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• 4191. [Article] Preparation of acetaminophen capsules containing beads prepared by hot-melt direct blend coating method

  The thrust of this thesis is to study oral solid dosage formulation using hot melt coating method and to use pharmacokinetic modeling and simulation (PK M&S) as a tool that can help to predict pharmacokinetics ...

  Citation × Citation Citation Abstract Copy Title: Preparation of acetaminophen capsules containing beads prepared by hot-melt direct blend coating method Author: Pham, Loan The thrust of this thesis is to study oral solid dosage formulation using hot melt coating method and to use pharmacokinetic modeling and simulation (PK M&S) as a tool that can help to predict pharmacokinetics of a drug in human and the probability of passing various bioequivalence criteria of the formulation based on the PK of the drug. Hot-melt coating using a new method, direct blending, was performed to create immediate and sustained release formulations (IR and SR). This new method was introduced to offer another choice to produce IR and SR drug delivery formulations using single and double coating layer of waxes onto sugar beads and/or drug loaded pellets. Twelve waxes were applied to coat sugar cores. The harder the wax the slower the drug was released from single coated beads. The wax coating can be deposited up to 28% of the weight of the core bead with 58% drug loading efficiency in the coating The cores were coated with single or double wax layers containing acetaminophen. Carnauba wax coated beads dissolved in approximately 6 hrs releasing 80% of loaded drug. However, when covered with another layer, the drug loaded beads released drug for over 20 hrs. When drug loaded pellets were used as cores, 33-58% drug loading was achieved. Double coated pellets exhibited a near zero order drug release for up to 16 hrs. Hot melt coating by direct blending using waxes is a simple process compared to conventional hot melt coating using coating pan or fluid bed coating machines. It offers an alternative way of making immediate, sustained drug release (IR, SR) and modified release (IR+SR) oral dosage forms of drugs which are stable at high temperature (100°C). The pellet-containing-drug coated formulations provide options when higher drug loading is warranted. It is required by the US Food and Drug Administration (FDA) that a new modified –release (MR) product or identical generic product be regarded as bioequivalent (BE) to the originators reference drug product. However, there are concerns that current regulatory criteria are not sufficient when evaluating bioequivalence (BE) for many MR products, and additional metrics for BE assessment of the products should be applied to ensure therapeutic equivalence. This study used pharmacokinetic modeling and simulation (M&S) to investigate 1) the probability of BE occurring between the MR test and reference products 2) the rates of false positive and true negative of the BE test; and 3) the estimation of the sample size in pivotal BE studies; all of which when partial area under the curves (pAUCs) were applied as additional BE criteria. Reference data of two MR forms of methylphenydate HCl (MPH) were simulated and obtained from literature (formulation Q and Metadate CD, respectively). Monte Carlo simulations were performed to simulate the test drug concentration profiles and BE assessment was carried out utilizing the mean (method 1) and individual concentration time curves (method 2). For formulation Q, adding pAUC₀₋[subscript Tmax] to current BE criteria reduced the possibility of passing BE from approximately 98% to 85%, with a true negative rate of 5%. The earlier the time points used to determine for pAUC before Tmax, the lower the chance of passing BE for the test product. The possibility of passing BE varied and depended on the coefficient of variations (CV) of T[subscript lag], K[subscript a] and K[subscript e] and that considerable variability in the parameters affected the earlier segments of the drug concentration profile curves more. Similar drug concentration time profiles between the test and reference products is recommended to ensure bioequivalence occurs with a reasonable subject sample size. A similar scenario was seen when Metadate CD was used as the reference product. PK M&S can help provide appropriate additional metrics to assure the BE test is a better tool ensuring therapeutic equivalence for MR products with little negative impact to generic manufacturers. Predictions can also be made about the required sample size and the chances of passing BE with any addition to the conventional three criteria for the test product. PK M&S was also used to predict drug concentrations of levofloxacin in tissue. Levofloxacin has been widely used in clinical practice as an effective broad-spectrum antimicrobial, however tendonitis and tendon rupture have been reported with increasing use of this agent. Here, these incidents will be assessed by investigating pharmacokinetic behavior of the compound to see if they are related to drug's tissue disposition. The PK model for levofloxacin was established. Mean concentration time profiles of single or multiple dosing of 500 mg levofloxacin following oral and IV infusion administration were simulated. Monte Carlo simulation was used to simulate the drug concentration time profiles in plasma (compartment 1) and tissue (compartment 2) after seven dosing regimens while varying the drug's elimination and distribution rates to see the effect of changing those rates have on the drug accumulation in tissue. Monte Carlo Simulation shows that low elimination rates affect the drug concentration in plasma and tissue significantly with the level in plasma rising up to 35 μg/mL at day 7. A normal elimination rate together with escalation of distribution rates from plasma to tissue could increase the tissue concentration after 7 doses to 9.5 µg/mL, a value that is more than twice that of normal. PK M&S can be used as an effective tool to evaluate drug concentration in different compartments (plasma and tissues, for example). The unexpectedly high concentration values in some cases may explain, at least in part, the reason of tendinopathy occurs in the clinical setting. Title: Preparation of acetaminophen capsules containing beads prepared by hot-melt direct blend coating method Author: Pham, Loan The thrust of this thesis is to study oral solid dosage formulation using hot melt coating method and to use pharmacokinetic modeling and simulation (PK M&S) as a tool that can help to predict pharmacokinetics of a drug in human and the probability of passing various bioequivalence criteria of the formulation based on the PK of the drug. Hot-melt coating using a new method, direct blending, was performed to create immediate and sustained release formulations (IR and SR). This new method was introduced to offer another choice to produce IR and SR drug delivery formulations using single and double coating layer of waxes onto sugar beads and/or drug loaded pellets. Twelve waxes were applied to coat sugar cores. The harder the wax the slower the drug was released from single coated beads. The wax coating can be deposited up to 28% of the weight of the core bead with 58% drug loading efficiency in the coating The cores were coated with single or double wax layers containing acetaminophen. Carnauba wax coated beads dissolved in approximately 6 hrs releasing 80% of loaded drug. However, when covered with another layer, the drug loaded beads released drug for over 20 hrs. When drug loaded pellets were used as cores, 33-58% drug loading was achieved. Double coated pellets exhibited a near zero order drug release for up to 16 hrs. Hot melt coating by direct blending using waxes is a simple process compared to conventional hot melt coating using coating pan or fluid bed coating machines. It offers an alternative way of making immediate, sustained drug release (IR, SR) and modified release (IR+SR) oral dosage forms of drugs which are stable at high temperature (100°C). The pellet-containing-drug coated formulations provide options when higher drug loading is warranted. It is required by the US Food and Drug Administration (FDA) that a new modified –release (MR) product or identical generic product be regarded as bioequivalent (BE) to the originators reference drug product. However, there are concerns that current regulatory criteria are not sufficient when evaluating bioequivalence (BE) for many MR products, and additional metrics for BE assessment of the products should be applied to ensure therapeutic equivalence. This study used pharmacokinetic modeling and simulation (M&S) to investigate 1) the probability of BE occurring between the MR test and reference products 2) the rates of false positive and true negative of the BE test; and 3) the estimation of the sample size in pivotal BE studies; all of which when partial area under the curves (pAUCs) were applied as additional BE criteria. Reference data of two MR forms of methylphenydate HCl (MPH) were simulated and obtained from literature (formulation Q and Metadate CD, respectively). Monte Carlo simulations were performed to simulate the test drug concentration profiles and BE assessment was carried out utilizing the mean (method 1) and individual concentration time curves (method 2). For formulation Q, adding pAUC₀₋[subscript Tmax] to current BE criteria reduced the possibility of passing BE from approximately 98% to 85%, with a true negative rate of 5%. The earlier the time points used to determine for pAUC before Tmax, the lower the chance of passing BE for the test product. The possibility of passing BE varied and depended on the coefficient of variations (CV) of T[subscript lag], K[subscript a] and K[subscript e] and that considerable variability in the parameters affected the earlier segments of the drug concentration profile curves more. Similar drug concentration time profiles between the test and reference products is recommended to ensure bioequivalence occurs with a reasonable subject sample size. A similar scenario was seen when Metadate CD was used as the reference product. PK M&S can help provide appropriate additional metrics to assure the BE test is a better tool ensuring therapeutic equivalence for MR products with little negative impact to generic manufacturers. Predictions can also be made about the required sample size and the chances of passing BE with any addition to the conventional three criteria for the test product. PK M&S was also used to predict drug concentrations of levofloxacin in tissue. Levofloxacin has been widely used in clinical practice as an effective broad-spectrum antimicrobial, however tendonitis and tendon rupture have been reported with increasing use of this agent. Here, these incidents will be assessed by investigating pharmacokinetic behavior of the compound to see if they are related to drug's tissue disposition. The PK model for levofloxacin was established. Mean concentration time profiles of single or multiple dosing of 500 mg levofloxacin following oral and IV infusion administration were simulated. Monte Carlo simulation was used to simulate the drug concentration time profiles in plasma (compartment 1) and tissue (compartment 2) after seven dosing regimens while varying the drug's elimination and distribution rates to see the effect of changing those rates have on the drug accumulation in tissue. Monte Carlo Simulation shows that low elimination rates affect the drug concentration in plasma and tissue significantly with the level in plasma rising up to 35 μg/mL at day 7. A normal elimination rate together with escalation of distribution rates from plasma to tissue could increase the tissue concentration after 7 doses to 9.5 µg/mL, a value that is more than twice that of normal. PK M&S can be used as an effective tool to evaluate drug concentration in different compartments (plasma and tissues, for example). The unexpectedly high concentration values in some cases may explain, at least in part, the reason of tendinopathy occurs in the clinical setting. Close

• 4192. [Article] Addressing wastewater epidemiology limitations with the use of dynamic population surrogates, complementary urinalyses and in-situ experiments

  Wastewater epidemiology is an emerging discipline that requires collaborative research involving analytical chemists, drug epidemiologists, and wastewater engineers. Wastewater epidemiology involves the ...

  Citation × Citation Citation Abstract Copy Title: Addressing wastewater epidemiology limitations with the use of dynamic population surrogates, complementary urinalyses and in-situ experiments Author: Brewer, Alex J. Wastewater epidemiology is an emerging discipline that requires collaborative research involving analytical chemists, drug epidemiologists, and wastewater engineers. Wastewater epidemiology involves the sampling and quantitative analysis of raw wastewaters from communities for illicit drugs and their metabolites. Mass loads (mass per day) and per capita (mg per day per person) are then calculated from concentrations and indicate the approximate quantity of illicit drugs used and excreted by the community. Limitations to wastewater epidemiology include that the population served by wastewater treatment plants within a day and between days is not well known. In addition, biodegradation of illicit drugs during transit in sewers may affect the concentration and mass flows that reach wastewater treatment plants. This thesis describes a series of studies conducted by an international collaboration between scientists and engineers from the United States and Switzerland to answer these two limitations. The experimental approaches for these studies used included high-frequency wastewater sampling strategies, the use of creatinine as a human urinary biomarker, as well as the use of unique locations as test sites including an open community, a prison in the state of Oregon, and a 5 km section of sewer in Zürich Switzerland. In Chapter 2, the diurnal study on the mass flows of illicit drugs or metabolites was formed over four days in a municipality with a population of approximately 55,000 people. The diurnal trends in illicit substances vary by substance. The high (g/day) mass flows of caffeine, methamphetamine, and creatinine indicate that lower-frequency sampling (approximately one sample per h) may representatively capture the use and excretion of these substances. However, lower and episodic mass flows of cocaine and its primary human metabolite, benzoylecgonine, indicate that higher-frequency is needed to accurately assess the use of the cocaine within the municipality. Normalization of illicit substances to creatinine gave between-day trends in illicit and legal substances that differed from non-normalized trends. Resident use of cocaine and methamphetamine were indicated by normalized mass flows that increased during early morning hours while commuters are largely absent from the community. Chapter 3 describes a series of experiments conducted at an Oregon state prison. The prison setting provided a unique opportunity to study a nearly-fixed population of individuals and their corresponding mass flows of illicit substances, the number of doses per person consumed, as well as an opportunity to quantify the level of agreement between numbers of individuals and the measured mass flows of creatinine. Methamphetamine use was more prevalent than cocaine/benzoylecgonine in the prison over the one month study in which single daily (24 h) composite samples of wastewater were collected. The hypothesis that the mass flows of methamphetamine and cocaine would be lower on days on which random urinalysis testing (RUA) is typically conducted by the prison (Monday-Thursday) was rejected. While the mass flows (mg/d) of methamphetamine were less than those for a nearby open community, the number of estimated doses per person was higher for the prison population. A higher number of positive RUA results were obtained for methamphetamine while none were positive for cocaine, which is consistent with the data obtained from wastewater. The hourly (diurnal) trend in methamphetamine mass loads indicated continual methamphetamine use/excretion inside the prison while cocaine and benzoylecgonine were detected in five hourly composite samples. Use of methamphetamine and cocaine by inmates could not be unambiguously distinguished from that of non-inmates (employees and visitors). The observed diurnal trends in creatinine mass loads were similar to those of an open community and are indicative of the general pattern of human wakefulness/activity. Predicted creatinine mass loads based on the total prison (inmates + non-inmates) were in good agreement with the measured mass loads, which indicates the potential use of creatinine as a quantitative population indicator. Additional research on the biodegradability of creatinine is needed because the prison setting was deliberately selected to minimize the potential for creatinine biodegradation. Chapter 4 addresses the data gap that exists on illicit drug transformation during in situ transit in sewers. The rates of in situ biodegradation have not yet been determined for conditions that are relevant to sewers, which include low to variable oxygen concentrations, the presence of a biofilm, and temperatures ≤ 20 °C. For this reason, two tracer tests were conducted in a 5 km stretch of sewer located near Zürich, Switzerland. The stable-isotope forms (deuterated) of cocaine and benzoylecgonine were injected into flowing wastewater and three locations up to 5 km downstream were sampled over time. Breakthrough curves were constructed from measurements of cocaine-d3 and benzoylecgonine-d3 concentration with time. The area under the curve (mass) was determined by integrating concentration over time. Benzoylecgonine-d3 was present in the injectate that should have only contained cocaine-d3; because the benzoylecgonine-d3 formation prior to injection is not known. The injected mass of cocaine-d3 did not decline over the 5 km distance. The observed mass of cocaine-d3 at 5 km was 10% greater than at 500 m, which indicates that the transformation of cocaine was not significant over the 1.5 h experiment. At 5 km downgradient, the apparent mass of benzoylecgonine-d3 had increased by 35% over that observed at 500 m. However, the apparent increase in benzoylecgonine-d3 mass was not accompanied by a corresponding loss of cocaine-d3. While uncertainty is apparent in the increase of both cocaine-d3 and benzoylecgonine-d3, the ratio of cocaine-d3/benzoylecgonine-d3 is subject only to analytical error because any errors associated with sampling and the integration of masses cancel out. The ratio of cocaine-d3/benzoylecgonine-d3 declined from 2.98 in the injectate to 1.66 at Location 3, which indicated a greater increase in benzoylecgonine-d3 relative to cocaine over the 5 km distance. Due to the benzoylecgonine-d3 that was present in the injectate, any biodegradation of cocaine-c3 to form benzoylecgonine-d3 could not be unambiguously distinguished. During the second tracer test in which benzoylecgonine-d3 was injected, the mass of benzoylecgonine-d3 did not significantly decline, which suggests that the apparent loss of benzoylecgonine-d3 during the cocaine-d3 test cannot be attributed to in-situ biodegradation. Overall, while uncertainty exists about the integrated masses for cocaine-d3 and benzoylecgonine-d3, the 5 km distance was too short in order to observe a significant loss of cocaine-d3 and formation of benzoylecgonine-d3. Recommendations for future research include conducting analysis on the injectate solution to ensure that only cocaine-d3 is introduced so that any formation of benzoylecgonine-d3 is readily apparent and quantifiable. In addition, the tracer tests should be repeated in a longer section of sewer to increase the residence time beyond 1.5 hr and degradation products of benzoylecgonine-d3 should be monitored including ecgonine and ecgonine methyl ester. Title: Addressing wastewater epidemiology limitations with the use of dynamic population surrogates, complementary urinalyses and in-situ experiments Author: Brewer, Alex J. Wastewater epidemiology is an emerging discipline that requires collaborative research involving analytical chemists, drug epidemiologists, and wastewater engineers. Wastewater epidemiology involves the sampling and quantitative analysis of raw wastewaters from communities for illicit drugs and their metabolites. Mass loads (mass per day) and per capita (mg per day per person) are then calculated from concentrations and indicate the approximate quantity of illicit drugs used and excreted by the community. Limitations to wastewater epidemiology include that the population served by wastewater treatment plants within a day and between days is not well known. In addition, biodegradation of illicit drugs during transit in sewers may affect the concentration and mass flows that reach wastewater treatment plants. This thesis describes a series of studies conducted by an international collaboration between scientists and engineers from the United States and Switzerland to answer these two limitations. The experimental approaches for these studies used included high-frequency wastewater sampling strategies, the use of creatinine as a human urinary biomarker, as well as the use of unique locations as test sites including an open community, a prison in the state of Oregon, and a 5 km section of sewer in Zürich Switzerland. In Chapter 2, the diurnal study on the mass flows of illicit drugs or metabolites was formed over four days in a municipality with a population of approximately 55,000 people. The diurnal trends in illicit substances vary by substance. The high (g/day) mass flows of caffeine, methamphetamine, and creatinine indicate that lower-frequency sampling (approximately one sample per h) may representatively capture the use and excretion of these substances. However, lower and episodic mass flows of cocaine and its primary human metabolite, benzoylecgonine, indicate that higher-frequency is needed to accurately assess the use of the cocaine within the municipality. Normalization of illicit substances to creatinine gave between-day trends in illicit and legal substances that differed from non-normalized trends. Resident use of cocaine and methamphetamine were indicated by normalized mass flows that increased during early morning hours while commuters are largely absent from the community. Chapter 3 describes a series of experiments conducted at an Oregon state prison. The prison setting provided a unique opportunity to study a nearly-fixed population of individuals and their corresponding mass flows of illicit substances, the number of doses per person consumed, as well as an opportunity to quantify the level of agreement between numbers of individuals and the measured mass flows of creatinine. Methamphetamine use was more prevalent than cocaine/benzoylecgonine in the prison over the one month study in which single daily (24 h) composite samples of wastewater were collected. The hypothesis that the mass flows of methamphetamine and cocaine would be lower on days on which random urinalysis testing (RUA) is typically conducted by the prison (Monday-Thursday) was rejected. While the mass flows (mg/d) of methamphetamine were less than those for a nearby open community, the number of estimated doses per person was higher for the prison population. A higher number of positive RUA results were obtained for methamphetamine while none were positive for cocaine, which is consistent with the data obtained from wastewater. The hourly (diurnal) trend in methamphetamine mass loads indicated continual methamphetamine use/excretion inside the prison while cocaine and benzoylecgonine were detected in five hourly composite samples. Use of methamphetamine and cocaine by inmates could not be unambiguously distinguished from that of non-inmates (employees and visitors). The observed diurnal trends in creatinine mass loads were similar to those of an open community and are indicative of the general pattern of human wakefulness/activity. Predicted creatinine mass loads based on the total prison (inmates + non-inmates) were in good agreement with the measured mass loads, which indicates the potential use of creatinine as a quantitative population indicator. Additional research on the biodegradability of creatinine is needed because the prison setting was deliberately selected to minimize the potential for creatinine biodegradation. Chapter 4 addresses the data gap that exists on illicit drug transformation during in situ transit in sewers. The rates of in situ biodegradation have not yet been determined for conditions that are relevant to sewers, which include low to variable oxygen concentrations, the presence of a biofilm, and temperatures ≤ 20 °C. For this reason, two tracer tests were conducted in a 5 km stretch of sewer located near Zürich, Switzerland. The stable-isotope forms (deuterated) of cocaine and benzoylecgonine were injected into flowing wastewater and three locations up to 5 km downstream were sampled over time. Breakthrough curves were constructed from measurements of cocaine-d3 and benzoylecgonine-d3 concentration with time. The area under the curve (mass) was determined by integrating concentration over time. Benzoylecgonine-d3 was present in the injectate that should have only contained cocaine-d3; because the benzoylecgonine-d3 formation prior to injection is not known. The injected mass of cocaine-d3 did not decline over the 5 km distance. The observed mass of cocaine-d3 at 5 km was 10% greater than at 500 m, which indicates that the transformation of cocaine was not significant over the 1.5 h experiment. At 5 km downgradient, the apparent mass of benzoylecgonine-d3 had increased by 35% over that observed at 500 m. However, the apparent increase in benzoylecgonine-d3 mass was not accompanied by a corresponding loss of cocaine-d3. While uncertainty is apparent in the increase of both cocaine-d3 and benzoylecgonine-d3, the ratio of cocaine-d3/benzoylecgonine-d3 is subject only to analytical error because any errors associated with sampling and the integration of masses cancel out. The ratio of cocaine-d3/benzoylecgonine-d3 declined from 2.98 in the injectate to 1.66 at Location 3, which indicated a greater increase in benzoylecgonine-d3 relative to cocaine over the 5 km distance. Due to the benzoylecgonine-d3 that was present in the injectate, any biodegradation of cocaine-c3 to form benzoylecgonine-d3 could not be unambiguously distinguished. During the second tracer test in which benzoylecgonine-d3 was injected, the mass of benzoylecgonine-d3 did not significantly decline, which suggests that the apparent loss of benzoylecgonine-d3 during the cocaine-d3 test cannot be attributed to in-situ biodegradation. Overall, while uncertainty exists about the integrated masses for cocaine-d3 and benzoylecgonine-d3, the 5 km distance was too short in order to observe a significant loss of cocaine-d3 and formation of benzoylecgonine-d3. Recommendations for future research include conducting analysis on the injectate solution to ensure that only cocaine-d3 is introduced so that any formation of benzoylecgonine-d3 is readily apparent and quantifiable. In addition, the tracer tests should be repeated in a longer section of sewer to increase the residence time beyond 1.5 hr and degradation products of benzoylecgonine-d3 should be monitored including ecgonine and ecgonine methyl ester. Close

• 4193. [Article] Biological Soil Crusts of the Great Basin : An Examination of their Distribution, Recovery from Disturbance and Restoration

  We are at risk of losing the sagebrush steppe in the floristic Great Basin to the invasion of Bromus tectorum L., cheatgrass. The floristic Great Basin includes the Central Basin and Range, the Northern ...

  Citation × Citation Citation Abstract Copy Title: Biological Soil Crusts of the Great Basin : An Examination of their Distribution, Recovery from Disturbance and Restoration Author: Condon, Lea A. We are at risk of losing the sagebrush steppe in the floristic Great Basin to the invasion of Bromus tectorum L., cheatgrass. The floristic Great Basin includes the Central Basin and Range, the Northern Basin and Range, and the Snake River Plain. The Great Basin receives most of its precipitation as winter snow and experiences hot and dry summers. Early accounts of invasion by cheatgrass associated it with farming and grazing practices. The non-farmed areas in the region are still actively grazed and referred to as rangelands. On invaded sites, cheatgrass changes the flammability of fuels on invaded landscapes, across the Great Basin, from coarser fuels that are widely spaced to fine fuels that are continuous, filling interspaces between perennial plants. The fuel load created by cheatgrass regenerates annually. This has resulted in a change in the fire regime of the Great Basin from infrequent, small fires to more frequent large fires. In arid lands globally, soil interspaces between perennial plants are typically filled by biological soil crusts (biocrusts). This is also true for ecoregions in and surrounding the Great Basin. Biocrusts are known to influence many ecosystem processes that cheatgrass influences, specifically nutrient cycling and availability of soil moisture. However, little work has been done on biocrusts of the Great Basin and to my knowledge, no one had restored biocrusts within the Great Basin. I attempt to fill some of this knowledge "interspace" by relating biocrust presence to disturbances and cheatgrass invasion and to demonstrate the potential for biocrust restoration within this region. Previous work in eastern Oregon demonstrated relationships between declines in biocrusts and increases in cheatgrass with increasing grazing intensity, soil temperature, and decreasing soil moisture. Grazing intensity influences the cover of biocrusts as well as the abundance and composition of native bunchgrasses. Native bunchgrasses influence the interspace gap size between perennial herbaceous vegetation which is directly associated with the cover of cheatgrass. In a region where grazing records may be incomplete and may exist in various forms of data, having a simple indicator of grazing impacts would be useful. It is also crucial that we have an understanding of what leads to loss of site resistance to cheatgrass. This previous work suggested that cover of biocrusts, in addition to bunchgrass composition, were associated with increased site resistance to cheatgrass. In Chapter 2, I used current grazing records from a range of suspected grazing intensities, to examine the ability of both biocrusts and perennial vegetation to maintain site resistance to cheatgrass after fire. I examined the ability of mosses and lichens to maintain site resistance separately given that these are two very different kinds of organisms. Mosses are non-vascular plants and early colonizers of sites in primary succession. Lichens have a symbiotic relationship between a fungus and a photosynthesizing partner, a cyanobacteria, an algae or both. Using structural equation models, I corroborated that perennial vegetation and lichens are associated with increased site resistance to cheatgrass and that mosses are associated with and may facilitate both lichens and perennial herbaceous vegetation. Also in Chapter 2, I identified that burned sites were associated with increased grazing pressure by livestock as shown by increases in cow dung density and increases in gap size between perennial herbaceous vegetation. The Great Basin is managed for cover of perennial vegetation but it could also be managed for morphogroups of biocrusts. Considering morphogroups of biocrusts, which were shown in the Chapter 2 to be important for site resilience and resistance, I wanted to determine if there were site characteristics associated with biocrust distribution and recovery from disturbance, across the Great Basin. Outside of the Great Basin on the Columbia Plateau, others had found that mosses were still present on disturbed sites whereas lichens were often lost. In addition, biocrust species were more associated with soil properties than with grazing by livestock. Given that grazing by livestock and fire are common disturbances across the region, I wanted to know if the same relationships between biocrusts, soil properties and disturbance were true in the Great Basin. I found that cover of the lichen component of biocrusts was higher on sites that were both ungrazed and unburned. Factors related to disturbance characteristics were correlated with the recovery of biocrusts, even after accounting for time since fire. Factors related to disturbance, a composite of grazing and fire, were more important for structuring the cover and composition of morphogroups as opposed to environmental conditions. Lichens were the most sensitive morphogroup, compared to tall mosses, followed by short mosses which were favored by some disturbance but reduced in cover immediately after fire. Perennial grasses were also favored by some disturbance and perennial forbs did not show an obvious relationship with a disturbance gradient. Chapter 3 highlights that grazing by livestock and fire are common disturbances across the region so much so that the effects of one on the abundances of morphogroups could not be separated from the other. Given the observed contributions of biocrusts to site resilience and resistance, I wanted to know if we could restore biocrusts in the field. Others have grown mosses in a lab setting but this was the first study to restore mosses in the Great Basin. I tested the influence of factors that are commonly used in the field of restoration for facilitating plant establishment. I tested the influence of season of inoculation (fall versus spring), the addition of organic matter (in the form of jute net), irrigation (in the spring season) and the climatic setting of moss the collection sites (for moss propagation), in comparison to the experiment site (warm, dry versus cool, moist) on moss growth. I used two moss species: a ruderal (Bryum argenteum) and a later successional species (Syntrichia ruralis). Moss cover increased when the climatic setting of the collection site matched the experiment site. Mosses were facilitated by the addition of the organic jute netting, putting on most of their growth in winter. Although there is still a great deal of work to be done developing moss material for restoration and working out inoculation rates of moss fragments, similar to seeding rates, land managers have another tool to consider when rehabilitating sites after disturbance. Managing the Great Basin for biocrusts in the presence of grazing and fire will not only increase site resistance to cheatgrass but it will add to the conservation of ecosystem functions related to nutrient cycling, hydrologic cycling and soil erosion. Site resistance will be improved with increased periods of rest from grazing following fire. The lichen component of biocrusts is a more sensitive indicator of disturbance when compared with mosses or perennial vegetation but we are currently actively managing for perennial vegetation and not biocrusts. The moss component of biocrusts can be successfully restored in the Great Basin, without irrigation. This dissertation shows that land managers should consider a suite of organisms, in addition to perennial plants to achieve management goals and maintain site resistance to cheatgrass. Title: Biological Soil Crusts of the Great Basin : An Examination of their Distribution, Recovery from Disturbance and Restoration Author: Condon, Lea A. We are at risk of losing the sagebrush steppe in the floristic Great Basin to the invasion of Bromus tectorum L., cheatgrass. The floristic Great Basin includes the Central Basin and Range, the Northern Basin and Range, and the Snake River Plain. The Great Basin receives most of its precipitation as winter snow and experiences hot and dry summers. Early accounts of invasion by cheatgrass associated it with farming and grazing practices. The non-farmed areas in the region are still actively grazed and referred to as rangelands. On invaded sites, cheatgrass changes the flammability of fuels on invaded landscapes, across the Great Basin, from coarser fuels that are widely spaced to fine fuels that are continuous, filling interspaces between perennial plants. The fuel load created by cheatgrass regenerates annually. This has resulted in a change in the fire regime of the Great Basin from infrequent, small fires to more frequent large fires. In arid lands globally, soil interspaces between perennial plants are typically filled by biological soil crusts (biocrusts). This is also true for ecoregions in and surrounding the Great Basin. Biocrusts are known to influence many ecosystem processes that cheatgrass influences, specifically nutrient cycling and availability of soil moisture. However, little work has been done on biocrusts of the Great Basin and to my knowledge, no one had restored biocrusts within the Great Basin. I attempt to fill some of this knowledge "interspace" by relating biocrust presence to disturbances and cheatgrass invasion and to demonstrate the potential for biocrust restoration within this region. Previous work in eastern Oregon demonstrated relationships between declines in biocrusts and increases in cheatgrass with increasing grazing intensity, soil temperature, and decreasing soil moisture. Grazing intensity influences the cover of biocrusts as well as the abundance and composition of native bunchgrasses. Native bunchgrasses influence the interspace gap size between perennial herbaceous vegetation which is directly associated with the cover of cheatgrass. In a region where grazing records may be incomplete and may exist in various forms of data, having a simple indicator of grazing impacts would be useful. It is also crucial that we have an understanding of what leads to loss of site resistance to cheatgrass. This previous work suggested that cover of biocrusts, in addition to bunchgrass composition, were associated with increased site resistance to cheatgrass. In Chapter 2, I used current grazing records from a range of suspected grazing intensities, to examine the ability of both biocrusts and perennial vegetation to maintain site resistance to cheatgrass after fire. I examined the ability of mosses and lichens to maintain site resistance separately given that these are two very different kinds of organisms. Mosses are non-vascular plants and early colonizers of sites in primary succession. Lichens have a symbiotic relationship between a fungus and a photosynthesizing partner, a cyanobacteria, an algae or both. Using structural equation models, I corroborated that perennial vegetation and lichens are associated with increased site resistance to cheatgrass and that mosses are associated with and may facilitate both lichens and perennial herbaceous vegetation. Also in Chapter 2, I identified that burned sites were associated with increased grazing pressure by livestock as shown by increases in cow dung density and increases in gap size between perennial herbaceous vegetation. The Great Basin is managed for cover of perennial vegetation but it could also be managed for morphogroups of biocrusts. Considering morphogroups of biocrusts, which were shown in the Chapter 2 to be important for site resilience and resistance, I wanted to determine if there were site characteristics associated with biocrust distribution and recovery from disturbance, across the Great Basin. Outside of the Great Basin on the Columbia Plateau, others had found that mosses were still present on disturbed sites whereas lichens were often lost. In addition, biocrust species were more associated with soil properties than with grazing by livestock. Given that grazing by livestock and fire are common disturbances across the region, I wanted to know if the same relationships between biocrusts, soil properties and disturbance were true in the Great Basin. I found that cover of the lichen component of biocrusts was higher on sites that were both ungrazed and unburned. Factors related to disturbance characteristics were correlated with the recovery of biocrusts, even after accounting for time since fire. Factors related to disturbance, a composite of grazing and fire, were more important for structuring the cover and composition of morphogroups as opposed to environmental conditions. Lichens were the most sensitive morphogroup, compared to tall mosses, followed by short mosses which were favored by some disturbance but reduced in cover immediately after fire. Perennial grasses were also favored by some disturbance and perennial forbs did not show an obvious relationship with a disturbance gradient. Chapter 3 highlights that grazing by livestock and fire are common disturbances across the region so much so that the effects of one on the abundances of morphogroups could not be separated from the other. Given the observed contributions of biocrusts to site resilience and resistance, I wanted to know if we could restore biocrusts in the field. Others have grown mosses in a lab setting but this was the first study to restore mosses in the Great Basin. I tested the influence of factors that are commonly used in the field of restoration for facilitating plant establishment. I tested the influence of season of inoculation (fall versus spring), the addition of organic matter (in the form of jute net), irrigation (in the spring season) and the climatic setting of moss the collection sites (for moss propagation), in comparison to the experiment site (warm, dry versus cool, moist) on moss growth. I used two moss species: a ruderal (Bryum argenteum) and a later successional species (Syntrichia ruralis). Moss cover increased when the climatic setting of the collection site matched the experiment site. Mosses were facilitated by the addition of the organic jute netting, putting on most of their growth in winter. Although there is still a great deal of work to be done developing moss material for restoration and working out inoculation rates of moss fragments, similar to seeding rates, land managers have another tool to consider when rehabilitating sites after disturbance. Managing the Great Basin for biocrusts in the presence of grazing and fire will not only increase site resistance to cheatgrass but it will add to the conservation of ecosystem functions related to nutrient cycling, hydrologic cycling and soil erosion. Site resistance will be improved with increased periods of rest from grazing following fire. The lichen component of biocrusts is a more sensitive indicator of disturbance when compared with mosses or perennial vegetation but we are currently actively managing for perennial vegetation and not biocrusts. The moss component of biocrusts can be successfully restored in the Great Basin, without irrigation. This dissertation shows that land managers should consider a suite of organisms, in addition to perennial plants to achieve management goals and maintain site resistance to cheatgrass. Close

• 4194. [Article] Structural investigations to understand the mechanisms of two proteins involved in sulfur chemistry : SsuE and cysteine dioxygenase

  Sulfur is one of the six elements required during the early stages of the evolution of life, and enzymes involved in sulfur transfer and oxidation are increasingly being recognized as potential drug targets ...

  Citation × Citation Citation Abstract Copy Title: Structural investigations to understand the mechanisms of two proteins involved in sulfur chemistry : SsuE and cysteine dioxygenase Author: Driggers, Camden McCullough Sulfur is one of the six elements required during the early stages of the evolution of life, and enzymes involved in sulfur transfer and oxidation are increasingly being recognized as potential drug targets for antimicrobials as well as for therapies for cancer, neurodegenerative and inflammatory diseases. Bacteria are able to carry out a much broader range of sulfur chemistry than mammals and are able to use sulfate as a sole sulfur source, meaning that they can synthesize cysteine from sulfate, whereas mammals must take in either homocysteine or cysteine in their diet. Some bacteria, such as Escherichia coli, are able to obtain sulfate from alkanesulfonates through the expression a sulfur starvation utilization system, which includes a two-component system composed of an NADPH-dependent FMN-reductase, SsuE, and a monooxygenase, SsuD. Once cysteine (Cys) is available to a cell, the careful regulation of its concentration is important because even though Cys is required for life, at high levels it is toxic, especially for mammals. In bacteria, metazoa and fungi the Cys levels are primarily regulated by cysteine dioxygenase (CDO). In mammals, CDO is known to form a Cys-Tyr crosslink that greatly increases its enzymatic activity. Here, I report structural studies and evolutionary considerations aimed at elucidating the mechanisms of SsuE and CDO. With regard to the work on SsuE, I report the first structures of any SsuE, and these crystal structures show that SsuE is structurally similar to closely related FMN-reductases. These structures revealed that SsuE forms a tetramer that is similar to related FMN-reductases, and an active site that is completed by the dimer interface. An evolutionarily conserved π-helix appears to link FMN binding with tetramer dissociation. Three different states of SsuE were captured at ~ 2.0 Å resolution: an apo enzyme, an FMN-bound enzyme and a FMNH2-bound enzyme. Based on these results, a reinterpretation of previous kinetics data on SsuE led to a novel proposal for the SsuE mechanism that is similar to those of its homologs. Furthermore, a general catalytic cycle was defined for NADPH-dependent FMN-reductases from the flavodoxin-like superfamily that provides a framework for understanding how the mechanism of these enzymes might change depending on cellular conditions and interactions with partner proteins. With regard to the work on CDO, over 30 structures have been solved of wild type and mutant rat CDOs. In one set of studies, 14 crystal structures between 1.25 and 2.15 Å resolution, including a room temperature structure, are used to clearly define the pH dependence of Cys-persulfenate complex formation in the crystal, and fortuitously also provide the first high resolution view of an unreacted Cys bound in the active site. The main conclusions from this work are that persulfenate formation is consistently seen at pH values between 5.5 and 7, that is it not an artifact of freezing or synchrotron radiation, and at pH≥8 the unliganded active site iron shifts from 4- to 5-coordinate. We are able to identify that important active site pKa values lie between 5.0-5.5 and 7.0-8.0. In a second study, 17 CDO crystal structures in the presence of Cys and inhibitors, ranging from 1.25 to 1.65 Å resolution and mostly of site-directed mutants, are used to define the role of an active site Cys-Tyr crosslink in catalysis as well as to determine the mechanisms of CDO inhibition by homocysteine and azide. Main conclusions are that a chloride ion binds to the active site iron in both the C93A and Y157F variants, even though the active site iron is still in the ferrous form. Upon exposure to Cys, the chloride is displaced but Cys does not bind in the same way as it does to wild-type CDO, with the Cys largely coordinating the iron only through its thiolate, and not through the α-amino group. Cys-persulfenate does not form in the C93A or Y157F active sites, indicating the crosslink is necessary for persulfenate formation in the crystalline enzyme. These results defines a key role for Tyr157 in Cys binding, through both positioning Cys in the active site and modulating the pKa of its α-amino group. The structures in the presence of homocysteine and azide revealed why the CDO-homocysteine complex is unproductive for catalysis, and and how azide binds to the wild-type CDO, associating with the iron and the hydroxyl of Tyr157 in the crosslinked enzyme. Finally, in a third CDO study, I describe the crystal structures of two bacterial CDO homologs that were originally solved by structural genomics groups but had not been reported in the literature. These are of great interest as they represent Gln-type and Arg-type classes of bacterial CDO homologs defined by the residue aligning with Arg60 of rat CDO. For the Arg-type CDO from Bacillus subtilis, we reproduced the crystals and were able to obtain a Cys-bound complex at 2.30 Å resolution that shows that its mode of Cys binding is comparable to mammalian CDOs. Also, we obtained the original diffraction images and further refined the structure of the Gln-type CDO homolog from Ralstonia eutropha to 1.65 Å resolution, and discovered the active site contained an unexpected iron-bound dioxygen. From this structure, we identified a novel active site Arg that is evolutionarily conserved among Gln-type CDO homologs and is positioned in a way that allows us to conclude that these enzymes cannot bind Cys and thus are not authentic CDOs. This is consistent with the observation that the one Gln-type CDO homolog with characterized substrate specificity has been identified as a 3-mercaptopropionate dioxygenases. Title: Structural investigations to understand the mechanisms of two proteins involved in sulfur chemistry : SsuE and cysteine dioxygenase Author: Driggers, Camden McCullough Sulfur is one of the six elements required during the early stages of the evolution of life, and enzymes involved in sulfur transfer and oxidation are increasingly being recognized as potential drug targets for antimicrobials as well as for therapies for cancer, neurodegenerative and inflammatory diseases. Bacteria are able to carry out a much broader range of sulfur chemistry than mammals and are able to use sulfate as a sole sulfur source, meaning that they can synthesize cysteine from sulfate, whereas mammals must take in either homocysteine or cysteine in their diet. Some bacteria, such as Escherichia coli, are able to obtain sulfate from alkanesulfonates through the expression a sulfur starvation utilization system, which includes a two-component system composed of an NADPH-dependent FMN-reductase, SsuE, and a monooxygenase, SsuD. Once cysteine (Cys) is available to a cell, the careful regulation of its concentration is important because even though Cys is required for life, at high levels it is toxic, especially for mammals. In bacteria, metazoa and fungi the Cys levels are primarily regulated by cysteine dioxygenase (CDO). In mammals, CDO is known to form a Cys-Tyr crosslink that greatly increases its enzymatic activity. Here, I report structural studies and evolutionary considerations aimed at elucidating the mechanisms of SsuE and CDO. With regard to the work on SsuE, I report the first structures of any SsuE, and these crystal structures show that SsuE is structurally similar to closely related FMN-reductases. These structures revealed that SsuE forms a tetramer that is similar to related FMN-reductases, and an active site that is completed by the dimer interface. An evolutionarily conserved π-helix appears to link FMN binding with tetramer dissociation. Three different states of SsuE were captured at ~ 2.0 Å resolution: an apo enzyme, an FMN-bound enzyme and a FMNH2-bound enzyme. Based on these results, a reinterpretation of previous kinetics data on SsuE led to a novel proposal for the SsuE mechanism that is similar to those of its homologs. Furthermore, a general catalytic cycle was defined for NADPH-dependent FMN-reductases from the flavodoxin-like superfamily that provides a framework for understanding how the mechanism of these enzymes might change depending on cellular conditions and interactions with partner proteins. With regard to the work on CDO, over 30 structures have been solved of wild type and mutant rat CDOs. In one set of studies, 14 crystal structures between 1.25 and 2.15 Å resolution, including a room temperature structure, are used to clearly define the pH dependence of Cys-persulfenate complex formation in the crystal, and fortuitously also provide the first high resolution view of an unreacted Cys bound in the active site. The main conclusions from this work are that persulfenate formation is consistently seen at pH values between 5.5 and 7, that is it not an artifact of freezing or synchrotron radiation, and at pH≥8 the unliganded active site iron shifts from 4- to 5-coordinate. We are able to identify that important active site pKa values lie between 5.0-5.5 and 7.0-8.0. In a second study, 17 CDO crystal structures in the presence of Cys and inhibitors, ranging from 1.25 to 1.65 Å resolution and mostly of site-directed mutants, are used to define the role of an active site Cys-Tyr crosslink in catalysis as well as to determine the mechanisms of CDO inhibition by homocysteine and azide. Main conclusions are that a chloride ion binds to the active site iron in both the C93A and Y157F variants, even though the active site iron is still in the ferrous form. Upon exposure to Cys, the chloride is displaced but Cys does not bind in the same way as it does to wild-type CDO, with the Cys largely coordinating the iron only through its thiolate, and not through the α-amino group. Cys-persulfenate does not form in the C93A or Y157F active sites, indicating the crosslink is necessary for persulfenate formation in the crystalline enzyme. These results defines a key role for Tyr157 in Cys binding, through both positioning Cys in the active site and modulating the pKa of its α-amino group. The structures in the presence of homocysteine and azide revealed why the CDO-homocysteine complex is unproductive for catalysis, and and how azide binds to the wild-type CDO, associating with the iron and the hydroxyl of Tyr157 in the crosslinked enzyme. Finally, in a third CDO study, I describe the crystal structures of two bacterial CDO homologs that were originally solved by structural genomics groups but had not been reported in the literature. These are of great interest as they represent Gln-type and Arg-type classes of bacterial CDO homologs defined by the residue aligning with Arg60 of rat CDO. For the Arg-type CDO from Bacillus subtilis, we reproduced the crystals and were able to obtain a Cys-bound complex at 2.30 Å resolution that shows that its mode of Cys binding is comparable to mammalian CDOs. Also, we obtained the original diffraction images and further refined the structure of the Gln-type CDO homolog from Ralstonia eutropha to 1.65 Å resolution, and discovered the active site contained an unexpected iron-bound dioxygen. From this structure, we identified a novel active site Arg that is evolutionarily conserved among Gln-type CDO homologs and is positioned in a way that allows us to conclude that these enzymes cannot bind Cys and thus are not authentic CDOs. This is consistent with the observation that the one Gln-type CDO homolog with characterized substrate specificity has been identified as a 3-mercaptopropionate dioxygenases. Close

• 4195. [Article] The geology, geochronology, and geochemistry of the Kaskanak Batholith, and other late Cretaceous to Eocene magmatism at the Pebble porphyry Cu-Au-Mo deposit, SW Alaska

  The Pebble porphyry Cu-Au-Mo deposit in Alaska is one of the world's largest Cu-Au mineral resources. Late Cretaceous magmatic evolution in the Pebble district culminated with the intrusion of the Kaskanak ...

  Citation × Citation Citation Abstract Copy Title: The geology, geochronology, and geochemistry of the Kaskanak Batholith, and other late Cretaceous to Eocene magmatism at the Pebble porphyry Cu-Au-Mo deposit, SW Alaska Author: Olson, Nansen H. The Pebble porphyry Cu-Au-Mo deposit in Alaska is one of the world's largest Cu-Au mineral resources. Late Cretaceous magmatic evolution in the Pebble district culminated with the intrusion of the Kaskanak Batholith and associated porphyry copper-gold-molybdenum mineralization. The Kaskanak Batholith is a multiphase granodiorite intrusion with an estimated footprint of ≥150 km². The batholith is exposed at surface west of the deposit and lies at >600 m depth in the East Zone. The geology, geochemistry, and geochronology of the Pebble district intrusions were investigated to better understand the magmatic processes and their relationship to the formation of this giant ore deposit. The principal zones of mineralization in the Pebble district are the West Zone and East Zone, with prospects containing mineralized porphyry intrusions located at the 38 Zone, 308 Zone, and 65 Zone. Together, they constitute an estimated resource of 1.08 Bt of ore containing 36.5 Mt Cu, 2.54 Mt Mo, and 3340 M gm Au. The main and equigranular granodiorite phase of the Kaskanak Batholith forms a series of porphyritic cupolas along its roof. These cupolas are cross-cut by three distinct porphyry dike sets associated with mineralization in the district. These are: 1) a voluminous granodiorite porphyry plug in the East zone, 2) quartz granite porphyry dikes in the West Zone, East Zone, and 38 Zone, and 3) narrow leucocratic quartz granite porphyry dikes in the 38 Zone and 65 Zone. New SHRIMP-RG and LA-ICP-MS U-Pb analyses of zircon from 25 samples, together with previously published ages establish a period of 9 m.y. for Late Cretaceous magmatism in the district from 98 to 89 Ma. Pre-ore diorite, alkalic monzodiorite and monzonite porphyry intrusions, and granodiorite sills were emplaced between ~98-95 Ma; the main equigranular granodiorite was emplaced at ~91 Ma; and the younger mineralized porphyries were emplaced at ~90-89 Ma (e.g., 90.3±1.0 Ma, 89.4±1.4 Ma; 89.2±1.2 Ma for these porphyries, respectively). New recognition of pre-mineral and post-mineral andesite porphyry dikes are observed in the 1 Gold Zone, the 308 Zone, and in the East Zone. New U-Pb ages on the pre-mineral andesite porphyry dikes in the 308 Zone are 90.6±1.5 Ma and 91.1±1.2 Ma. Following the cessation of hydrothermal activity, the area was magmatically quiescent until the end of the Cretaceous. The age of renewed magmatism is established by new U-Pb age determinations of dacite and andesite dikes of 63.9±1.0 Ma and 65.9±1.4 Ma, respectively. These dikes cross-cut mineralization in the East Zone, but are eroded by a Paleocene erosional unconformity. The maximum age of the erosional unconformity is constrained by the 11 youngest detrital zircons from a sample of basal conglomerates of the Copper Lake Formation that immediately overlies the unconformity (e.g., 61.2±0.8 Ma). Based on petrographic and geochemical evidence, the Kaskanak Batholith was apparently more oxidized and hydrous than earlier local intrusions that predate mineralization. The Kaskanak Batholith is characterized by high ratios of whole rock V/Sc (up to 160), zircon Eu/Eu[subscript CN]* (>0.4) and zircon Ce/Nd[subscript CN] (>40). Zircon trace element compositions of the Kaskanak Batholith are distinct from pre-ore or post-ore Paleocene-Eocene intrusions. Ti-in-zircon geothermometry indicates pre-ore intrusions were hotter (~750-940° C) than Kaskanak Batholith phases (~685-760° C). Zircon Ce/Ce[subscript CN]* and Eu/Eu[subscript CN]* values are elevated in all phases of the Kaskanak Batholith as well as in pre-ore granodiorite sills, and within some zircons of late monzonite porphyry dikes, which reflects an increase in ƒO₂ and H₂O content from early pre-ore intrusions to emplacement of the Kaskanak Batholith. Investigation of apatite SO₃ and halogen concentrations suggest that the Kaskanak Granodiorite melt initially contained 0.1-0.3 wt. % SO₃ and had initial Cl/H₂O ratios of 0.3-0.6. The presence of SO₃-rich apatites hosted in primary biotite, occasionally in magnetite, and commonly within interstitial quartz and K-feldspar was observed. These sulfur-rich apatites may have crystallized from hot andesitic melts that subsequently mixed with the Kaskanak Granodiorite, or by breakdown and release of magmatic anhydrite upon volatile exsolution, or a combination of both. Mafic enclaves have been observed locally within the Kaskanak Granodiorite, but observed andesitic melts in the district make up much less than a fraction of a percent of the volume of the Kaskanak Batholith. On the basis of whole rock major and trace element compositions, the Kaskanak Batholith likely differentiated from hydrous and oxidized calc-alkaline andesitic melts. Compositions of andesitic porphyries from the 1 Gold Zone are inferred to represent parental melt compositions. Raleigh fractionation modeling suggests the Kaskanak Granodiorite can be produced by 10-12 wt. % crystal fractionation of amphibole, biotite, magnetite, apatite, and zircon, and the evolved porphyry dikes could have been produced by an additional 10-14 wt. % fractionation of amphibole, titanite, apatite, and zircon. Titanite fractionation at relatively low temperatures was apparently important for sharply depleting evolved porphyry melts of REEs, Nb, and Ta. Jurassic to Eocene age Pebble district intrusions of basaltic to granitic compositions, all have non-radiogenic initial isotopic signatures (⁸⁷Sr/⁸⁶Sr[subscript i] = 0.70329 - 0.70424 and ¹⁴³Nd/¹⁴⁴Nd[subscript i] = 0.51278 - 0.51284 (ƐNdi = +4.9 - +6.1); t = 180, 90, & 65 Ma) reflecting the age and bulk composition of the crustal section. These intrusions are interpreted to have been derived from homogenous shoshonitic and calc-alkaline andesites parental melts generated by melting of the mantle wedge and overlying lower crust yielding similar Sr and Nd isotopic compositions to those of the Peninsular Terrane in the Talkeetna Mountains. Xenocrystic zircon from Pebble district intrusions were derived primarily from the Kahiltna Basin sediments upon emplacement, but a greater component of Paleozoic and Proterozoic grains have also been observed that are unlikely derived in whole from the Kahiltna Basin sediments, consistent with xenocrystic zircons found in some Talkeetna Arc volcanics along the Alaska Peninsula that pre-date the formation of the Kahiltna Basin sediments. These zircons may have been derived from late Triassic - early Jurassic metamorphosed sediments and volcanics that predate Talkeetna Arc magmatism. During the lifespan of the Kaskanak Batholith, it is estimated that ~2 km of cover rocks were unroofed which produced telescoping advanced argillic ledges on top of potassic alteration in the East Zone. By latest Late Cretaceous – Paleocene time (~67-58 Ma), an additional ~2.5 km were rapidly eroded and subsequently buried by volcanoclastic rocks and tuff deposits of the Copper Lake Formation, which may have been initiated by the subduction of the Kula-Farallon mid-ocean ridge. The bulk of the displacement of the ZG Fault that down-dropped high-grade copper ore in the East Graben likely occurred at this time, and much of the mineralized advanced argillic alteration and epithermal-style mineralization overlying the Pebble deposit had been removed. Subsequent eastward Eocene - Quaternary tilting (~20°) has exposed the Kaskanak Batholith to the bedrock surface at Kaskanak Mountain and in the West Zone. Title: The geology, geochronology, and geochemistry of the Kaskanak Batholith, and other late Cretaceous to Eocene magmatism at the Pebble porphyry Cu-Au-Mo deposit, SW Alaska Author: Olson, Nansen H. The Pebble porphyry Cu-Au-Mo deposit in Alaska is one of the world's largest Cu-Au mineral resources. Late Cretaceous magmatic evolution in the Pebble district culminated with the intrusion of the Kaskanak Batholith and associated porphyry copper-gold-molybdenum mineralization. The Kaskanak Batholith is a multiphase granodiorite intrusion with an estimated footprint of ≥150 km². The batholith is exposed at surface west of the deposit and lies at >600 m depth in the East Zone. The geology, geochemistry, and geochronology of the Pebble district intrusions were investigated to better understand the magmatic processes and their relationship to the formation of this giant ore deposit. The principal zones of mineralization in the Pebble district are the West Zone and East Zone, with prospects containing mineralized porphyry intrusions located at the 38 Zone, 308 Zone, and 65 Zone. Together, they constitute an estimated resource of 1.08 Bt of ore containing 36.5 Mt Cu, 2.54 Mt Mo, and 3340 M gm Au. The main and equigranular granodiorite phase of the Kaskanak Batholith forms a series of porphyritic cupolas along its roof. These cupolas are cross-cut by three distinct porphyry dike sets associated with mineralization in the district. These are: 1) a voluminous granodiorite porphyry plug in the East zone, 2) quartz granite porphyry dikes in the West Zone, East Zone, and 38 Zone, and 3) narrow leucocratic quartz granite porphyry dikes in the 38 Zone and 65 Zone. New SHRIMP-RG and LA-ICP-MS U-Pb analyses of zircon from 25 samples, together with previously published ages establish a period of 9 m.y. for Late Cretaceous magmatism in the district from 98 to 89 Ma. Pre-ore diorite, alkalic monzodiorite and monzonite porphyry intrusions, and granodiorite sills were emplaced between ~98-95 Ma; the main equigranular granodiorite was emplaced at ~91 Ma; and the younger mineralized porphyries were emplaced at ~90-89 Ma (e.g., 90.3±1.0 Ma, 89.4±1.4 Ma; 89.2±1.2 Ma for these porphyries, respectively). New recognition of pre-mineral and post-mineral andesite porphyry dikes are observed in the 1 Gold Zone, the 308 Zone, and in the East Zone. New U-Pb ages on the pre-mineral andesite porphyry dikes in the 308 Zone are 90.6±1.5 Ma and 91.1±1.2 Ma. Following the cessation of hydrothermal activity, the area was magmatically quiescent until the end of the Cretaceous. The age of renewed magmatism is established by new U-Pb age determinations of dacite and andesite dikes of 63.9±1.0 Ma and 65.9±1.4 Ma, respectively. These dikes cross-cut mineralization in the East Zone, but are eroded by a Paleocene erosional unconformity. The maximum age of the erosional unconformity is constrained by the 11 youngest detrital zircons from a sample of basal conglomerates of the Copper Lake Formation that immediately overlies the unconformity (e.g., 61.2±0.8 Ma). Based on petrographic and geochemical evidence, the Kaskanak Batholith was apparently more oxidized and hydrous than earlier local intrusions that predate mineralization. The Kaskanak Batholith is characterized by high ratios of whole rock V/Sc (up to 160), zircon Eu/Eu[subscript CN]* (>0.4) and zircon Ce/Nd[subscript CN] (>40). Zircon trace element compositions of the Kaskanak Batholith are distinct from pre-ore or post-ore Paleocene-Eocene intrusions. Ti-in-zircon geothermometry indicates pre-ore intrusions were hotter (~750-940° C) than Kaskanak Batholith phases (~685-760° C). Zircon Ce/Ce[subscript CN]* and Eu/Eu[subscript CN]* values are elevated in all phases of the Kaskanak Batholith as well as in pre-ore granodiorite sills, and within some zircons of late monzonite porphyry dikes, which reflects an increase in ƒO₂ and H₂O content from early pre-ore intrusions to emplacement of the Kaskanak Batholith. Investigation of apatite SO₃ and halogen concentrations suggest that the Kaskanak Granodiorite melt initially contained 0.1-0.3 wt. % SO₃ and had initial Cl/H₂O ratios of 0.3-0.6. The presence of SO₃-rich apatites hosted in primary biotite, occasionally in magnetite, and commonly within interstitial quartz and K-feldspar was observed. These sulfur-rich apatites may have crystallized from hot andesitic melts that subsequently mixed with the Kaskanak Granodiorite, or by breakdown and release of magmatic anhydrite upon volatile exsolution, or a combination of both. Mafic enclaves have been observed locally within the Kaskanak Granodiorite, but observed andesitic melts in the district make up much less than a fraction of a percent of the volume of the Kaskanak Batholith. On the basis of whole rock major and trace element compositions, the Kaskanak Batholith likely differentiated from hydrous and oxidized calc-alkaline andesitic melts. Compositions of andesitic porphyries from the 1 Gold Zone are inferred to represent parental melt compositions. Raleigh fractionation modeling suggests the Kaskanak Granodiorite can be produced by 10-12 wt. % crystal fractionation of amphibole, biotite, magnetite, apatite, and zircon, and the evolved porphyry dikes could have been produced by an additional 10-14 wt. % fractionation of amphibole, titanite, apatite, and zircon. Titanite fractionation at relatively low temperatures was apparently important for sharply depleting evolved porphyry melts of REEs, Nb, and Ta. Jurassic to Eocene age Pebble district intrusions of basaltic to granitic compositions, all have non-radiogenic initial isotopic signatures (⁸⁷Sr/⁸⁶Sr[subscript i] = 0.70329 - 0.70424 and ¹⁴³Nd/¹⁴⁴Nd[subscript i] = 0.51278 - 0.51284 (ƐNdi = +4.9 - +6.1); t = 180, 90, & 65 Ma) reflecting the age and bulk composition of the crustal section. These intrusions are interpreted to have been derived from homogenous shoshonitic and calc-alkaline andesites parental melts generated by melting of the mantle wedge and overlying lower crust yielding similar Sr and Nd isotopic compositions to those of the Peninsular Terrane in the Talkeetna Mountains. Xenocrystic zircon from Pebble district intrusions were derived primarily from the Kahiltna Basin sediments upon emplacement, but a greater component of Paleozoic and Proterozoic grains have also been observed that are unlikely derived in whole from the Kahiltna Basin sediments, consistent with xenocrystic zircons found in some Talkeetna Arc volcanics along the Alaska Peninsula that pre-date the formation of the Kahiltna Basin sediments. These zircons may have been derived from late Triassic - early Jurassic metamorphosed sediments and volcanics that predate Talkeetna Arc magmatism. During the lifespan of the Kaskanak Batholith, it is estimated that ~2 km of cover rocks were unroofed which produced telescoping advanced argillic ledges on top of potassic alteration in the East Zone. By latest Late Cretaceous – Paleocene time (~67-58 Ma), an additional ~2.5 km were rapidly eroded and subsequently buried by volcanoclastic rocks and tuff deposits of the Copper Lake Formation, which may have been initiated by the subduction of the Kula-Farallon mid-ocean ridge. The bulk of the displacement of the ZG Fault that down-dropped high-grade copper ore in the East Graben likely occurred at this time, and much of the mineralized advanced argillic alteration and epithermal-style mineralization overlying the Pebble deposit had been removed. Subsequent eastward Eocene - Quaternary tilting (~20°) has exposed the Kaskanak Batholith to the bedrock surface at Kaskanak Mountain and in the West Zone. Close

• 4196. [Article] Molecular mechanism of germination of Clostridium perfringens spores

  Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these ...

  Citation × Citation Citation Abstract Copy Title: Molecular mechanism of germination of Clostridium perfringens spores Author: Paredes-Sabja, Daniel Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these toxins. Although a small percentage of C. perfringens isolates (mostly belonging to type A) produce C. perfringens enterotoxin (CPE), these are very important human gastrointestinal (GI) pathogens, causing C. perfringens type A food poisoning (FP) and nonfood-borne GI diseases (NFBGID). Due to its anaerobic nature and the ability to form extremely resistant spores found ubiquitously in the environment, to cause the wide array of C. perfringens-associated diseases (CPAD), these C. perfringens spores must germinate, release the nascent cell, grow and produce their toxins. Therefore, germination of C. perfringens spores is the initial and perhaps most important step for the progression of diseases in animals and humans. Although extensive research has been conducted on the mechanism of spore germination of Bacillus species, very few studies of spore germination have been conducted in Clostridium species mainly due to the lack of molecular genetic tools. Genomic comparisons reveal significant differences in the backbone of the germination apparatus between Bacillus and Clostridium species. Consequently, a detail understanding of the molecular mechanism of germination of C. perfringens spores is essential for the development of novel preventive strategies for CPAD as well as diseases caused by other pathogenic Clostridium species. The first focus of this work was to identify and characterize the germinants and the receptors involved in C. perfringens spore germination. Result from these studies found differential germination requirements between spores of FP and NFBGID isolates in that: (i) while a mixture of L-asparagine and KCl was a good germinant for spores of FP and NFBGID isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in FP isolates only; and ii) L-alanine and L-valine induced significant germination of spores of NFBGID but not FP isolates. In contrast to B. subtilis, C. perfrinegns genomes sequenced to date possess no tricistronic gerA-like operon, but has a monocistronic gerAA that is far from a gerK locus. The gerK locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream and in the opposite orientation to gerKA-gerKC. Consequently, through the construction of mutations into strain SM101, a C. perfringens FP isolate, the role of gerAA, gerKA-gerKC and gerKB genes in C. perfringens spore germination were investigated. Results indicated that KCl, L-asparagine and Ca-DPA required GerKA and/or GerKC receptors, while GerAA and GerKB played an auxiliary role in germination. Lack of GerKA and/or GerKC, and GerKB significantly reduced spores colony forming efficiency, indicating a role in spore viability. The fact that C. perfringens spores lacking the main germinant receptor(s) proteins, GerKA and/or GerKC, are still able to germinate albeit poorly compared to wild-type, and that C. perfringens spores germinate with K+ ions alone, raises the hypothesis that GrmA-like antiporters might also play some role in C. perfringens spore germination. Two putative GrmA-like antiporters (i.e., GerO and GerQ) are encoded in the genome of all C. perfringens sequenced to date. This study shows that gerO and gerQ genes are expressed uniquely during sporulation and the mother cell compartment of the sporulating cell. Complementation studies of K+ uptake and Na+ sensitive E. coli mutants indicate that while GerO is capable of translocating K+ and Na+, GerQ is only capable of translocating, to a small extent, Na+ ions. Spores lacking GerO had defective germination in rich medium, KCl, L-asparagine, and Ca-DPA, but not with dodecylamine, defect that might be prior to DPA release during germination. In contrast, loss of GerQ had a much smaller effect on spore germination. Two adjacent Asp residues, important in ion transcloation of the E. coli Na+/H+ antiporter NhaA were also present in GerO, but not GerQ, and replacement of these residues for Asn reduced the protein’s ability to complement gerO spores. Although results from this study indicate that putative antiporters have some role on C. perfringens spore germination, it is unclear whether their role is direct or during spore formation. C. perfringens type A FP spores are capable of germinating with K+ ions, an intrinsic mineral of meats commonly associated with FP. Inorganic phosphate (Pi) is also intrinsically found in meat products. Consequently, we hypothesized that FP spores are capable of germinating in presence of Pi. Results from this study show that spores of the majority of FP, but not NFBGID isolates, are able to germinate in presence of Pi. Pi-induced germination of FP spores is primarily through the GerKA and/or GerKC protein, while GerAA and to a much lesser extent, GerKB, play auxiliary roles. The putative Na+/K+-H+ antiporter, GerO, is also required for normal Pi-induced germination. These results suggest that the differential germination phenotypes between spores of FP and NFGID isolates is tightly regulated by their adaptation to different environmental niches. A second focus of this work was to investigate the mechanism of signal transduction between the germinant receptors and the downstream effectors. In B. subtilis, the SpoVA proteins have been associated with Ca-DPA uptake and subsequent release during sporulation and germination, respectively. In addition, Ca-DPA acts as a signal molecule for cortex hydrolysis during B. subtilis spore germination, activating the cortex lytic enzyme (CLE) CwlJ. Results from this study show that in contrast to B. subtilis spoVA mutants, where spores lyse quickly during purification, C. perfringens spoVA spores were stable and germinated similarly as wild-type spores. These results suggest major differences in the regulation of the germination pathway between C. perfringens and B. subtilis, and suggest that activation of CLEs in C. perfringens might be through a different pathway than the Ca-DPA pathway of B. subtilis. A third focus of this work was to investigate the in vivo role of the CLE involved in peptidoglycan (PG) spore cortex hydrolysis during C. perfringens spore germination. Two C. perfringens CLEs (i.e., SleC and SleM) degrade PG spore cortex hydrolysis in vitro, however, due to lack of genetic tools, their in vivo role in spore germination remains unclear. Results from this study show that C. perfringens sleC spores released their DPA slower than wild-type and were not able to germinate with nutrients and non-nutrient germinants. In contrast, sleM spores germinated similar as wild type in presence of nutrient and non-nutrient germinants, indicating that while SleC is essential for cortex hydrolysis and viability of C. perfringens spores, SleM although can degrade cortex PG in vitro, is not essential. A fourth focus of this work was to investigate the in vivo role of the Csp proteases in the initiation of cortex hydrolysis. In vitro work has shown that Csp proteins process the inactive proSleC into the mature enzyme, SleC. However, the in vivo role of the Csp proteins has not been established. In this study, spores a C. perfringens cspB mutant exhibited significantly less viability than wild-type spores, and were unable to germinate with either rich medium or Ca-DPA. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis. Results from this study indicate that CspB is essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. In contrast to B. subtilis, Ca-DPA did not activate the CLEs during spore germination present in cspB spores supporting previous results that Ca-DPA acts trough the GerKA and/or GerKC receptor. A final focus of this work was to develop a strategy to inactivate C. perfringens spores in meat products. C. perfringens spores posses high heat and pressure resistance, however, they loss their resistance properties during early stages of germination. In contrast to B. subtilis spores, germination of C. perfringens spores could not be triggered with low pressures. However, they germinated efficiently when heat activated in presence of L-asparagine and KCl at temperatures lethal for vegetative cells, and these germinated spores were efficiently inactivated by subsequent treatment with pressure assisted thermal processing (586 MPa at 73ºC for 10 min). This study shows the feasibility of a novel strategy to inactivate C. perfringens spores in meat products formulated with germinants. Collectively, the present study contributes to the understanding of the mechanism of spore germination in the pathogenic bacterium C. perfringens, and developed an alternative and novel strategy to inactivate C. perfringens spores in meat products. Title: Molecular mechanism of germination of Clostridium perfringens spores Author: Paredes-Sabja, Daniel Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these toxins. Although a small percentage of C. perfringens isolates (mostly belonging to type A) produce C. perfringens enterotoxin (CPE), these are very important human gastrointestinal (GI) pathogens, causing C. perfringens type A food poisoning (FP) and nonfood-borne GI diseases (NFBGID). Due to its anaerobic nature and the ability to form extremely resistant spores found ubiquitously in the environment, to cause the wide array of C. perfringens-associated diseases (CPAD), these C. perfringens spores must germinate, release the nascent cell, grow and produce their toxins. Therefore, germination of C. perfringens spores is the initial and perhaps most important step for the progression of diseases in animals and humans. Although extensive research has been conducted on the mechanism of spore germination of Bacillus species, very few studies of spore germination have been conducted in Clostridium species mainly due to the lack of molecular genetic tools. Genomic comparisons reveal significant differences in the backbone of the germination apparatus between Bacillus and Clostridium species. Consequently, a detail understanding of the molecular mechanism of germination of C. perfringens spores is essential for the development of novel preventive strategies for CPAD as well as diseases caused by other pathogenic Clostridium species. The first focus of this work was to identify and characterize the germinants and the receptors involved in C. perfringens spore germination. Result from these studies found differential germination requirements between spores of FP and NFBGID isolates in that: (i) while a mixture of L-asparagine and KCl was a good germinant for spores of FP and NFBGID isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in FP isolates only; and ii) L-alanine and L-valine induced significant germination of spores of NFBGID but not FP isolates. In contrast to B. subtilis, C. perfrinegns genomes sequenced to date possess no tricistronic gerA-like operon, but has a monocistronic gerAA that is far from a gerK locus. The gerK locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream and in the opposite orientation to gerKA-gerKC. Consequently, through the construction of mutations into strain SM101, a C. perfringens FP isolate, the role of gerAA, gerKA-gerKC and gerKB genes in C. perfringens spore germination were investigated. Results indicated that KCl, L-asparagine and Ca-DPA required GerKA and/or GerKC receptors, while GerAA and GerKB played an auxiliary role in germination. Lack of GerKA and/or GerKC, and GerKB significantly reduced spores colony forming efficiency, indicating a role in spore viability. The fact that C. perfringens spores lacking the main germinant receptor(s) proteins, GerKA and/or GerKC, are still able to germinate albeit poorly compared to wild-type, and that C. perfringens spores germinate with K+ ions alone, raises the hypothesis that GrmA-like antiporters might also play some role in C. perfringens spore germination. Two putative GrmA-like antiporters (i.e., GerO and GerQ) are encoded in the genome of all C. perfringens sequenced to date. This study shows that gerO and gerQ genes are expressed uniquely during sporulation and the mother cell compartment of the sporulating cell. Complementation studies of K+ uptake and Na+ sensitive E. coli mutants indicate that while GerO is capable of translocating K+ and Na+, GerQ is only capable of translocating, to a small extent, Na+ ions. Spores lacking GerO had defective germination in rich medium, KCl, L-asparagine, and Ca-DPA, but not with dodecylamine, defect that might be prior to DPA release during germination. In contrast, loss of GerQ had a much smaller effect on spore germination. Two adjacent Asp residues, important in ion transcloation of the E. coli Na+/H+ antiporter NhaA were also present in GerO, but not GerQ, and replacement of these residues for Asn reduced the protein’s ability to complement gerO spores. Although results from this study indicate that putative antiporters have some role on C. perfringens spore germination, it is unclear whether their role is direct or during spore formation. C. perfringens type A FP spores are capable of germinating with K+ ions, an intrinsic mineral of meats commonly associated with FP. Inorganic phosphate (Pi) is also intrinsically found in meat products. Consequently, we hypothesized that FP spores are capable of germinating in presence of Pi. Results from this study show that spores of the majority of FP, but not NFBGID isolates, are able to germinate in presence of Pi. Pi-induced germination of FP spores is primarily through the GerKA and/or GerKC protein, while GerAA and to a much lesser extent, GerKB, play auxiliary roles. The putative Na+/K+-H+ antiporter, GerO, is also required for normal Pi-induced germination. These results suggest that the differential germination phenotypes between spores of FP and NFGID isolates is tightly regulated by their adaptation to different environmental niches. A second focus of this work was to investigate the mechanism of signal transduction between the germinant receptors and the downstream effectors. In B. subtilis, the SpoVA proteins have been associated with Ca-DPA uptake and subsequent release during sporulation and germination, respectively. In addition, Ca-DPA acts as a signal molecule for cortex hydrolysis during B. subtilis spore germination, activating the cortex lytic enzyme (CLE) CwlJ. Results from this study show that in contrast to B. subtilis spoVA mutants, where spores lyse quickly during purification, C. perfringens spoVA spores were stable and germinated similarly as wild-type spores. These results suggest major differences in the regulation of the germination pathway between C. perfringens and B. subtilis, and suggest that activation of CLEs in C. perfringens might be through a different pathway than the Ca-DPA pathway of B. subtilis. A third focus of this work was to investigate the in vivo role of the CLE involved in peptidoglycan (PG) spore cortex hydrolysis during C. perfringens spore germination. Two C. perfringens CLEs (i.e., SleC and SleM) degrade PG spore cortex hydrolysis in vitro, however, due to lack of genetic tools, their in vivo role in spore germination remains unclear. Results from this study show that C. perfringens sleC spores released their DPA slower than wild-type and were not able to germinate with nutrients and non-nutrient germinants. In contrast, sleM spores germinated similar as wild type in presence of nutrient and non-nutrient germinants, indicating that while SleC is essential for cortex hydrolysis and viability of C. perfringens spores, SleM although can degrade cortex PG in vitro, is not essential. A fourth focus of this work was to investigate the in vivo role of the Csp proteases in the initiation of cortex hydrolysis. In vitro work has shown that Csp proteins process the inactive proSleC into the mature enzyme, SleC. However, the in vivo role of the Csp proteins has not been established. In this study, spores a C. perfringens cspB mutant exhibited significantly less viability than wild-type spores, and were unable to germinate with either rich medium or Ca-DPA. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis. Results from this study indicate that CspB is essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. In contrast to B. subtilis, Ca-DPA did not activate the CLEs during spore germination present in cspB spores supporting previous results that Ca-DPA acts trough the GerKA and/or GerKC receptor. A final focus of this work was to develop a strategy to inactivate C. perfringens spores in meat products. C. perfringens spores posses high heat and pressure resistance, however, they loss their resistance properties during early stages of germination. In contrast to B. subtilis spores, germination of C. perfringens spores could not be triggered with low pressures. However, they germinated efficiently when heat activated in presence of L-asparagine and KCl at temperatures lethal for vegetative cells, and these germinated spores were efficiently inactivated by subsequent treatment with pressure assisted thermal processing (586 MPa at 73ºC for 10 min). This study shows the feasibility of a novel strategy to inactivate C. perfringens spores in meat products formulated with germinants. Collectively, the present study contributes to the understanding of the mechanism of spore germination in the pathogenic bacterium C. perfringens, and developed an alternative and novel strategy to inactivate C. perfringens spores in meat products. Close

• 4197. [Article] Polyunsaturated fatty acid metabolism in broiler chickens : effects of maternal diet

  Three experiments were conducted in broiler hens to study the influence of dietary n-3 polyunsaturated fatty acids (PUFA) on egg quality, antioxidant status in progeny, and eicosanoid production in tissue. The ...

  Citation × Citation Citation Abstract Copy Title: Polyunsaturated fatty acid metabolism in broiler chickens : effects of maternal diet Author: Bautista Ortega, Jaime Three experiments were conducted in broiler hens to study the influence of dietary n-3 polyunsaturated fatty acids (PUFA) on egg quality, antioxidant status in progeny, and eicosanoid production in tissue. The objective of experiment 1 was to determine the effect of hen age and dietary n-3 PUFA on egg quality and hatchability. Two-hundred-twenty breeder chicks (males and females) (Cobb Breeders) were raised until 20 weeks of age following the company guidelines. At this age, 3 groups of birds (24 breeder hens and 3 roosters) were randomly allocated to one of the following dietary treatments: 3.5% sunflower oil (Low n-3 diet), 1.75% sunflower oil + 1.75% fish oil (Medium n-3 diet) or 3.5% fish oil (High n-3 diet). Egg quality was evaluated at 29, 37 and 45 weeks of age by determining total egg weight, its components (albumen, yolk and shell) and shell thickness. Total fat content in the yolk and its fatty acid profile was also determined. Egg production was recorded daily. Breeder hens fed the High n-3 diet laid lighter eggs with lighter yolks, albumens and shells than those fed the Medium and Low n-3 diets (p<0.05). Eggs laid by hens fed the Medium n-3 diet had thicker shells than those laid by hens fed the Low n-3 diet (p<0.05). Egg weight, yolk weight, albumen weight, shell weight and shell thickness increased significantly with hen age (p<0.05). Total fat content in the yolk was significantly higher in the eggs laid by 37-week-old and 45-week-old hens than in those laid by 29-week-old hens. Hens fed the high n-3 diet laid eggs with significantly higher n-3 PUFA and lower n-6 PUFA content than hens from the other treatments (p<0.05). Hen age did not affect the n-3 or n-6 PUFA content. Fertility and hatchability were not affected by maternal diet. Total egg weight, yolk weight, albumen weight and shell weight was decreased by feeding n-3 PUFA to breeder hens. The decreased n6:n3 ratio brought about by maternal dietary n-3 PUFA was further investigated in connection with possible effects on antioxidant and eicosanoid status in newly hatched chicks. The objective of experiment 2 was to determine the effect of maternal diet (Low, Medium and High n-3) on the antioxidant and eicosanoid status, tissue fatty acid profile and lipid peroxidation in the newly hatched chick. Two-hundred-ninety-eight eggs were collected from the 29-week-old breeder hens mentioned in experiment 1. After incubation, day-old chicks were randomly selected from a pool of eggs laid by hens fed the three experimental diets. Antioxidant status was established by measuring activity of antioxidant enzymes (glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase) and the content of total glutathione. Hatchability and total fat content in the tissues were not affected by maternal diet. n-3 PUFA content increased and n-6 PUFA decreased significantly in chick’s tissue (p<0.05) hatched from hens fed the fish-oil supplemented diet compared to those hatched from hens fed the Low n-3 diet (p<0.05). Total glutathione and antioxidant enzyme activity was not affected by maternal diet, except for catalase, whose activity was significantly lower in chicks hatched from hens fed the High n-3 and the Medium n-3 diets than in those hatched from hens fed the Low n-3 diets (p<0.05). Malondialdehyde, a measure of lipid peroxidation, was significantly lower in the liver of chicks hatched from eggs laid by hens fed the High n-3 diet than in those hatched from hens fed the Medium n-3 diet. Maternal dietary n-3 PUFA was successfully transferred to the newly hatched chicks without compromising their antioxidant status. The decreased n-6/n-3 ratio observed in chicks hatched from hens fed the fish-oil supplemented diets needs was further investigated relative to its downstream modulatory effects in connection with fat metabolism. The objective of experiment 3 was to establish the effect of maternal diet on fatty acid accretion in heart tissue, and the production of eicosanoids by heart tissue homogenates and peripheral blood mononuclear cells (PBMNC) from broilers fed diet devoid of long-chain PUFA. Broilers were hatched from hens fed the Low, Medium or High n-3 diet. One-hundred-fortyfour 1-day-old chicks were weighed and randomly allocated to four pens housed in three rooms of similar dimensions. Temperature was controlled according to specifications by Cobb Breeders during the 42 days that the experiment lasted. A cardiac morphological study was conducted on a weekly basis starting at 14 days of age to assess the heart weight relative to body weight. Also, the ventricular weights index (right ventricular weight divided by the total ventricular weight) was determined weekly from 14 days onwards. Day-0 chicks hatched from hens fed the High n-3 diet were significantly lighter than those hatched from hens fed the Low n-3 diet. After accounting for age, chicks hatched from hens fed the Low n-3 diet were significantly heavier than those hatched from hens fed the High n-3 diet (p<0.05). Maternal diet did not affect heart weight, after accounting for age. The heart percentage (heart weight relative to body weight) was significantly higher in chicks hatched from hens fed the High n-3 diet than in those hatched from hens fed the Low n-3 diet (p<0.05). The ventricular weights index was not affected by maternal diet. At 7 and 14 days of age, arachidonic acid (AA) content in heart tissue was significantly lower in chicks hatched from hens fed the High n-3 diet than those hatched from hens fed the Low n-3 diet (p<0.05). At 7 days of age, AA content in the heart tissue of chicks hatched from hens fed the High n-3 diet was significantly lower than in those hatched from hens fed the Medium n-3 diet (p<0.05). At day 0, the heart tissue production of prostaglandin E2 (PGE2) was significantly higher in the chicks hatched from hens fed the Low n-3 diet than in those hatched from hens fed the Medium or High n-3 diets (p<0.05). At the same age, thromboxane A3 (TXA3) production was significantly lower in the heart tissue of chicks hatched from hens fed the Low n-3 diet than in that of chicks hatched from hens fed either the Medium or Low n-3 diets (p<0.05). At day 7, PBMNCs isolated from chickens hatched from hens fed the Low and Medium n-3 diets produced significantly higher PGE2 and TXA2 concentrations than those isolated from birds hatched from eggs laid by hens fed the High n-3 diet (p<0.05). At day 31, PBMNCs isolated from chickens hatched from hens fed the Medium and High n-3 diets produced significantly higher PGE2 and TXA2 concentrations than those isolated from chicks hatched from eggs laid by hens fed the Low n-3 diet (p<0.05). Chicks hatched from hens fed the High n-3 diet had a higher heart/body weight than those hatched from the Low n-3 diet. Thus, chicks hatched from hens fed the Low n-3 diet may be at higher risk of developing cardiovascular complications related to high AA concentrations in the heart and blood cells during the first week of age. Further research is encouraged in which the three subpopulations of broilers (i.e.hatched from hens fed the Low, Medium and High n-3 diets) are raised under commercial conditions to investigate how the economic loss, due to a reduction in body weight observed in the High n-3 chickens compares with the potential reduction in mortality during the first week of age. Finally, the mechanistic action of n-3 PUFA needs further investigation, especially molecular aspects related to modulatory effects on gene expression. Title: Polyunsaturated fatty acid metabolism in broiler chickens : effects of maternal diet Author: Bautista Ortega, Jaime Three experiments were conducted in broiler hens to study the influence of dietary n-3 polyunsaturated fatty acids (PUFA) on egg quality, antioxidant status in progeny, and eicosanoid production in tissue. The objective of experiment 1 was to determine the effect of hen age and dietary n-3 PUFA on egg quality and hatchability. Two-hundred-twenty breeder chicks (males and females) (Cobb Breeders) were raised until 20 weeks of age following the company guidelines. At this age, 3 groups of birds (24 breeder hens and 3 roosters) were randomly allocated to one of the following dietary treatments: 3.5% sunflower oil (Low n-3 diet), 1.75% sunflower oil + 1.75% fish oil (Medium n-3 diet) or 3.5% fish oil (High n-3 diet). Egg quality was evaluated at 29, 37 and 45 weeks of age by determining total egg weight, its components (albumen, yolk and shell) and shell thickness. Total fat content in the yolk and its fatty acid profile was also determined. Egg production was recorded daily. Breeder hens fed the High n-3 diet laid lighter eggs with lighter yolks, albumens and shells than those fed the Medium and Low n-3 diets (p<0.05). Eggs laid by hens fed the Medium n-3 diet had thicker shells than those laid by hens fed the Low n-3 diet (p<0.05). Egg weight, yolk weight, albumen weight, shell weight and shell thickness increased significantly with hen age (p<0.05). Total fat content in the yolk was significantly higher in the eggs laid by 37-week-old and 45-week-old hens than in those laid by 29-week-old hens. Hens fed the high n-3 diet laid eggs with significantly higher n-3 PUFA and lower n-6 PUFA content than hens from the other treatments (p<0.05). Hen age did not affect the n-3 or n-6 PUFA content. Fertility and hatchability were not affected by maternal diet. Total egg weight, yolk weight, albumen weight and shell weight was decreased by feeding n-3 PUFA to breeder hens. The decreased n6:n3 ratio brought about by maternal dietary n-3 PUFA was further investigated in connection with possible effects on antioxidant and eicosanoid status in newly hatched chicks. The objective of experiment 2 was to determine the effect of maternal diet (Low, Medium and High n-3) on the antioxidant and eicosanoid status, tissue fatty acid profile and lipid peroxidation in the newly hatched chick. Two-hundred-ninety-eight eggs were collected from the 29-week-old breeder hens mentioned in experiment 1. After incubation, day-old chicks were randomly selected from a pool of eggs laid by hens fed the three experimental diets. Antioxidant status was established by measuring activity of antioxidant enzymes (glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase) and the content of total glutathione. Hatchability and total fat content in the tissues were not affected by maternal diet. n-3 PUFA content increased and n-6 PUFA decreased significantly in chick’s tissue (p<0.05) hatched from hens fed the fish-oil supplemented diet compared to those hatched from hens fed the Low n-3 diet (p<0.05). Total glutathione and antioxidant enzyme activity was not affected by maternal diet, except for catalase, whose activity was significantly lower in chicks hatched from hens fed the High n-3 and the Medium n-3 diets than in those hatched from hens fed the Low n-3 diets (p<0.05). Malondialdehyde, a measure of lipid peroxidation, was significantly lower in the liver of chicks hatched from eggs laid by hens fed the High n-3 diet than in those hatched from hens fed the Medium n-3 diet. Maternal dietary n-3 PUFA was successfully transferred to the newly hatched chicks without compromising their antioxidant status. The decreased n-6/n-3 ratio observed in chicks hatched from hens fed the fish-oil supplemented diets needs was further investigated relative to its downstream modulatory effects in connection with fat metabolism. The objective of experiment 3 was to establish the effect of maternal diet on fatty acid accretion in heart tissue, and the production of eicosanoids by heart tissue homogenates and peripheral blood mononuclear cells (PBMNC) from broilers fed diet devoid of long-chain PUFA. Broilers were hatched from hens fed the Low, Medium or High n-3 diet. One-hundred-fortyfour 1-day-old chicks were weighed and randomly allocated to four pens housed in three rooms of similar dimensions. Temperature was controlled according to specifications by Cobb Breeders during the 42 days that the experiment lasted. A cardiac morphological study was conducted on a weekly basis starting at 14 days of age to assess the heart weight relative to body weight. Also, the ventricular weights index (right ventricular weight divided by the total ventricular weight) was determined weekly from 14 days onwards. Day-0 chicks hatched from hens fed the High n-3 diet were significantly lighter than those hatched from hens fed the Low n-3 diet. After accounting for age, chicks hatched from hens fed the Low n-3 diet were significantly heavier than those hatched from hens fed the High n-3 diet (p<0.05). Maternal diet did not affect heart weight, after accounting for age. The heart percentage (heart weight relative to body weight) was significantly higher in chicks hatched from hens fed the High n-3 diet than in those hatched from hens fed the Low n-3 diet (p<0.05). The ventricular weights index was not affected by maternal diet. At 7 and 14 days of age, arachidonic acid (AA) content in heart tissue was significantly lower in chicks hatched from hens fed the High n-3 diet than those hatched from hens fed the Low n-3 diet (p<0.05). At 7 days of age, AA content in the heart tissue of chicks hatched from hens fed the High n-3 diet was significantly lower than in those hatched from hens fed the Medium n-3 diet (p<0.05). At day 0, the heart tissue production of prostaglandin E2 (PGE2) was significantly higher in the chicks hatched from hens fed the Low n-3 diet than in those hatched from hens fed the Medium or High n-3 diets (p<0.05). At the same age, thromboxane A3 (TXA3) production was significantly lower in the heart tissue of chicks hatched from hens fed the Low n-3 diet than in that of chicks hatched from hens fed either the Medium or Low n-3 diets (p<0.05). At day 7, PBMNCs isolated from chickens hatched from hens fed the Low and Medium n-3 diets produced significantly higher PGE2 and TXA2 concentrations than those isolated from birds hatched from eggs laid by hens fed the High n-3 diet (p<0.05). At day 31, PBMNCs isolated from chickens hatched from hens fed the Medium and High n-3 diets produced significantly higher PGE2 and TXA2 concentrations than those isolated from chicks hatched from eggs laid by hens fed the Low n-3 diet (p<0.05). Chicks hatched from hens fed the High n-3 diet had a higher heart/body weight than those hatched from the Low n-3 diet. Thus, chicks hatched from hens fed the Low n-3 diet may be at higher risk of developing cardiovascular complications related to high AA concentrations in the heart and blood cells during the first week of age. Further research is encouraged in which the three subpopulations of broilers (i.e.hatched from hens fed the Low, Medium and High n-3 diets) are raised under commercial conditions to investigate how the economic loss, due to a reduction in body weight observed in the High n-3 chickens compares with the potential reduction in mortality during the first week of age. Finally, the mechanistic action of n-3 PUFA needs further investigation, especially molecular aspects related to modulatory effects on gene expression. Close