping gene actin (Altincicek & Vilcinskas, 2006). All samples were analyzed in triplicate, and the amount of mRNA detected normalized to control actin mRNA values. Relative quantification of genes expression was calculated by using the ∆∆CT method (Livak & Schmittgen, 2001). All experiments were performed a minimum of three times. Relative comparisons were done between corrected values with anova test for significance.

A P-value Amisulpride < 0.05 was considered statistically significant. The antibacterial activity of eight LCUFAs with various carboxyl lengths (18 carbons or more) was quantitatively evaluated against B. cenocepacia K56-2. The growth was monitored for 24 h at 37 °C in the presence of 20 mM of each LCUFA by measuring the OD640 nm. Of the eight lipids tested, only 3 [Petroselinic acid 18:1 (n-6), DHA 22:6 (n-3) and nervonic acid 24:1 (n-9)] showed growth inhibition effects. DHA caused the greatest growth inhibition (50–60%) compared with the control (Fig. 1), so it was selected for further studies. A control assay with 2.7% ethanol had no effect on the growth of B. cenocepacia K56-2 (Fig. 1). DHA against B. cenocepacia K56-2 recorded a MIC range of 40–50 mM, determined after 24 h by the reference broth microdilution method.

Xylella fastidiosa can be graft transmitted to healthy plants or vectored by several species GSK1120212 in vivo of xylem-feeding leafhoppers (Redak et al., 2004). Antimicrobial peptides (AMPs) are defensive substances widespread in nature, being produced from bacteria to mammals (Sang & Blecha, 2008). The majority display common features such as a low molecular

mass, a positive net charge at physiological pH and an amphipathic structure (Bulet et al., 2004). Generally, AMPs disrupt the plasmatic membrane, causing the rapid death of microorganisms. Nevertheless, some AMPs can cross the microbial membrane acting on internal cellular targets (Brogden, 2005). The elucidation of the exact mode of action of AMPs is essential to allow the use of these substances to develop a new generation of antibiotics to control infections of both plants and animals. Gomesin is a short β-hairpin AMP (2270.4 Da) isolated from the hemocytes of the tarantula spider Acanthoscurria gomesiana (Silva et al.,

2000). Similar to http://www.selleckchem.com/products/17-AAG(Geldanamycin).html most AMPs, gomesin is cationic and possesses an amphipatic structure (Mandard et al., 2002). Bacteria and fungi synthesize and secrete AMPs to inhibit the growth of neighboring microorganisms that compete for both space and nutrients (Sang & Blecha, 2008). Several bacteria and fungi species have been reported to compose an endophytic community that cohabits the xylem vessels of citrus plants along with X. fastidiosa (Araujo et al., 2002). Moreover, many tissues of insects, including salivary glands and the lining epithelial cells, produce AMPs to prevent infection (Bulet et al., 2004). Therefore, it is likely that X. fastidiosa may be exposed to AMPs during colonization of both the host plant and the insect vector. To verify whether and how X. fastidiosa would respond to AMPs, we evaluated the effect of gomesin on its global gene expression profile and on Baf-A1 supplier X. fastidiosa colonization success of

tobacco plants. Ampicillin, streptomycin and tetracycline were from Sigma-Aldrich (St. Louis, MO). Gomesin (Silva et al., 2000) was synthesized as described previously (Fazio et al., 2006). Lyophilized gomesin and conventional antibiotic were reconstituted in sterile deionized water immediately before use. Virulent 9a5c and avirulent J1a12 X. fastidiosa strains (Li et al., 1999; Koide et al., 2004) were grown in periwinkle wilt (PW) broth medium (Davis et al., 1981) supplemented with 20% glucose as described previously (Zaini et al., 2008). Mid-log suspensions (OD600 nm=0.400) of either 9a5c or J1a12 strains were incubated with different concentrations of gomesin (0.14–9 μg mL−1), ampicillin (0.31 × 103–5 × 103 μg mL−1), streptomycin (0.19–100 μg mL−1), tetracycline (0.97–500 μg mL−1) or water as control. After 18 h at 28 °C, cells were harvested by centrifugation at 4000 g for 5 min at 25 °C, suspended in fresh PW medium and plated on 2% agar PW.

Xylella fastidiosa can be graft transmitted to healthy plants or vectored by several species find protocol of xylem-feeding leafhoppers (Redak et al., 2004). Antimicrobial peptides (AMPs) are defensive substances widespread in nature, being produced from bacteria to mammals (Sang & Blecha, 2008). The majority display common features such as a low molecular

mass, a positive net charge at physiological pH and an amphipathic structure (Bulet et al., 2004). Generally, AMPs disrupt the plasmatic membrane, causing the rapid death of microorganisms. Nevertheless, some AMPs can cross the microbial membrane acting on internal cellular targets (Brogden, 2005). The elucidation of the exact mode of action of AMPs is essential to allow the use of these substances to develop a new generation of antibiotics to control infections of both plants and animals. Gomesin is a short β-hairpin AMP (2270.4 Da) isolated from the hemocytes of the tarantula spider Acanthoscurria gomesiana (Silva et al.,

2000). Similar to 5-FU mouse most AMPs, gomesin is cationic and possesses an amphipatic structure (Mandard et al., 2002). Bacteria and fungi synthesize and secrete AMPs to inhibit the growth of neighboring microorganisms that compete for both space and nutrients (Sang & Blecha, 2008). Several bacteria and fungi species have been reported to compose an endophytic community that cohabits the xylem vessels of citrus plants along with X. fastidiosa (Araujo et al., 2002). Moreover, many tissues of insects, including salivary glands and the lining epithelial cells, produce AMPs to prevent infection (Bulet et al., 2004). Therefore, it is likely that X. fastidiosa may be exposed to AMPs during colonization of both the host plant and the insect vector. To verify whether and how X. fastidiosa would respond to AMPs, we evaluated the effect of gomesin on its global gene expression profile and on Tau-protein kinase X. fastidiosa colonization success of

tobacco plants. Ampicillin, streptomycin and tetracycline were from Sigma-Aldrich (St. Louis, MO). Gomesin (Silva et al., 2000) was synthesized as described previously (Fazio et al., 2006). Lyophilized gomesin and conventional antibiotic were reconstituted in sterile deionized water immediately before use. Virulent 9a5c and avirulent J1a12 X. fastidiosa strains (Li et al., 1999; Koide et al., 2004) were grown in periwinkle wilt (PW) broth medium (Davis et al., 1981) supplemented with 20% glucose as described previously (Zaini et al., 2008). Mid-log suspensions (OD600 nm=0.400) of either 9a5c or J1a12 strains were incubated with different concentrations of gomesin (0.14–9 μg mL−1), ampicillin (0.31 × 103–5 × 103 μg mL−1), streptomycin (0.19–100 μg mL−1), tetracycline (0.97–500 μg mL−1) or water as control. After 18 h at 28 °C, cells were harvested by centrifugation at 4000 g for 5 min at 25 °C, suspended in fresh PW medium and plated on 2% agar PW.

In addition, a coherent

system of co-operation between the hospital and community services is also essential. Advocacy, communication and social mobilization are vital issues to bridge pre-existing gaps between the health system and the community by enhancing knowledge, attitude and practice related to TB, especially in pregnant women. There remain several major knowledge gaps in the management see more of TB during pregnancy. Interaction between poverty and undernutrition on one hand, and combination of pregnancy and TB, on the other, deserve thorough exploration by a large-scale analytical study in South Asian countries. A multicenter comparative cohort study could also overcome the current knowledge gaps in the perinatal implications of maternal TB, which remains a widely deserted and neglected area. N.J. TSA HDAC supplier conceived the idea of this article and provided the framework. All authors collected and analyzed the relevant information. N.J. wrote the first draft, and A.K.S. added perinatal management. Initial draft was modified by S.B., N.A. and A.K.S. with critical inputs. All authors read and approved the final manuscript. None required. None. None declared. “
“To report on improved perinatal states

in Japan, governmental and United Nations Children’s Fund reports were analyzed. Initial maternal mortality, which was 409.8 in 1899, decreased to 4.1 in 2010, with a reduction Thiamet G rate of

409.8/4.1 (102.4) in 111 years: 2.5 in the initial 50 years in home delivery and 39.3 in the later 60 years in hospital births. The difference between 2.5 versus 39.3 was attributed to the medicine and medical care provided in hospital births. The total reduction of neonatal mortality was 77.9/1.1 (70.8), and the rate in the initial 50 versus later 60 years was 2.8/25. Also, there was a big difference after introduction of extensive neonatal care. Virtual perinatal mortality after 22 weeks was estimated to be 428 in 1000 births in 1900 (i.e. those infants born at 22–28 weeks were unlikely to survive at that time), while the perinatal mortality was reported to be 22 weeks or more in 1979 (i.e. premature babies born at ≥22 weeks survived in 1979 because of the improved neonatal care). Actually, 60% of premature infants of 400–500 g survived in the neonatal intensive care unit. In a recent report, 36% of infants born at 22 weeks survived to 3 years. Although there were neurodevelopmental impairments, outcomes were improved. In conclusion, perinatal states have remarkably improved in Japan. Perinatal medicine started in Japan in the last year of the 19th century, 1899, with the first official reports of maternal mortality (409.8/100 000 total births) and neonatal mortality (77.9/1000 live births), and the first official midwife license.

Arsenate was added to M. penetrans cells to determine whether ATP hydrolysis by a motor-associated component directly provides

energy for gliding, as proposed for M. mobile, upon whose gliding motility arsenate has an immediate negative impact (Jaffe et al., 2004). M. penetrans continued to glide Selleckchem Androgen Receptor Antagonist in the presence of 50 mM arsenate, five times the amount in which growth was prevented (see above), at incubation times ranging from 1 to 8 h. In 50 mM arsenate, the gliding speeds of both M. mobile [F(1, 144) = 13331, P < 0.0003] and M. penetrans [F(1, 144) = 7670, P < 0.0003] were significantly reduced. However, the 37% decrease in M. penetrans was much smaller than that in M. mobile, which exhibited an 89% decrease in speed (Fig. 2), essentially in agreement with the observations of an absence of M. mobile cells moving faster than 10% of normal gliding speed after 10 min under similar conditions (Jaffe et al., 2004). Although the change in speed of M. penetrans was statistically significant, the moderate value of the decrease and the continued movement of the cells after 8 h (not shown) suggest that direct inhibition of the motor by ATP depletion was unlikely. Increasing the arsenate concentration fivefold further, to 250 mM, had a negligible effect on M. penetrans motility (Fig. 2). Thus, ATP hydrolysis is an unlikely energy buy HKI-272 source for gliding by M. penetrans. The

presence of membrane potential has been reported in a variety of mycoplasma species (Benyoucef et al., 1981; Schiefer & Schummer, 1982). To determine whether PMF supplies the energy needed for M. penetrans gliding motility, we observed motility

in the presence of the ionophore CCCP, which collapses the proton gradient. Cells were incubated for 1 h in the presence of 10 mM CCCP in DMSO and in PBS-G2K containing the same volume of DMSO used in the test buffer. After 1 h, gliding speed actually increased by 29% compared to the control buffer (P < 0.0001) (Fig. 2), ruling out PMF as an energy source for gliding motility of M. penetrans. Selleckchem Fluorouracil To test SMF as a potential energy source for M. penetrans gliding, cells were observed in the presence of amiloride, an inhibitor of Na+/H+ antiporters and sodium channels, which competes with Na+ in the medium (Benos, 1982). Mycoplasma penetrans gliding speed was not significantly affected by 1 h of incubation in amiloride (P = 0.6) (Fig. 2), ruling out SMF as an energy source. To determine the role of thermal energy in the motility mechanism of M. penetrans, we analyzed its gliding speed under conditions of differing temperature. If radiant energy from ambient heat is a significant power source, then we would predict increased speed even at temperatures in excess of those normally encountered physiologically. We analyzed gliding speed at temperatures ranging from 30 to 40 °C and pH levels ranging from 5.8 to 8.8 (Fig. 3). Speed increased with temperature, but at acidic or alkaline pH, the trend was less distinct.

Arsenate was added to M. penetrans cells to determine whether ATP hydrolysis by a motor-associated component directly provides

energy for gliding, as proposed for M. mobile, upon whose gliding motility arsenate has an immediate negative impact (Jaffe et al., 2004). M. penetrans continued to glide Epacadostat manufacturer in the presence of 50 mM arsenate, five times the amount in which growth was prevented (see above), at incubation times ranging from 1 to 8 h. In 50 mM arsenate, the gliding speeds of both M. mobile [F(1, 144) = 13331, P < 0.0003] and M. penetrans [F(1, 144) = 7670, P < 0.0003] were significantly reduced. However, the 37% decrease in M. penetrans was much smaller than that in M. mobile, which exhibited an 89% decrease in speed (Fig. 2), essentially in agreement with the observations of an absence of M. mobile cells moving faster than 10% of normal gliding speed after 10 min under similar conditions (Jaffe et al., 2004). Although the change in speed of M. penetrans was statistically significant, the moderate value of the decrease and the continued movement of the cells after 8 h (not shown) suggest that direct inhibition of the motor by ATP depletion was unlikely. Increasing the arsenate concentration fivefold further, to 250 mM, had a negligible effect on M. penetrans motility (Fig. 2). Thus, ATP hydrolysis is an unlikely energy Ruxolitinib mouse source for gliding by M. penetrans. The

presence of membrane potential has been reported in a variety of mycoplasma species (Benyoucef et al., 1981; Schiefer & Schummer, 1982). To determine whether PMF supplies the energy needed for M. penetrans gliding motility, we observed motility

in the presence of the ionophore CCCP, which collapses the proton gradient. Cells were incubated for 1 h in the presence of 10 mM CCCP in DMSO and in PBS-G2K containing the same volume of DMSO used in the test buffer. After 1 h, gliding speed actually increased by 29% compared to the control buffer (P < 0.0001) (Fig. 2), ruling out PMF as an energy source for gliding motility of M. penetrans. Teicoplanin To test SMF as a potential energy source for M. penetrans gliding, cells were observed in the presence of amiloride, an inhibitor of Na+/H+ antiporters and sodium channels, which competes with Na+ in the medium (Benos, 1982). Mycoplasma penetrans gliding speed was not significantly affected by 1 h of incubation in amiloride (P = 0.6) (Fig. 2), ruling out SMF as an energy source. To determine the role of thermal energy in the motility mechanism of M. penetrans, we analyzed its gliding speed under conditions of differing temperature. If radiant energy from ambient heat is a significant power source, then we would predict increased speed even at temperatures in excess of those normally encountered physiologically. We analyzed gliding speed at temperatures ranging from 30 to 40 °C and pH levels ranging from 5.8 to 8.8 (Fig. 3). Speed increased with temperature, but at acidic or alkaline pH, the trend was less distinct.

In vitro studies have shown buy GSK2118436 increased expression of HPV E1 and L1 viral genes in the presence of HIV transactivator

of transcription (tat) proteins [17]. Our analysis using an older set of high-risk HPV types suggested that higher VL may be associated with HPV detection and hinted at a role of HIV VL in HPV acquisition. However, such an association was not suggested using the latest set of high-risk HPV types. Hence, our research demonstrates the importance of considering the HPV types used when reviewing the literature. It has been postulated that immune reconstitution associated with HAART may lead to clearance of HPV, as has been the case with other viral or nonviral opportunistic infections,

and this is consistent with the results of our analyses, where higher CD4 cell count was associated with a higher probability of HPV clearance. There are a couple of limitations to our analyses. There was a trend for earlier discontinuation in subjects starting with HPV infection, suggesting possible informative censoring, and the small sample size did not allow the use of models that adjust for covariates such as age, cigarette smoking, HPV type and sexual activity. There are several advantages of the statistical methods that we used. The multi-state modelling approach accommodates multiple and recurrent events using intermittent Tanespimycin data. The hazard rates are estimated simultaneously in the model, eliminating the need to subset the data to estimate the HPV detection rate among not HPV-negative subjects

and separately to estimate clearance among the HPV-positive subjects. Also, while other HPV studies have used the midpoint between visit times as the event time (e.g. the time of HPV detection or clearance) or the time of the visit, the methods we used are appropriate when the exact event times are unknown. The approach utilized the incomplete data efficiently and provided a more comprehensive description of the HPV detection and clearance process. We thank the clinicians, study coordinators and study subjects at A5029 sites for their participation and the A5029 study team, headed by Ken Fife, for sharing the data. We also thank Stephen Lagakos, Janet Andersen and Michael Hughes for their thoughtful comments on the analysis. The authors are supported by the AIDS Clinical Trials Group and K24 Mid-Career Research Mentoring Award funded by the National Institute of Allergy and Infectious Diseases (Grants 1U01AI068636-01, 1U01AI068634-01 and K24AI066884).

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 Epacadostat mw 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 Selleck Dabrafenib 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 Farnesyltransferase 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).