Immobilization of enzymes into nanocavities for the improvement of biosensor stability

Nanoporous materials with different pore sizes are evaluated as immobilization and stabilization matrices of proteins for the development of highly stable biosensors. It has been proven experimentally that confinement of proteins in cages with a diameter that is 2-6 times larger than their size increases considerably the stability of the biomolecules, as has been shown earlier by theoretical calculations. Porous silica beads with pore sizes of 10nm were utilized for the immobilization of the enzymes HRP and GOx with diameters in the order of 5 and 7 nm, respectively. The sensitivity of the corresponding biosensor systems was monitored for 70 h under continuous operation conditions (+600 mV) and it was found that the stabilization factor of GOx is 1.7 times higher compared to HRP. Also the stabilization efficiency of enzymes against leaching and inactivation in porous polymer beads with pore diameters of 10 and 30 nm was examined. The leaching rate of the enzyme AChE from the 30 nm polymer beads was found to be 1.1 times higher than that from the 10nm beads. At the same time the remaining activity of GOx biosensors after 5 days of continuous operation conditions (+600 mV) was found to be 2.1 times higher when the enzyme had been immobilized in the 10nm beads compared to the 30 nm beads. It is thus evident that the matching between the pore size of nanoporous materials and the molecular size of enzymes is essential for the development of biosensors with extended shelf and operational lifetimes.     

Pesticide detection with a liposome-based nano-biosensor

Monitoring of the organophosphorus pesticides dichlorvos and paraoxon at very low levels has been achieved with liposome-based nano-biosensors. The enzyme acetylcholinesterase was effectively stabilized within the internal nano-environment of the liposomes. Within the liposomes, the pH sensitive fluorescent indicator pyranine was also immobilized for the optical transduction of the enzymatic activity. Increasing amounts of pesticides lead to the decrease of the enzymatic activity for the hydrolysis of the acetylcholine and thus to a decrease in the fluorescent signal of the pH indicator. The decrease of the liposome biosensors signal is relative to the concentration of dichlorvos and paraoxon down to 10⁻¹⁰ M levels. This biosensor system has been applied successfully to the detection of total toxicity in drinking water samples. Also a colorimetric screening device for pesticide analysis has been evaluated.     

Spermatinamine, the first natural product inhibitor of isoprenylcysteine carboxyl methyltransferase, a new cancer target

Isoprenylcysteine methyltransferase (Icmt) catalyzes the carboxyl methylation of oncogenic proteins in the final step of a series of post-translational modifications. The inhibition of Icmt provides an attractive and novel anticancer target. A natural product high-throughput screening campaign was conducted to discover inhibitors of Icmt. The Australian marine sponge, Pseudoceratina sp., yielded spermatinamine, a novel alkaloid with a bromotyrosyl-spermine-bromotyrosyl sequence, as the bioactive constituent. Its structure was determined by 1D and 2D NMR spectroscopy. Spermatinamine is the first natural product inhibitor of Icmt.     

Diversity of Helicobacter pylori cagA and vacA genes in Costa Rica: its relationship with atrophic gastritis and gastric cancer

BACKGROUND: Associations between Helicobacter pylori gene diversity and gastric cancer have not been reported on in Costa Rica, despite its being one of the countries with the highest gastric cancer incidence and mortality rates in the world. The aim of this study was to determine the prevalence of H. pylori cagA and vacA genes and investigate whether it could be correlated with atrophic gastritis (AG) and gastric cancer (GC) in Costa Rica. MATERIALS AND METHODS: Genomic DNAs from isolates of 104 patients classified into two groups: non-atrophic gastritis group (n = 68) and atrophic gastritis group (n = 36), were subjected to PCR-based genotyping of cagA and vacA genes and their correlation with clinical outcome was investigated. Total DNA extractions from gastric tissues of 25 H. pylori-infected gastric cancer patients were utilized for comparative purposes. RESULTS: The presence of cagA (75.3%), vacA s1b (75.3%), and vacA m1 (74.2%) was detected, and colonization by strains with different vacA genotypes in the same stomach was found in 9.7% of the patients. Age- and sex-adjusted vacA s1b and vacA m1 were associated with GC while only vacA m1 was significantly associated with AG. A tendency for association between cagA and vacA s1b, and AG was reported. CONCLUSIONS: The prevalence status of the cagA and vacA (s1/m1) genes in Costa Rica seems to fall between that found in European/North American and East Asian countries, and both cagA and vacA seem to have clinical relevance in this country.     

Molecular marker development and linkage analysis in three low phytic acid barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) mutant lines

Phytate is the primary form of phosphorus found in mature cereal grain. This form of phosphorus is not available to monogastric animals due to a lack of the enzyme phytase in their digestive tract. Several barley low phytic acid (lpa) mutants have been identified that contain substantial decreases in seed phytate accompanied by concomitant increases in inorganic phosphorus. Seed homozygous for low phytic acid 1-1 (lpa1-1) or low phytic acid 2-1 (lpa2-1) has a 50% and 70% decrease in seed phytate respectively. These mutations were previously mapped to chromosomes 2HL and 7HL respectively. The RFLP marker ABC153 located in the same region of 2H was converted to a sequence-characterized-amplified-region (SCAR) marker. Segregation analysis of the CDC McGwire × Lp422 doubled haploid population confirmed linkage between the SCAR marker and the lpa1-1 locus with 15% recombination. A third low phytic acid mutant, M635, has a 75% decrease in phytate. This mutation was located to chromosome 1HL by linkage with an inter-simple sequence repeat (ISSR) based marker (LP75) identified through bulked-segregant analysis, and has been designated lpa3-1. Based on analysis of recombination between marker LP75 and low phytic acid in an additional mutant line M955 (95% phytate decrease), lpa3-1 and the mutation in M955 are in the same region on chromosome 1HL, and may be allelic.     

A novel bacterial community index to assess stream ecological health

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Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells

Melanoma cells can adopt two functionally distinct forms, amoeboid and mesenchymal, which facilitates their ability to invade and colonize diverse environments during the metastatic process. Using quantitative imaging of single living tumor cells invading three-dimensional collagen matrices, in tandem with unsupervised computational analysis, we found that melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space—an apolar and polar route. We show that whereas particular Rho-family GTPases are required for the morphogenesis of amoeboid and mesenchymal forms, others are required for transitions via the apolar or polar route and not amoeboid or mesenchymal morphogenesis per se. Altering the transition rates between particular routes by depleting Rho-family GTPases can change the morphological heterogeneity of cell populations. The apolar and polar routes may have evolved in order to facilitate conversion between amoeboid and mesenchymal forms, as cells are either searching for, or attracted to, particular migratory cues, respectively.     

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus

Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid–derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a “macrophage-rich” model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.     

The Cedar Project: risk factors for transition to injection drug use among young, urban Aboriginal people

Background:

Studies suggest that Aboriginal people in Canada are over-represented among people using injection drugs. The factors associated with transitioning to the use of injection drugs among young Aboriginal people in Canada are not well understood.

Methods:

The Cedar Project is a prospective cohort study (2003–2007) involving young Aboriginal people in Vancouver and Prince George, British Columbia, who use illicit drugs. Participants’ venous blood samples were tested for antibodies to HIV and the hepatitis C virus, and drug use was confirmed using saliva screens. The primary outcomes were use of injection drugs at baseline and tranisition to injection drug use in the six months before each follow-up interview.

Results:

Of 605 participants, 335 (55.4%) reported using injection drugs at baseline. Young people who used injection drugs tended to be older than those who did not, female and in a relationship. Participants who injected drugs were also more likely than those who did not to have been denied shelter because of their drug use, to have been incarcerated, to have a mental illness and to have been involved in sex work. Transition to injection drug use occurred among 39 (14.4%) participants, yielding a crude incidence rate of 19.8% and an incidence density of 11.5 participants per 100 person-years. In unadjusted analysis, transition to injection drug use was associated with being female (odds ratio [OR] 1.98, 95% confidence interval (CI) 1.06–3.72), involved in sex work (OR 3.35, 95% CI 1.75–6.40), having a history of sexually transmitted infection (OR 2.01, 95% CI 1.07–3.78) and using drugs with sex-work clients (OR 2.51, 95% CI 1.19–5.32). In adjusted analysis, transition to injection drug use remained associated with involvement in sex work (adjusted OR 3.94, 95% CI 1.45–10.71).

Interpretation:

The initiation rate for injection drug use of 11.5 participants per 100 person-years among participants in the Cedar Project is distressing. Young Aboriginal women in our study were twice as likely to inject drugs as men, and participants who injected drugs at baseline were more than twice as likely as those who did not to be involved in sex work.Aboriginal leadership in Canada is deeply concerned about substance use, more specifically injection drug use and its association with the spread of HIV and the hepatitis C virus among Aboriginal young people.^1,2 Recent studies in Canada suggest that Aboriginal people are over-represented among people who use injection drugs.^3,4 For Aboriginal young people in Canada under the age of 24 years, injection drug use accounts for the majority of infections with the hepatitis C virus (70%–80%)^5,6 and over half (59%) of HIV infections.⁷Indigenous scholars have stated that research on substance use within Aboriginal communities must consider the context of colonization, including the intergenerational impacts of the residential school and child welfare systems.^8–11 It is now well documented that Aboriginal children experienced extensive psychological, sexual, physical and emotional abuses within those systems.^12,13 As former students of residential schools raise children and grandchildren, the intergenerational effects of abuse and familial fragmentation are evident in communities where interpersonal violence and drug dependence are pervasive.^14–16A priority for preventing infections with HIV and hepatitis C among young Aboriginal people is the development of programs and rights-based,^18,19 youth-informed¹⁷ policies aimed at preventing the use of injection drugs. However, research to date has not provided sufficient evidence to inform such development.^2,19 Concerns over this paucity of information led to the launch of a two-city cohort study in 2003 to address HIV-related vulnerabilities among young Aboriginal people in British Columbia — a unique study centred on at-risk youth and supported by and partnered with Aboriginal investigators and collaborators.We report here baseline and longitudinal data on the factors associated with injection drug use and the transition to injection drug use to inform the development of prevention programs and policies.     

Jaw and hyolingual movements during prey transport in varanid lizards: effects of prey type

The ability to modulate feeding kinematics in response to prey items with different functional properties is likely a prerequisite for most organisms that feed on a variety of food items. Variation in prey properties is expected to reveal variation in feeding function and the functional role of the different phases in a transport cycle. Here we describe the kinematics of prey transport of two varanid species, Varanus niloticus and Varanus ornatus. These species were selected for analysis because of their highly specialised hyolingual system and food transport mechanism (inertial food transport). In these animals, tongue and hyoid movements are expected to make no, or only a minor, contribution to prey transport. We observed statistically significant prey type effects that could be associated with prey properties such as mass, size and mobility. These data show that both species are capable of modulating the kinematics of food transport in response to different prey types. Moreover, not only the kinematics of the jaws were modulated in response to prey characteristics but also the anterior/posterior movements of the tongue and hyoid. This suggests a more important role of the tongue and hyolingual movements in these animals than previously suspected. In contrast, head movements were rather stereotyped and were not modulated in response to changes in prey type.