Enhanced Antibacterial Action of Bacteriocin Producing Cells by Binding to the Target Pathogen

Bacteriocins are natural weapons of bacterial inter-species competition in food preservation arsenal. Bacteriocins produced by lactic acid bacteria have gained particular attention owing to their potential application as the substitute of artificial chemical preservatives. This research made use of genetic engineering technologies to clone the class IIa bacteriocin genes and construct bacteriocin structural gene expression systems, aiming at solving the problem of low bacteriocin production in wild type lactic acid bacteria strains and achieving efficient killing of Listeria monocytogenes. The total DNA of Pediococcus acidilactici PA003 was used as the template to amplify the structural gene pedA, which was inserted into pET32a(+) vector and transformed into Escherichia coli. The recombinant plasmid containing the pedA gene was verified by DNA sequencing. This recombinant strain was induced with IPTG and it efficiently expressed a 22 kDa Trx-PedA fusion protein as inclusion bodies. One protein band corresponding to the predicted molecular mass of pediocin was obtained after renaturation and enterokinase treatment. The agar diffusion assay revealed that 512 arbitrary unit (AU) antilisterial activities were obtained from 1 ml culture of recombinant E. coli. The same strategy was adopted using pET20b(+) as the expression vector. The PelB signal peptide in this vector resulted in soluble expression of fusion protein both in the intracellular and periplasmic space with totally 384 AU/ml production. Lactococcus lactis cells were engineered to bind to cellulose by fusing cellulose-binding domain of Cellvibrio japonicus with PrtP, NisP and AcmA anchors for surface display. The CBD-PrtP showed the most efficient immobilization. Expression of sortase with the CBD-PrtP fusion did not improve binding of the anchor to the cell wall. Next, the surface display technique was aimed to be combined with secretion of antilisterial bacteriocins in order to construct an E. coli strain with capacity to bind and kill L. monocytogenes cells. Such cells could be used to test the hypothesis that antilisterial bacteriocin secreting cells kill listerial cells more efficiently if they also have the capacity to bind to listerial cells. Therefore, the CBD500 and CBDP35 cell-wall binding domains from Listeria phage endolysins were used to engineer E. coli cells to bind to L. monocytogenes cells using different cell anchoring domains. First CBD500 was fused to the outer membrane anchor of Yersinia enterocolitica adhesin YadA for potential surface display. Whole-cell ELISA showed that CBD-YadA fusion was displayed on the cell surface. However, production of the fusion protein was detrimental to the growth of recombinant cells. Therefore, a fragment of the E. coli outer membrane protein OmpA was selected for fused expression of CBD500 in E. coli. Western blot revealed the OmpA-CBD was mainly localized on the external surface of recombinant cells. However, the accessibility of the CBD on the cell envelope to cells of Listeria could not be shown. For an improved surface display, CBD was expressed as FliC CBD chimeric protein in flagella. CBD500 and CBDP35 domain coding sequences were inserted into vector pBluescript/fliCH7. CBD insertion in flagella was confirmed by Western blot. The FliC CBDP35 flagella were isolated and shown to bind to L. monocytogenes WSLC 1019 cells. To test the hypothesis that bacteriocin-secreting cells kill target cells more efficiently by binding to the target cells, bacteriocin-secreting strains with binding ability to Listeria cells were constructed. Antilisterial E. coli was obtained either by transferring pediocin production from Lactobacillus plantarum WHE 92 or leucocin C production from Leuconostoc carnosum 4010. The Listeria-binding cells producing pediocin decreased approximately 40 per cent of the Listeria cells during three hours, whereas the cell-free medium with the corresponding amount of pediocin could only inhibit cell growth but did not decrease the number of viable Listeria cells after the three hours incubation. The cell-mediated leucocin C killing resulted in a two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. These results indicate that close contact between Listeria and bacteriocin-producing cells is beneficial for the killing effect by preventing its dilution in the environment and adsorption onto particles before taking effect to the target cells.Bakteriosiinit ovat luonnollisia aseita lajienvälisessä kilpailussa elintarvikkeissa. Maitohappobakteerien tuottamat bakteriosiinit ovat olleet erityisen huomion kohteena niiden keinotekoisten kemiallisten säilöntäaineiden korvaamispotentiaalin takia. Tutkimuksissani hyödynsin geenimuokkausta kloonatessani ja tehdessäni luokka IIa bakteriosiiniituottosysteemit yrittäessäni ratkoa villityypin maitohappobakteerikannan matalan bakteriosiinintuottotason ongelmaa saaden tehokasta Listeria monocytogenes bakteerin tappoa aikaiseksi. Käytin Pediococcus acidilactici PA003 kannan totaali-DNA:ta templaattina amplifioiden rakennegeeniä pedA, jonka insertoin pET32a(+) vektoriin ja transformoin Escherichia coli -bakteeriin. DNA-sekvensoinnilla varmennettua pedA- rekombinattiplasmidia indusoin IPTG:llä johtaen 22 kDa Trx-PedA fuusioproteiinin tuottoon inkluusiopartikkeleina. Inkluusiopartikkelit käsittelin enterokinaasilla ja renaturoin, jolloin SDS-PAGE-analyysilla osoitin yhden proteiinikertymän liikkuneen geelissä korreloiden ennustetun proteiinin molekyylimassan mukaisesti. Yksi ml rekombinantti-E. coli viljelmää tuotti 512 yksikköä (AU) antilisteria-aktiviteettia. Samaa strategiaa käytin pET20b(+) PelB-signaalisekvenssiä hyödyntävällä sekreetiovektorilla. Tällä vektorilla fuusioproteiini säilyi liukoisena tuottaen yhteensä 384 AU/ml pediosiiniaktiviteettia syto- ja periplasmaan. Muokkasin Lactococcus lactis -soluja sitoutumaan selluloosaan fuusiomalla selluloosaasitovaan osan (CBD) Cellvibrio japonicus bakteerista PrtP-, NisP- ja AcmA-ankkuriosiin pintaekspressiota varten. CBD-PrtP-osa johti parhaaseen immobilisaatioon. Sortaasin ja CBD-PrtP-fuusion yhteistuotto ei lisännyt fuusioproteiinin ankkuroitumista soluseinään. Seuraavaksi yhdistin pintaekspressioteknikoita antilisteriabakteriosiinien sekreetioon voidakseeni konstruoida E. coli kanta, joka sitoutuu L. monocytogenes soluihin ja tappaa ne. Sellaisilla soluilla voidaan testata listeriasoluihin sitoutuva antilisteriabakteriosiinia sekretoiva solu tappaa tehokkaammin kuin vastaava solu, joka ei sitoudu listeriasoluihin hypoteesi. Tämän takia käytin CBD500 and CBDP35 soluseinään sitoutuvat osat Listeria-faagin endolysiineistä muokatakseni E. coli soluja sitoutumaan L. monocytogenes bakteeriin. Ensin fuusioin CBD500 Yersinia enterocolitica bakteerin ulkomembraaniproteiini adhesiini-YadA:han pintaekspressiota varten. Kokosolu-ELISA osoitti että CBD-YadA fuusio tuottui solujen pintaan. Toisaalta fuusioproteiinin tuotto haittasi solujen toimintaa heikentäen solujen kasvua. Tämän takia valitsin toisen ulkomembraaniproteiiniosan, E. coli bakteerin OmpA-osan, CBD500 fuusiopartneriksi E. coli isännässä. Western blot analyysi osoitti että OmpA-CBD tuottui hyvin solujen ulkomembraaniin, mutta niiden hyvää esilläoloa ei voitu osoittaa. Saavuttaakseni parempaa listeriasoluun tarttuvan CBD-osan presentaatiota, tuotin CBD FliC CBD-kimeerinä flagellassa. Insertoin CBD500- ja CBDP35-koodaavat osat vektoriin pBluescript/fliCH7. Varmistin CBD-insertion flagellassa Western analyysilla. Eristin FliC CBDP35-flagellat ja osoitin niiden sitoutuvan L. monocytogenes WSLC 1019 -soluihin. Listeriasoluihin sitoutuva antilisteriabakteriosiinia sekretoiva solu tappaa tehokkaammin kuin vastaava solu, joka ei sitoudu listeriasoluihin hypoteesin paikkaansapitävyyden testaukseen siirsin vielä listeriasoluihin tarttuviin E. coli kantoihin antilisteriabakteriosiinin sekreetiokyvyn. Antilisteria E. coli kannat sain aikaiseksi siirtämällä pediosiinituottoa Lactobacillus plantarum WHE 92 tai leukoksiini C tuottoa Leuconostoc carnosum 4010 -kannoista. Listeriaan sitoutuvat ja pediosiiniä tuottavat solut vähensivät elävien listeriasolujen määrää 40 prosenttia kolmessa tunnissa, kun kolmen tunnin inkubointi vastaavan määrän pediosiiniä sisältävässä soluvapaassa kasvatusalustassa ainoastaan inhiboi kasvua muttei vähentänyt elävien listeriasolujen määrää. Soluvälitteinen leukoksiini C tappo johti kahden logaritmin listeriavähennykseen, kun vastaava määrä leukoksiini C:tä soluvapaassa kasvatusliemessa vain inhiboi listerioiden kasvua vailla bakteriosidistä vaikutusta. Nämä tulokset viittaavat siihen, että lähikontakti listerian ja bakteriosiiniatuovien solujen kanssa edesauttavat tappovaikutusta, johtuen mahdollisesti vähäisemmästä bakteriosiinin laimenemisesta ympäristöön ja adsoptiosta muihin pintoihin ennen sitoutumista kohdesoluihiinsa

Phenotypic comparison and DNA sequencing analysis of a wild-type and a pediocin-resistant mutant of Listeria ivanovii

Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being L. monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.Peer reviewe

Multimodal Medical Image Fusion by Adaptive Manifold Filter

Medical image fusion plays an important role in diagnosis and treatment of diseases such as image-guided radiotherapy and surgery. The modified local contrast information is proposed to fuse multimodal medical images. Firstly, the adaptive manifold filter is introduced into filtering source images as the low-frequency part in the modified local contrast. Secondly, the modified spatial frequency of the source images is adopted as the high-frequency part in the modified local contrast. Finally, the pixel with larger modified local contrast is selected into the fused image. The presented scheme outperforms the guided filter method in spatial domain, the dual-tree complex wavelet transform-based method, nonsubsampled contourlet transform-based method, and four classic fusion methods in terms of visual quality. Furthermore, the mutual information values by the presented method are averagely 55%, 41%, and 62% higher than the three methods and those values of edge based similarity measure by the presented method are averagely 13%, 33%, and 14% higher than the three methods for the six pairs of source images

Field verification of low-level biochar applications as effective ameliorants to mitigate cadmium accumulation into Brassica campestris L from polluted soils

Introduction: Cadmium (Cd) has been recognized as a significant contributor to the pollution of farmland soils in China, and biochars have been reported to be effective in mitigating soil Cd pollution. However, most studies have been conducted in laboratory or greenhouse settings, not at a field scale, and the biochars used have been applied at unrealistically high amounts (>10 t/ha). Methods: In this research, three biochars: rice straw biochar (RSB), pig manure biochar (PMB) and rice husk biochar (RHB) were produced from readily available farm residues. Then the effects at low-level application (1.8 and 3.6 t/ha) on Cd were investigated in a field experiment cropped with rape (Brassica campestris L.). Results: Batch adsorption experiments indicated Cd adsorption capacity of three biochars followed the order of RSB (43.5 mg/g) > PMB (33.3 mg/g) > RHB (24.4 mg/g). Field experiment indicated biochar amendments could slightly change soil pH and cation exchange capacity (CEC); yet led to considerable and significant decreases in extractable Cd concentrations [reductions of: 43%–51% (PMB), 29%– 35% (RSB) and 17%–19% (RHB)]. Reduced extractable Cd correlated with lower Cd concentrations in rape plants. PMB and RSB were more effective in decreasing Cd phytoaccumulation into edible parts of rape (>68% reduction) than RHB. Discussion: Low-level application of PMB or RSB could efficiently decrease the phytoaccumulation of Cd from soils into crops. These results demonstrate the reality of biochar-based remediation solutions to contribute to the mitigation of diffuse Cd contamination in farmland. The results also highlight the need to trail biochars in the presence of the soil to be targeted for remediation

Semen quality in relation to biomarkers of pesticide exposure.

We previously reported reduced sperm concentration and motility in fertile men in a U.S. agrarian area (Columbia, MO) relative to men from U.S. urban centers (Minneapolis, MN; Los Angeles, CA; New York, NY). In the present study we address the hypothesis that pesticides currently used in agriculture in the Midwest contributed to these differences in semen quality. We selected men in whom all semen parameters (concentration, percentage sperm with normal morphology, and percentage motile sperm) were low (cases) and men in whom all semen parameters were within normal limits (controls) within Missouri and Minnesota (sample sizes of 50 and 36, respectively) and measured metabolites of eight current-use pesticides in urine samples provided at the time of semen collection. All pesticide analyses were conducted blind with respect to center and case-control status. Pesticide metabolite levels were elevated in Missouri cases, compared with controls, for the herbicides alachlor and atrazine and for the insecticide diazinon [2-isopropoxy-4-methyl-pyrimidinol (IMPY)]; for Wilcoxon rank test, p = 0.0007, 0.012, and 0.0004 for alachlor, atrazine, and IMPY, respectively. Men from Missouri with high levels of alachlor or IMPY were significantly more likely to be cases than were men with low levels [odds ratios (ORs) = 30.0 and 16.7 for alachlor and IMPY, respectively], as were men with atrazine levels higher than the limit of detection (OR = 11.3). The herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and metolachlor were also associated with poor semen quality in some analyses, whereas acetochlor levels were lower in cases than in controls (p = 0.04). No significant associations were seen for any pesticides within Minnesota, where levels of agricultural pesticides were low, or for the insect repellent DEET (N,N-diethyl-m-toluamide) or the malathion metabolite malathion dicarboxylic acid. These associations between current-use pesticides and reduced semen quality suggest that agricultural chemicals may have contributed to the reduction in semen quality in fertile men from mid-Missouri we reported previously

Field verification of low-level biochar applications as effective ameliorants to mitigate cadmium accumulation into Brassica campestris L from polluted soils

Introduction: Cadmium (Cd) has been recognized as a significant contributor to the pollution of farmland soils in China, and biochars have been reported to be effective in mitigating soil Cd pollution. However, most studies have been conducted in laboratory or greenhouse settings, not at a field scale, and the biochars used have been applied at unrealistically high amounts (>10 t/ha).Methods: In this research, three biochars: rice straw biochar (RSB), pig manure biochar (PMB) and rice husk biochar (RHB) were produced from readily available farm residues. Then the effects at low-level application (1.8 and 3.6 t/ha) on Cd were investigated in a field experiment cropped with rape (Brassica campestris L.).Results: Batch adsorption experiments indicated Cd adsorption capacity of three biochars followed the order of RSB (43.5 mg/g) > PMB (33.3 mg/g) > RHB (24.4 mg/g). Field experiment indicated biochar amendments could slightly change soil pH and cation exchange capacity (CEC); yet led to considerable and significant decreases in extractable Cd concentrations [reductions of: 43%–51% (PMB), 29%–35% (RSB) and 17%–19% (RHB)]. Reduced extractable Cd correlated with lower Cd concentrations in rape plants. PMB and RSB were more effective in decreasing Cd phytoaccumulation into edible parts of rape (>68% reduction) than RHB.Discussion: Low-level application of PMB or RSB could efficiently decrease the phytoaccumulation of Cd from soils into crops. These results demonstrate the reality of biochar-based remediation solutions to contribute to the mitigation of diffuse Cd contamination in farmland. The results also highlight the need to trail biochars in the presence of the soil to be targeted for remediation

Geographic differences in semen quality of fertile U.S. males.

Although geographic variation in semen quality has been reported, this is the first study in the United States to compare semen quality among study centers using standardized methods and strict quality control. We evaluated semen specimens from partners of 512 pregnant women recruited through prenatal clinics in four U.S. cities during 1999-2001; 91% of men provided two specimens. Sperm concentration, semen volume, and motility were determined at the centers, and morphology was assessed at a central laboratory. Study protocols were identical across centers, and quality control was rigorously maintained. Sperm concentration was significantly lower in Columbia, Missouri, than in New York, New York; Minneapolis, Minnesota; and Los Angeles, California. Mean counts were 58.7, 102.9, 98.6, and 80.8 X 10(6)/mL (medians 53.5, 88.5, 81.8, and 64.8 X 10(6)/mL) in Missouri, New York, Minnesota, and California, respectively. The total number of motile sperm was also lower in Missouri than in other centers: 113, 196, 201, and 162 X 10(6) in Missouri, New York, Minnesota, and California, respectively. Semen volume and the percent morphologically normal sperm did not differ appreciably among centers. These between-center differences remained significant in multivariate models that controlled for abstinence time, semen analysis time, age, race, smoking, history of sexually transmitted disease, and recent fever (all p-values < 0.01). Confounding factors and differences in study methods are unlikely to account for the lower semen quality seen in this mid-Missouri population. These data suggest that sperm concentration and motility may be reduced in semirural and agricultural areas relative to more urban and less agriculturally exposed areas

Decrease in Anogenital Distance among Male Infants with Prenatal Phthalate Exposure

Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in male rodents. We present data from the first study to examine AGD and other genital measurements in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was obtained in 134 boys 2–36 months of age. AGD was significantly correlated with penile volume (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20, p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors of age-adjusted AGI in regression and categorical analyses that included all participants with prenatal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP concentration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age-adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009). The associations between male genital development and phthalate exposure seen here are consistent with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally exposed rodents. The median concentrations of phthalate metabolites that are associated with short AGI and incomplete testicular descent are below those found in one-quarter of the female population of the United States, based on a nationwide sample. These data support the hypothesis that prenatal phthalate exposure at environmental levels can adversely affect male reproductive development in humans

Are Environmental Levels of Bisphenol A Associated with Reproductive Function in Fertile Men?

Mendiola J, Jørgensen N, Andersson A-M, Calafat AM, Ye X, et al. 2010 Are Environmental Levels of Bisphenol A Associated with Reproductive Function in Fertile Men?. Environ Health Perspect 118(9): doi:10.1289/ehp.1002037Rodent and in vitro studies have demonstrated the estrogenicity of bisphenol A (BPA). However, few studies have examined the relationship between human exposure to BPA and male reproductive function. We investigated the relationships between environmental BPA exposure and reproductive parameters, including semen quality and male reproductive hormones, in prospectively recruited fertile men

ESTs, cDNA microarrays, and gene expression profiling : tools for dissecting plant physiology and development

Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit