Immobilization of whole cells of Lactococcus lactis containing high levels of a hyperthermostable ß-galactosidase enzyme in chitosan beads for efficient galacto-oligosaccharide production.

Galacto-oligosaccharides (GOS) are prebiotic food ingredients that are proposed to stimulate the growth of beneficial gut microorganisms, particularly bifidobacteria. Previously, we developed a method for efficient GOS production using whole cells of Lactococcus lactis containing high levels of a hyper-thermostable ß-galactosidase enzyme from Sulfolobus solfataricus. In this study, a recombinant DNA removal and whole-cell enzyme immobilization process was developed to produce GOS from lactose before removal of the immobilized whole-cell enzyme, which could be reused for subsequent applications. Chitosan was found to be a superior immobilization material compared with alginate, as it retained its bead structure during the high temperature (90°C) used here for GOS production. Prior to immobilization, the recombinant DNA was degraded in the whole cells using UV treatment, resulting in an immobilized whole-cell enzyme that was free of recombinant DNA and with minimum effect on the efficiency of the enzyme. The optimum pH and temperature for GOS synthesis using the chitosan beads was pH = 5.5 and 90°C. The highest GOS production using the chitosan beads occurred with 40% initial lactose resulting in 150 g/L of GOS (tri-oligosaccharides and tetra-oligosaccharides) in addition to di-oligosaccharide GOS products that were not quantified. Notably, the highest lactose conversion rate was found using lower starting lactose concentrations, with more than 60% conversion into tri-oligosaccharides and tetra-oligosaccharides. The immobilized enzyme retained ∼50% activity after 2 cycles of GOS production. In conclusion, the chitosan-immobilized whole-cell enzyme can be used for efficient GOS production that is free of the whole-cell enzyme as well as detectable recombinant DNA.

The frequency of snowline-region planets from four-years of OGLE-MOA-Wise second-generation microlensing.

We present a statistical analysis of the first four seasons from a "second-generation" microlensing survey for extrasolar planets, consisting of near-continuous time coverage of 8 deg2 of the Galactic bulge by the OGLE, MOA, and Wise microlensing surveys. During this period, 224 microlensing events were observed by all three groups. Over 12% of the events showed a deviation from single-lens microlensing, and for ~1/3 of those the anomaly is likely caused by a planetary companion. For each of the 224 events we have performed numerical ray-tracing simulations to calculate the detection efficiency of possible companions as a function of companion-to-host mass ratio and separation. Accounting for the detection efficiency, we find that 55 - 22 + 34 % of microlensed stars host a snowline planet. Moreover, we find that Neptunes-mass planets are ~ 10 times more common than Jupiter-mass planets. The companion-to-host mass ratio distribution shows a deficit at q ~ 10-2, separating the distribution into two companion populations, analogous to the stellar-companion and planet populations, seen in radial-velocity surveys around solar-like stars. Our survey, however, which probes mainly lower-mass stars, suggests a minimum in the distribution in the super-Jupiter mass range, and a relatively high occurrence of brown-dwarf companions.

Air and surface contamination patterns of meticillin-resistant Staphylococcus aureus on eight acute hospital wards.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) can be recovered from hospital air and from environmental surfaces. This poses a potential risk of transmission to patients. AIM: To investigate associations between MRSA isolates recovered from air and environmental surfaces with those from patients when undertaking extensive patient and environmental sampling. METHODS: This was a prospective observational study of patients and their environment in eight wards of a 700-bed tertiary care hospital during 2010 and 2011. Sampling of patients, air and surfaces was carried out on all ward bays, with more extended environmental sampling in ward high-dependency bays and at particular times of the day. The genetic relatedness of isolates was determined by DNA microarray profiling and spa typing. FINDINGS: MRSA was recovered from 30/706 (4.3%) patients and from 19/132 (14.4%) air samples. On 9/132 (6.8%) occasions both patient and air samples yielded MRSA. In 32 high-dependency bays, MRSA was recovered from 12/161 (7.4%) patients, 8/32 (25%) air samples, and 21/644 (3.3%) environmental surface samples. On 10/132 (7.6%) occasions, MRSA was isolated from air in the absence of MRSA-positive patients. Patient demographic data combined with spa typing and DNA microarray profiling revealed four likely transmission clusters, where patient and environmental isolates were deemed to be very closely related. CONCLUSION: Air sampling yielded MRSA on frequent occasions, especially in high-dependency bays. Environmental and air sampling combined with patient demographic data, spa typing and DNA microarray profiling indicated the presence of clusters that were not otherwise apparent.

Production of galactooligosaccharides using a hyperthermophilic ß-galactosidase in permeabilized whole cells of Lactococcus lactis.

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using ß-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable ß-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria.

Freeze-drying is a common method for preservation of probiotics, including bifidobacteria, for further industrial applications. However, the stability of freeze-dried bifidobacteria varies depending on the freeze-drying method and subsequent storage conditions. The primary goals of this study were to develop an optimized freeze-drying procedure and to determine the effects of temperature, water activity, and atmosphere on survival of freeze-dried bifidobacteria. To address these goals, a commercially used bifidobacteria strain that is resilient to stress, Bifidobacterium animalis ssp. lactis Bb-12, and a characterized intestinal strain that is more sensitive to stress conditions, Bifidobacterium longum DJO10A, were used. A freeze-drying protocol was developed using trehalose as the cryoprotectant, which resulted in almost no loss of viability during freeze-drying. Resuscitation medium, temperature, and time did not significantly influence recovery rates when this cryoprotectant was used. The effects of temperature (-80 to 45°C), water activity (0.02 to 0.92), and atmosphere (air, vacuum, and nitrogen) were evaluated for the stability of the freeze-dried powders during storage. Freeze-dried B. animalis ssp. lactis Bb-12 was found to survive under all conditions tested, with optimum survival at temperatures up to 21°C, water activities up to 0.44, and all 3 atmospheres tested. The intestinal-adapted strain B. longum DJO10A was much more sensitive to the different storage conditions, but could be adequately maintained using optimum conditions. These optimum storage conditions included frozen storage, replacement of oxygen with nitrogen, and water activities between 0.11 and 0.22. These results indicated that an optimized storage environment is required to maintain viability of stress-sensitive bifidobacteria strains, whereas stress-resilient bifidobacteria strains can maintain viability over a wide range of storage conditions, which is practical in countries where controlled cold storage conditions may not be readily available.

Transmission of endemic ST22-MRSA-IV on four acute hospital wards investigated using a combination of spa, dru and pulsed-field gel electrophoresis typing.

The transmission of meticillin-resistant Staphylococcus aureus (MRSA) between individual patients is difficult to track in institutions where MRSA is endemic. We investigated the transmission of MRSA where ST22-MRSA-IV is endemic on four wards using demographic data, patient and environmental screening, and molecular typing of isolates. A total of 939 patients were screened, 636 within 72 h of admission (on admission) and 303 >72 h after admission, and 1,252 environmental samples were obtained. Isolates were typed by spa, dru and pulsed-field gel electrophoresis (PFGE) typing. A composite dendrogram generated from the three sets of typing data was used to divide isolates into 'dendrogram groups' (DGs). Ten percent of patients (92/939) were MRSA-positive; 7 % (44/636) on admission and 16 % (48/303) >72 h after admission (p = 0.0007). MRSA was recovered from 5 % of environmental specimens (65/1,252). Most isolates from patients (97 %, 85/88) and the environment (97 %, 63/65) exhibited the ST22-MRSA-IV genotype. Four DGs (DG1, DG4, DG16 and DG17) accounted for 58 % of ST22-MRSA-IV isolates from patients. Epidemiological evidence suggested cross-transmission among 44/92 patients (48 %) but molecular typing confirmed probable cross-transmission in only 11 instances (13 %, 11/88), with the majority of cross-transmission (64 %; 7/11) occurring on one ward. In the setting of highly clonal endemic MRSA, the combination of local epidemiology, PFGE, spa and dru typing provided valuable insights into MRSA transmission.

Triclosan antagonizes fluconazole activity against Candida albicans.

Triclosan is a broad-spectrum antimicrobial compound commonly used in oral hygiene products. Investigation of its activity against Candida albicans showed that triclosan was fungicidal at concentrations of 16 mg/L. However, at subinhibitory concentrations (0.5-2 mg/L), triclosan antagonized the activity of fluconazole. Although triclosan induced CDR1 expression in C. albicans, antagonism was still observed in cdr1Δ and cdr2Δ strains. Triclosan did not affect fluconazole uptake or alter total membrane sterol content, but did induce the expression of FAS1 and FAS2, indicating that its mode of action may involve inhibition of fatty acid synthesis, as it does in prokaryotes. However, FAS2 mutants did not exhibit increased susceptibility to triclosan, and overexpression of both FAS1 and FAS2 alleles did not alter triclosan susceptibility. Unexpectedly, the antagonistic effect was specific for C. albicans under hypha-inducing conditions and was absent in the non-filamentous efg1Δ strain. This antagonism may be due to the membranotropic activity of triclosan and the unique composition of hyphal membranes.

Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of Bifidobacterium animalis subspecies lactis in Cheddar cheese.

Bifidobacteria cultures were incorporated into Cheddar cheeses to conduct a comparative analysis between the commercially available strain Bifidobacterium animalis ssp. lactis Bb-12 and the wild-type intestinal isolate, Bifidobacterium longum DJO10A. They were incorporated as starter adjuncts in separate vats and as a mixed culture, and survival through manufacturing and cheese ripening was assessed. For cheese using only Bb-12, the cells may have grown during cheese manufacture as 133% of the inoculum was incorporated into the cheese, resulting in 8.00 log cfu/g. Counts remained high during ripening showing less than 1 log decrease over a 12-mo period. For cheese using a mixed culture of Bb-12 and DJO10A, both strains were incorporated at much lower levels: 3.02 and 1.11%, respectively. This resulted in cheese with 6.00 and 5.04 log cfu/g for Bb-12 and DJO10A, respectively. Bifidobacteria survival rates were low, most likely due to the moisture of the cheese being below 38%. The Bb-12 demonstrated almost 100% viability during ripening. Numbers of DJO10A started to decline after 2 mo of ripening and dropped below the level of detection (2 log cfu/g) after 4.5 mo of ripening. Neither DJO10A nor Bb-12 fortified cheeses produced detectable amounts of organic acids during ripening other than lactic acid, indicating the lack of detectable metabolic contribution from bifidobacteria during cheese production and ripening such as production of acetic acid. To determine if sublethal stresses could improve the viability of DJO10A, 2 more vats were made, 1 with DJO10A exposed to sublethal acid, cold, and centrifugation stresses, and 1 exposed to none of these stresses. Although stress-primed DJO10A survived cheese manufacture better, as 72.8% were incorporated into the cheese compared with 41.1% of the unprimed, the statistical significance of this difference is unknown. In addition, the difference in moisture levels in the cheese cannot be excluded as influencing this difference. However, the rate of decline during ripening was similar for both. After 6 mo of ripening, cell counts in cheese were 4.68 and 4.24 log cfu/g for primed and unprimed cultures, respectively. These results suggest that whereas priming bifidobacteria with sublethal stresses before incorporation in a cheese fermentation may improve the number of viable cells that get incorporated into the cheese, it does not affect viability during cheese ripening.

A survey of undergraduate education in dental implantology in UK dental schools.

The aim of this study was to ascertain knowledge on current teaching of implant dentistry in the undergraduate curriculum of Dental Schools in the UK. Information on the teaching modalities, including year of introduction of implant dentistry into undergraduate curriculum, departments involved in teaching, format of teaching, use of adjunctive teaching aids, and types of implant systems used in undergraduate teaching was collected by means of a questionnaire, which was sent to all undergraduate dental schools in the UK. Based on a 100% response rate, the findings indicate that all dental schools in the UK reported that they included dental implantology in their undergraduate curriculum; however there were marked variations in the content and delivery of the teaching.

CYP56 (Dit2p) in Candida albicans: characterization and investigation of its role in growth and antifungal drug susceptibility.

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth.

BACKGROUND: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. RESULTS: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events. CONCLUSION: Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

Discovery of a Jupiter/Saturn analog with gravitational microlensing.

Searches for extrasolar planets have uncovered an astonishing diversity of planetary systems, yet the frequency of solar system analogs remains unknown. The gravitational microlensing planet search method is potentially sensitive to multiple-planet systems containing analogs of all the solar system planets except Mercury. We report the detection of a multiple-planet system with microlensing. We identify two planets with masses of approximately 0.71 and approximately 0.27 times the mass of Jupiter and orbital separations of approximately 2.3 and approximately 4.6 astronomical units orbiting a primary star of mass approximately 0.50 solar mass at a distance of approximately 1.5 kiloparsecs. This system resembles a scaled version of our solar system in that the mass ratio, separation ratio, and equilibrium temperatures of the planets are similar to those of Jupiter and Saturn. These planets could not have been detected with other techniques; their discovery from only six confirmed microlensing planet detections suggests that solar system analogs may be common.

The effect of a range of disinfectants on the dimensional accuracy and stability of some impression materials.

Disinfection of dental impressions should be considered as a routine procedure in dental surgeries and dental laboratories. Disinfectants can have deleterious effects on some properties of impression materials. The aim of this study was to evaluate the dimensional accuracy and dimensional stability of a model dental stone, reproduced from five commonly used impression materials (Aquasil soft putty/Aquasil Ultra LV; Aquasil Monophase; Aquasil Ultra Heavy; Impregum F and Provil putty/Provil Light CD wash) retained by their adhesives in acrylic resin trays and exposed to three disinfectant solutions (Perform ID; Haz-Tabs and MD 520). Two hundred models were used to investigate the effect of the three disinfectants on the dimensional accuracy of the five impression materials. Five impressions were taken for each impression material for each disinfection treatment group. Measurements were carried out using a High Precision Reflex Microscope. All materials demonstrated a percentage change in dimensions when subjected to no disinfection when compared to the brass master die and all materials demonstrated a percentage change in dimension when subjected to the different disinfection procedures. The results of this study have demonstrated that for all of the materials investigated, the changes in dimensional stability were small in the order of microns. These changes may however be of clinical significance for procedures requiring a high degree of accuracy, for example fixed prosthodontics. The materials respond differently depending on the disinfectant used and it may therefore be appropriate that manufacturers recommend the use of particular disinfectants for their products in order to ensure optimum dimensional accuracy and stability.

Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans.

Candida albicans and C. dubliniensis are very closely related yeast species. In this study, we have conducted a thorough comparison of the ability of the two species to produce hyphae and their virulence in two infection models. Under all induction conditions tested C. albicans consistently produced hyphae more efficiently than C. dubliniensis. In the oral reconstituted human epithelial model, C. dubliniensis isolates grew exclusively in the yeast form, while the C. albicans strains produced abundant hyphae that invaded and caused significant damage to the epithelial tissue. In the oral-intragastric infant mouse infection model, C. dubliniensis strains were more rapidly cleared from the gastrointestinal tract than C. albicans. Immunosuppression of Candida-infected mice caused dissemination to internal organs by both species, but C. albicans was found to be far more effective at dissemination than C. dubliniensis. These data suggest that a major reason for the comparatively low virulence of C. dubliniensis is its lower capacity to produce hyphae.

An in vitro investigation of the effect of some analgesics on human enamel.

The sale of over-the-counter pain relief medication has increased dramatically in recent years, and typically amounts to several hundred thousands of pounds per year in the UK. Many soluble analgesic preparations contain citric acid, and it has been suggested that these formulations may cause dental erosion. The aim of this study was to investigate the effect of some over-the-counter analgesics on tooth surface loss from human enamel. Six commonly available analgesics were chosen for this study and the effect of immersing unerupted human enamel was examined using non-contact optical profilometry. Two of the six analgesics investigated caused no detectable erosion (Boots soluble aspirin and Anadin Extra). Three caused statistically significant enamel erosion, but this was very slight and is thought to be clinically insignificant (Alka Seltzer, Panadol and Solpadeine). Only one analgesic caused possible potentially clinical significant enamel erosion. Further studies are needed to determine whether Aspro causes clinically significant enamel erosion.

An in vitro investigation of the effect of the addition of untreated and surface treated silica on the transverse and impact strength of poly(methyl methacrylate) acrylic resin.

Silica is a commonly used filler in dental materials and as a reinforcing agent in industry. The aim of this study was to further investigate the effect of the addition of untreated and a novel surface treated silica on the transverse bend and impact strength of acrylic resin denture base material. It was hypothesized that the silica/resin composite materials would have an improved flexural and impact strength than the conventional heat-cured acrylic resin. Three types of untreated and two of treated silica powder were used in this study. The range of percentages used were 1%, 0.5%, 0.2%, 0.1%. The treated particles were coated with hexamethyldisilazane or dimethyldichloridesilazane. Conventional heat cured acrylic resin was used as a control. The modulus of rupture for all groups of acrylic resin containing silica was significantly lower than for the control. The modulus of elasticity was not significantly greater than the control group. For the impact strength statistical analysis revealed a significant difference between the groups. There was a nonsignificant increase in the impact strength for specimens compared to the control. In conclusion the addition of silica to poly(methyl methacrylate) denture base materials did not produce a significant improvement in the transverse bend or impact strength compared to conventional heat-cured acrylic resin. The incorporation of untreated and surface treated silica cannot be recommended as a method of reinforcement.

Bioavailable iron and heme metabolism in Plasmodium falciparum.

Iron metabolism is essential for cell function and potentially toxic because iron can catalyze oxygen radical production. Malaria-attributable anemia and iron deficiency anemia coincide as being treatable diseases in the developing world. In absolute amounts, more than 95% of Plasmodium metal biochemistry occurs in the acidic digestive vacuole where heme released from hemoglobin catabolism forms heme crystals. The antimalarial quinolines interfere with crystallization. Despite the completion of the Plasmodium genome, many 'gene gaps' exist in components of the metal pathways described in mammalian or yeast cells. Present evidence suggests that parasite bioavailable iron originates from a labile erythrocyte cytosolic pool rather than from abundant heme iron. Indeed the parasite has to make its own heme within two separate organelles, the mitochondrion and the apicomplast. Paradoxically, despite the abundance of iron within the erythrocyte, iron chelators are cytocidal to the Plasmodium parasite. Hemozoin has become a sensitive biomarker for laser desorption mass spectrometry detection of Plasmodium infection in both mice and humans.

Azole susceptibility and resistance in Candida dubliniensis.

Candida dubliniensis is a recently described species of pathogenic yeast that shares many phenotypic features with Candida albicans. It is primarily associated with oral colonization and infection in HIV-infected individuals. Isolates of C. dubliniensis are generally susceptible to commonly used azole antifungal agents; however, resistance has been observed in clinical isolates and can be induced by in vitro exposure. Molecular mechanisms of azole resistance in C. dubliniensis include increased drug efflux, modifications of the target enzyme and alterations in the ergosterol biosynthetic pathway.

Reproductive performance in anestrous dairy cows following treatment with two protocols and two doses of progesterone.

The objectives of this study were to evaluate the effect using two doses of progesterone (P4) releasing devices in two different programs on reproductive performance of anestrous dairy cows. Cows (n = 1555) not detected in estrus by 10 d before the planned start of the seasonal breeding program and in which no CL was palpable were treated with an intravaginal P4-releasing device ('Single'; approximately 1.56 g of P4) or a modified device with triple the normal P4 dose ('Triple'; approximately 4.7 g of P4). The devices were in place for either 6 d ('Short') or 8 d ('Long'), with 1mg estradiol benzoate (EB) given 24 h after device removal. The 'Long' program also included treatment with 2 mg EB at device insertion. The Long program resulted in a higher first service conception rate (RR = 1.18 (95% CI = 1.03-1.33); P = 0.02), but had no effect on the 28-d, 56-d or final pregnancy rate compared to the Short program. There were no effects of dose of P4 on any outcome. In conclusion, the Long compared to the Short program, but not the dose of P4, improved first service conception rates in anestrous cows.