We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide （phylloseptin, PSN-1）. Chromatographic separation was accompl...We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide （phylloseptin, PSN-1）. Chromatographic separation was accomplished on a Waters bridged ethyl hybrid （BEH） C18 （50mm× 2.1 mm, 1.7 μm） column with acetonitrile-water （25：75, v/v） as isocratic mobile phase. Mass spectrometry detection was performed in the positive electrospray ionization mode and by monitoring of the transitions at m/z 679.6/120, 509.6/120 （PSN-1） and m/z 340.7/165 （Thymopentin, IS）. Protein precipitation was investigated and the recovery was satisfactory （above 82%）. The method was shown to be reproducible and reliable with intra-day precision below 5.3%, inter-day precision below 14.2%, and linear range from 0.02 to 2 lag/mL with r〉0.994. The method was successfully applied to a pharmacokinetic study of PSN-1 in rats after intravenous administration.展开更多
AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expres...AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia coli.Site directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and purified.Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42.These interactions were quanti- fied using surface plasmon resonance measurements.Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and purification.It is also capable of binding to CDC42,as judged by crosslinking experiments.Interaction appears to be strongest in the presence of added GTP.The three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild type.This was caused largely by a dramatic reduction in the associa- tion rate constant.In contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type.The cancer-associated variant,M1231I, also had a similar affinity to wild type.However,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately 10-fold.Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type.One such region is a loop linking the proposed CDC42 bind- ing site with the helix containing the altered residue.It is suggested that this increase in rigidity is responsible for the observed changes in association and dissocia- tion rate constants. CONCLUSION:The consequences of introducing nega- tive charge at Ser-1441 or Ser-1443 in IQGAP1 are dif- ferent.The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.展开更多
The development of photocatalytic technology has grown significantly since its initial report and as such,a number of screening methods have been developed to assess activity. In the field of environmental remediation...The development of photocatalytic technology has grown significantly since its initial report and as such,a number of screening methods have been developed to assess activity. In the field of environmental remediation, a crucial factor is the formation of highly oxidising species such as OH radicals. These radicals are often the primary driving force for the removal and breakdown of organic and inorganic contaminants. The quantification of such compounds is challenging due to the nature of the radical,however indirect methods which deploy a chemical probe to essentially capture the radical have been shown to be effective. As discussed in the work presented here, optimisation of such a method is fundamental to the efficiency of the method. A starting concentration range of coumarin from 50 mmol/L to 1000 mmol/L was used along with a catalyst loading of 0.01 g/L to 1 g/L Ti TiO2 to identify that 250 mmol/L and 0.5 g/L Ti TiO2 were the optimum conditions for production. Under these parameters a maximum production rate of 35.91 mmol/L（Rmax= 0.4 mmol/L OH＊ min-1） was achieved which yielded at photonic efficiency of 4.88 OH＊moles photon-1 under UV irradiation. The data set presented also highlighted the limitations which are associated with the method which included; rapid exhaustion of the probe molecule and process inhibition through UV light saturation. Identifying both the optimum conditions and the potential limitations of the process were concluded to be key for the efficient deployment of the photocatalytic screening method.展开更多
Globally,the Rising Plate Meter(RPM)is a device used to measure compressed sward height,to enable estimation of herbage mass.Despite improved farm management practices aided by a variety of technological advances,the ...Globally,the Rising Plate Meter(RPM)is a device used to measure compressed sward height,to enable estimation of herbage mass.Despite improved farm management practices aided by a variety of technological advances,the standard design of a RPM has remained relatively unchanged.Recently,however,a RPM utilising a micro-sonic sensor,with digital data capture capability via a Bluetooth communications link to a smart device application,has been developed.Here,we assess the comparable ability of both a standard cumulative ratchet counter RPM and the micro-sonic sensor RPM,to accurately and precisely measure fixed heights.Moreover,as correct allocation of grazing area requires accurate geolocation positioning,we assess the associated GPS technology.The micro-sonic sensor RPM was significantly more accurate for height capture than the cumulative ratchet counter RPM.Overall,across all heights,the cumulative ratchet counter RPM underestimated height by 7.68±0.06mm(mean±SE).Alternatively,the micro-sonic sensor RPM overestimated height by 0.18±0.08 mm.In relation to a practical applications,these discrepancies can result in an under-and overestimation of dry matter yield by 13.71%and 0.32%kilograms per hectare,respectively.The performance of the on-board GPS did not significantly differ from that of a tertiary device.Overall,the wireless technology,integrated mapping,and decision support tools offered by the innovative micro-sonic sensor RPM provides for a highly efficacious grassland management tool.展开更多
Background:Overcrowding in Emergency Departments(EDs)is a significant global concern with negative consequences for patients,healthcare staff,and healthcare systems.The use of EDs by parents of children with non-urgen...Background:Overcrowding in Emergency Departments(EDs)is a significant global concern with negative consequences for patients,healthcare staff,and healthcare systems.The use of EDs by parents of children with non-urgent conditions is associated with overcrowding,higher healthcare costs,lower quality of care,and longer waiting times.Research in this area has largely been conducted in high-income countries,with a dearth of work originating in middle and low-income regions.The aim of this study was to explore the reasons for parents attending EDs with their child for non-urgent conditions in Turkey.Method:Semi-structuredinterviews wereconductedwith 13parents,15 EDstaff,and10GeneralPractitioners(GPs)in2 regions of Turkey between March and May 2017.Data were analyzed using the principles of grounded theory.Results:The findings were classified into 5 core categories:(1)parents’feelings,knowledge,and perceived inability to provide self-care;(2)perceived limitationsofhealthcare services,system,and staff;(3)parents’preferencesforhospital andED services;(4)adverse impact on ED services;and(5)perceived needs for care.Conclusion:This is the first study conducted in a middle-income country regarding parental reasons for using the ED for non-urgent conditions.Greater efforts must be made to reduce unnecessary visits to the ED to better meet service user needs and to increase the satisfaction of both parents and healthcare staff.The findings of this study may inform healthcare providers,policymakers,healthcare staff,and researchers to design interventions in order to mitigate overcrowding in the ED.展开更多
文摘We described the first results of a quantitative ultra performance liquid chromatographytandem mass spectrometry method for a novel antimicrobial peptide （phylloseptin, PSN-1）. Chromatographic separation was accomplished on a Waters bridged ethyl hybrid （BEH） C18 （50mm× 2.1 mm, 1.7 μm） column with acetonitrile-water （25：75, v/v） as isocratic mobile phase. Mass spectrometry detection was performed in the positive electrospray ionization mode and by monitoring of the transitions at m/z 679.6/120, 509.6/120 （PSN-1） and m/z 340.7/165 （Thymopentin, IS）. Protein precipitation was investigated and the recovery was satisfactory （above 82%）. The method was shown to be reproducible and reliable with intra-day precision below 5.3%, inter-day precision below 14.2%, and linear range from 0.02 to 2 lag/mL with r〉0.994. The method was successfully applied to a pharmacokinetic study of PSN-1 in rats after intravenous administration.
基金Supported by The Biotechnology and Biological Sciences Research Council(BBSRC),United Kingdom,No.BB/D000394/1Action Cancer,Northern Ireland,United Kingdom,No.PG2 2005
文摘AIM:To understand the interaction of human IQGAP1 and CDC42,especially the effects of phosphorylation and a cancer-associated mutation. METHODS:Recombinant CDC42 and a novel C-termi- nal fragment of IQGAP1 were expressed in,and puri- fied from,Escherichia coli.Site directed mutagenesis was used to create coding sequences for three phos- phomimicking variants(S1441E,S1443D and S1441E/ S1443D)and to recapitulate a cancer-associated mu- tation(M1231I).These variant proteins were also ex- pressed and purified.Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42.These interactions were quanti- fied using surface plasmon resonance measurements.Molecular modelling was employed to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant. RESULTS:The novel,C-terminal region of human IQGAP1 (residues 877-1558)is soluble following expression and purification.It is also capable of binding to CDC42,as judged by crosslinking experiments.Interaction appears to be strongest in the presence of added GTP.The three phosphomimicking mutants had different affini- ties for CDC42.S1441E had an approximately 200-fold reduction in affinity compared to wild type.This was caused largely by a dramatic reduction in the associa- tion rate constant.In contrast,both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type.The cancer-associated variant,M1231I, also had a similar affinity to wild type.However,in the case of this variant,both the association and dis- sociation rate constants were reduced approximately 10-fold.Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region,revealed no gross structural changes compared to wild type(root mean square deviation of 0.564over 5556 equivalent atoms).However,pre- dictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type.One such region is a loop linking the proposed CDC42 bind- ing site with the helix containing the altered residue.It is suggested that this increase in rigidity is responsible for the observed changes in association and dissocia- tion rate constants. CONCLUSION:The consequences of introducing nega- tive charge at Ser-1441 or Ser-1443 in IQGAP1 are dif- ferent.The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.
基金the financial support of Northern Irelands Department of Education and Learning for funding Caitlin Buck’s Ph DQueen’s University Belfast Pioneering Research Programme (PRP) for funding the research of Dr Nathan Skillen
文摘The development of photocatalytic technology has grown significantly since its initial report and as such,a number of screening methods have been developed to assess activity. In the field of environmental remediation, a crucial factor is the formation of highly oxidising species such as OH radicals. These radicals are often the primary driving force for the removal and breakdown of organic and inorganic contaminants. The quantification of such compounds is challenging due to the nature of the radical,however indirect methods which deploy a chemical probe to essentially capture the radical have been shown to be effective. As discussed in the work presented here, optimisation of such a method is fundamental to the efficiency of the method. A starting concentration range of coumarin from 50 mmol/L to 1000 mmol/L was used along with a catalyst loading of 0.01 g/L to 1 g/L Ti TiO2 to identify that 250 mmol/L and 0.5 g/L Ti TiO2 were the optimum conditions for production. Under these parameters a maximum production rate of 35.91 mmol/L（Rmax= 0.4 mmol/L OH＊ min-1） was achieved which yielded at photonic efficiency of 4.88 OH＊moles photon-1 under UV irradiation. The data set presented also highlighted the limitations which are associated with the method which included; rapid exhaustion of the probe molecule and process inhibition through UV light saturation. Identifying both the optimum conditions and the potential limitations of the process were concluded to be key for the efficient deployment of the photocatalytic screening method.
文摘Globally,the Rising Plate Meter(RPM)is a device used to measure compressed sward height,to enable estimation of herbage mass.Despite improved farm management practices aided by a variety of technological advances,the standard design of a RPM has remained relatively unchanged.Recently,however,a RPM utilising a micro-sonic sensor,with digital data capture capability via a Bluetooth communications link to a smart device application,has been developed.Here,we assess the comparable ability of both a standard cumulative ratchet counter RPM and the micro-sonic sensor RPM,to accurately and precisely measure fixed heights.Moreover,as correct allocation of grazing area requires accurate geolocation positioning,we assess the associated GPS technology.The micro-sonic sensor RPM was significantly more accurate for height capture than the cumulative ratchet counter RPM.Overall,across all heights,the cumulative ratchet counter RPM underestimated height by 7.68±0.06mm(mean±SE).Alternatively,the micro-sonic sensor RPM overestimated height by 0.18±0.08 mm.In relation to a practical applications,these discrepancies can result in an under-and overestimation of dry matter yield by 13.71%and 0.32%kilograms per hectare,respectively.The performance of the on-board GPS did not significantly differ from that of a tertiary device.Overall,the wireless technology,integrated mapping,and decision support tools offered by the innovative micro-sonic sensor RPM provides for a highly efficacious grassland management tool.
基金PhD funding was provided by the Republic of Turkey’s Ministry of National Education.
文摘Background:Overcrowding in Emergency Departments(EDs)is a significant global concern with negative consequences for patients,healthcare staff,and healthcare systems.The use of EDs by parents of children with non-urgent conditions is associated with overcrowding,higher healthcare costs,lower quality of care,and longer waiting times.Research in this area has largely been conducted in high-income countries,with a dearth of work originating in middle and low-income regions.The aim of this study was to explore the reasons for parents attending EDs with their child for non-urgent conditions in Turkey.Method:Semi-structuredinterviews wereconductedwith 13parents,15 EDstaff,and10GeneralPractitioners(GPs)in2 regions of Turkey between March and May 2017.Data were analyzed using the principles of grounded theory.Results:The findings were classified into 5 core categories:(1)parents’feelings,knowledge,and perceived inability to provide self-care;(2)perceived limitationsofhealthcare services,system,and staff;(3)parents’preferencesforhospital andED services;(4)adverse impact on ED services;and(5)perceived needs for care.Conclusion:This is the first study conducted in a middle-income country regarding parental reasons for using the ED for non-urgent conditions.Greater efforts must be made to reduce unnecessary visits to the ED to better meet service user needs and to increase the satisfaction of both parents and healthcare staff.The findings of this study may inform healthcare providers,policymakers,healthcare staff,and researchers to design interventions in order to mitigate overcrowding in the ED.
