This multicenter study was designed to assess the accuracy and reproducibility of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system for ...identification of
and
species compared to DNA sequencing. A total of 963 clinical isolates representing 51 taxa were evaluated. In all, 663 isolates were correctly identified to the species level (69%), with another 231 (24%) correctly identified to the complex or group level. Fifty-five isolates (6%) could not be identified despite repeat testing. All of the tuberculous mycobacteria (45/45; 100%) and most of the nontuberculous mycobacteria (569/606; 94%) were correctly identified at least to the group or complex level. However, not all species or subspecies within the
,
, and
complexes and within the
and
groups could be differentiated. Among the 312
isolates tested, 236 (76%) were correctly identified to the species level, with an additional 44 (14%) correctly identified to the complex level. Species within the
and
complexes could not always be differentiated. Eleven percent of the isolates (103/963) underwent repeat testing in order to get a final result. Identification of a representative set of
and
species was highly reproducible, with 297 of 300 (99%) replicates correctly identified using multiple kit lots, instruments, analysts, and sites. These findings demonstrate that the system is robust and has utility for the routine identification of mycobacteria and
in clinical practice.
Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic ...degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined.
Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds.
This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.
...while many toxic molecules have been described in various pathogenic bacteria, toxic molecules have not been reported for Toxoplasma. ...a single low-dose vaccination with NRTUAs rapidly elicits a ...lifelong CD8+ T-cell-dependent protective immunity against reinfection by pathogenic strains of Toxoplasma 17, 18, 22, 23. ...NRTUA treatment rapidly promoted production of the T-cell-recruiting chemokines CXCL9 and CXCL10 in the tumor environment 21. ...analysis of the CD8+ T-cell populations after NRTUA treatment revealed dramatic increases in activated tumor antigen-specific CD8+ T cells that could specifically recognize and kill tumor cells. ...NRTUAs in essence behave as a safe and potent intracellular TH1 adjuvant for dendritic cells. Since NRTUAs are easily genetically engineered to express heterologous antigens that are vigorously presented by MHCI to activate antigen-specific CD8+ T cells 18, 23, NRTUAs represent a novel and broadly applicable TH1 vaccine platform with the ability to elicit potent CD8+ T-cell-dependent protective immune responses not only against cancer but perhaps also against various intracellular protozoan (malaria), bacterial (tuberculosis), or viral (HIV) pathogens where a more effective TH1 cellular immunity could be beneficial to prevent infection or eradicate existing infection. NRTUAs safely triggered effective antitumor immunity regardless of whether the tumor-bearing animals were immune to Toxoplasma prior to NRTUA treatment 20. ...to existing bacterial and viral anticancer platforms that are rendered less effective by immunity 35, pre-existing immunity to Toxoplasma is not a barrier to this potential cancer therapy.
Measurement of total suspended solids (TSS) and total dissolved solids (TDS) loadings in streams is often used to assess potential impacts from mining on surface water quality within a drainage ...basin. It has been suggested that TSS could be used as an indicator to estimate TDS loading through the use of a TDS/TSS ratio. The reliability of this approach was tested by examining empirical linear relationships between TSS and TDS loads at three locations within a mining-influenced watershed in Colorado. Predictive accuracy of the relationships was assessed using an independent source of data for the same locations, with comparisons between estimated and observed TDS loads for periods within and outside the years used to develop relationships. Evaluations were conducted over the entire flow regime and for separate periods of low- and high-flow. Improved representation of the data was achieved by incorporation of a regressed average baseflow contribution, indicating the relationship between TDS and TSS loads is not accurately represented by a simple ratio. High variability in data between locations prevents application of a basin scale relationship to all locations within that basin. Local-scale relationships developed under specific flow regimes resulted in more accurate predictions, but results suggest that factors additional to flow may need to be considered to improve predictability. Usefulness of linear empirical relationships to predict TDS load from TSS load will depend on the magnitude of uncertainty that is tolerable for a given situation.
Source control of mine drainage and leachate from mining wastes is ideal, but there is limited use of control methods aside from removal of wastes to repositories or capping wastes in situ. In some ...locations, neither removal nor capping is possible; thus, there is renewed interest in understanding technologies for coating mining wastes. This paper presents the current state of knowledge from a critical review of the few field case studies where chemical coating technologies have been applied to mined surfaces (e.g. a highwall) or mining wastes. It also includes laboratory studies where the same technology was examined on the same type of waste material as the field studies. The chemicals used in field studies for coating mining wastes were magnesium oxide, potassium permanganate, silicate compounds, phosphate compounds, and a furfuryl alcohol resin sealant. Descriptions of the methods employed, and results obtained are provided. Limitations of the individual studies and of the technologies in general are discussed. Potential reasons why coating technologies have not been commonly used are presented, along with further research needs.
Long-day photoperiod is known to positively affect milk production in confinement dairy systems, and it has been hypothesized that pineal melatonin (MT) secretion plays a substantial role in this ...process. Specialized mammalian photoreceptors that regulate MT secretion are optimally stimulated by short wavelength blue light. We investigated the blue light intensity administered to one eye required to suppress MT secretion in nonlactating dairy cows, and subsequently examined effects on milk production in grazing dairy cows. Following a 14-d light-dark 8:16 h environmental conditioning period, 5 nonlactating Holstein-Friesian cows were exposed to treatments of <1, 70, 125, 175, and 225 lx for 8 additional hours using a 5 × 5 Latin square design. Light was administered via headpieces fitted with light-emitting diodes emitting blue light (465 nm) to the right eye. All cows were then exposed to a light-dark 16:8 h cycle for one night via the indoor lighting system (>200 lx white light). Plasma samples collected at regular intervals were assayed for MT. A dose-dependent effect of light treatment on mean circulating MT concentrations (and 95% CI) was observed 9.4 (7.2, 12.3), 5.0 (3.8, 6.6), 4.4 (3.3, 5.7), 3.3 (2.5, 4.3) and 1.7 (1.3, 2.3) pg/mL for treatments of 0, 70, 125, 175, and 225 lx, respectively. Only the 225 lx treatment acutely suppressed plasma melatonin concentration to levels similar to the light-dark 16:8 h treatment 1.9 (1.4, 2.5) pg/mL. Forty spring-calving cows were blocked on parity, calving date and Economic Breeding Index for milk production and assigned to the control treatment or blue light to a single eye (LT) treatment from calving through 32 wk of lactation. The cows assigned to LT treatment were fitted with headpieces providing 225 lx of blue light to the right eye from 1700 until 0000 h. Mean milk production (and 95% CI) during 32 wk of lactation was not affected by treatment 20.3 (19.3, 21.3) vs. 20.9 (19.8, 22.0) kg/d, control and LT, respectively. Within multiparous cows, a treatment by week interaction was detected, whereby LT treatment increased milk production during the first 12 wk of lactation 25.8 (24.3, 27.3) vs. 28.0 (26.5, 29.5) kg/d; +8.5%, but had no effect thereafter. Treatment did not affect plasma insulin-like growth factor 1. We identified the blue light intensity to one eye required to acutely suppress MT concentrations. Transient favorable effects on milk production were observed in multiparous cows. It remains unclear how single-eye blue light treatment affects galactopoiesis in grazing dairy cows, and further research is needed to explore whether this modality of light delivery represents a useful means to aid productivity in pasture-based dairy systems.
Acid-mine drainage (AMD) input to a stream typically results in the stream having a reduced pH, increased concentrations of metals and salts, and decreased biological productivity. Removal and/or ...treatment of these AMD sources is desired to return the impacted stream(s) to initial conditions, or at least to conditions suitable for restoration of the aquatic ecosystem. Some expected changes in the water chemistry of the stream following removal of AMD input include an increase in pH, a decrease in ionic strength, and an increase in dissolved organic carbon (DOC) concentrations from increased biological activity in the absence of toxic metals concentrations. These changes in water chemistry may cause the existing contaminated bed sediments to become a source of metals to the stream water. Streambed sediments, collected from North Fork Clear Creek (NFCC), Colorado, currently impacted by AMD, were assessed for the effects of pH, ionic strength, DOC concentration, time, and particle size on metals release using a factorial design. The design included two levels for each chemical parameter (ionic strength
=
40 and 80% lower than ambient; pH
=
6 and 8; and DOC
=
1 and 3
mg/l higher than ambient), ten sampling times (from zero to 48
h), and two size fractions of sediments (63
μm
≤
x
<
2
mm and <63
μm). Greater concentrations of metals were released from the smaller sized sediments compared with the larger, with the exception of Cu. A mild acid digestion (0.6
M HCl) evaluated the amount of each metal that could be removed easily from each of the sediment size fractions. Release of all metals over all time points, treatments, and from both sediment sizes was less than 1% of the extractable concentrations, with the exception of Mn, which ranged from 4 to 7% from the smaller sized sediment. Greater percentages of the 0.6
M HCl-extractable concentrations of Cu, Fe, and Zn were released from the larger sized sediment, while this was true for release of Cd and Mn from the smaller sized sediment. Thus, at least for Cd and Mn, the observed higher concentrations released from the smaller sized sediment with each treatment solution is not simply a function of these particles having higher concentrations available for release, but that these metals also are more readily released from the smaller sediment particles versus the larger. DOC concentration strongly influenced the release of Cu; ionic strength strongly influenced the release of Cd, Mn, and Zn; and interaction effects were observed with the release of Cu, Mn, and Zn from the larger size fraction and with the release of Zn from the smaller size fraction. Overall, results suggest that the expected changes in water chemistry following removal/treatment of the AMD sources would result in a release of metals from the existing sediments, with a greater effect on the release of Cu and Fe, than on the release of Cd, Mn, and Zn.
Oral delivery, while a highly desirable form of nanoparticle‐drug administration, is limited by challenges associated with overcoming several biological barriers. Here, the authors study how ...fluorescent and poly(ethylene glycol)‐coated (PEGylated) core‐shell silica nanoparticles sized 5 to 50 nm interact with major barriers including intestinal mucus, intestinal epithelium, and stomach acid. From imaging fluorescence correlation spectroscopy studies using quasi‐total internal reflection fluorescence microscopy, diffusion of nanoparticles through highly scattering mucus is progressively hindered above a critical hydrodynamic size around 20 nm. By studying Caco‐2 cell monolayers mimicking the intestinal epithelia, it is observed that ultrasmall nanoparticles below 10 nm diameter (Cornell prime dots, C’ dots) show permeabilities correlated with high absorption in humans from primarily enhanced passive passage through tight junctions. Particles above 20 nm diameter exclusively show active transport through cells. After establishing C’ dot stability in artificial gastric juice, in vivo oral gavage experiments in mice demonstrate successful passage through the body followed by renal clearance without protein corona formation. Results suggest C’ dots as viable candidates for oral administration to patients with a proven pathway towards clinical translation and may generate renewed interest in examining silica as a food additive and its effects on nutrition and health.
Interactions between poly(ethylene glycol)‐coated (PEGylated) core‐shell silica nanoparticles (5–50) nm and major barriers associated with oral delivery are studied. Ultrasmall nanoparticles (<10 nm) rapidly diffuse through intestinal mucus, passively permeate intestinal epithelial cell layers, and are found in mouse urine after oral gavage. Together with earlier in vivo results, suggest these nanoparticles are promising candidates for oral administration.
State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with ...traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.
secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8
T cells. Here, ...we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8
T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8
T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8
T cell recognition and clearance of
infected host cells.
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