Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 °C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes

Effects of mutations in the helix G region of horseradish peroxidase

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F′ (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state Vm/E values occurred with reducing substrate ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site

Tranexamic acid in bleeding trauma patients: an exploration of benefits and harms.

BACKGROUND: The CRASH-2 trial showed that tranexamic acid (TXA) administration reduces mortality in bleeding trauma patients. However, the effect appeared to depend on how soon after injury TXA treatment was started. Treatment within 3 h reduced bleeding deaths whereas treatment after 3 h increased the risk. We examine how patient characteristics vary by time to treatment and explore whether any such variations explain the time-dependent treatment effect. METHODS: Exploratory analysis were carried out, including per-protocol analyses, of data from the CRASH-2 trial, a randomised placebo-controlled trial of the effect of TXA on mortality in 20,211 trauma patients with, or at risk of, significant bleeding. We examine how patient characteristics (age, type of injury, presence or absence of head injury, Glasgow coma scale (GCS), systolic blood pressure and capillary refill time) vary with time to treatment and use univariable (restriction) and multivariable methods to examine whether any such variations explain the time-dependent effect of TXA. If not explained by differences in patient characteristics, we planned to conduct separate prespecified subgroup analyses for the early benefit and late harm. RESULTS: There was no substantial variation in age or capillary refill by time to treatment. However, the proportion of patients with blunt trauma, the proportion with head injury and mean systolic blood pressure increased as time to treatment increased. Mean GCS decreased as time to treatment increased. Analyses restricted to patients with blunt trauma, those without head injury and those with a systolic blood pressure 89 mmHg); GCS (severe 3-8, moderate 9-12, mild 13-15); and type of injury (penetrating versus blunt) showed no significant heterogeneity. CONCLUSIONS: The time-dependent effect of TXA in bleeding trauma patients is not explained by the type of injury, the presence or absence of head injury or systolic blood pressure. When given within 3 h of injury, TXA reduces death due to bleeding regardless of type of injury, GCS or blood pressure. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00375258 . Registered on 11 September 2006

Operational Experience and Control Strategies for a Stand-Alone Power System based on Renewable Energy and Hydrogen

The topic of this thesis is investigation of a small-scale stand-alone power system, based on both experimental work and computer simulations. The power system in this study uses solar energy as energy input, lead-acid batteries as short-term energy storage, and hydrogen as long-term energy storage. The main focus is upon operation and control of the hydrogen subsystem, as a robust controller is needed in order to prevent excessive use of the components in this subsystem. The laboratory power system comprises of: Hydrogen subsystem (PEM electrolyser, metal hydride, and PEM fuel cell), a lead-acid battery, programmable power supply for emulation of PV arrays, wind turbines, and controlled characterisation of the individual system components, and a programmable electronic load. The intention was to build the laboratory power system as simple and energy efficient as possible. The components were connected directly in parallel on a common 48 V DC bus bar, no power electronics were applied between the components. Furthermore, the metal hydride and the fuel cell were air-cooled, avoiding auxiliaries required for water- cooling. The electrolyser, however, needed water-cooling. But with the electrolyser delivering hydrogen at 16 bars to a low pressure metal hydride, no use of compressor was required. On the other hand, metal hydrides needs purified hydrogen gas, > 99.999 %, in order to maintain its capacity as specified by the manufacturer. The actual work in this thesis is divided in three main parts: 1. Design, construction, and operation of a laboratory hydrogen power system 2. Establishment of a computer model of the laboratory hydrogen power system, which interpolates and extrapolates its outputs based on experimental data collected from the laboratory system 3. Establishment of control algorithms for high-level energy management of the laboratory hydrogen power system based on the developed computer model. It is a goal to make the implementation and maintenance of these control algorithms as simple as possible. Furthermore, the control algorithms must enable efficient usage of the system components and secure energy supply to the end user The results of this thesis are divided in two main parts: The first part of the main results relates to the proposal and development of two types of control algorithms for high-level energy management, which will be denoted as the Control Matrix and the Fuzzy controller in the thesis. These control algorithms are suggested as opposed to the more traditional battery five-step charge controller. Identification of important system parameters and choosing proper settings for control parameters must be implemented into the control algorithms in order to finalise a complete control strategy. It will be shown that the electrolyser annual runtime decreases while the electrolyser annual hydrogen production remains the same by using the proposed control strategies, thus running the electrolyser more efficient. Furthermore, with a reduction in the total number of electrolyser start-ups, a more stable system operation is achieved. The second part of the main results relates to the operational experience of the small-scale laboratory hydrogen power system. Due to the amount of power required by the local control system integrated into the fuel cell and the electrolyser, the energy efficiency of the fuel cell and the electrolyser is lower at partial loads. Thus, with the additional energy needed for hydrogen purification, the round-trip efficiency of the hydrogen subsystem is found to be rather low (< 30 %), when the fuel cell and the electrolyser runs at low partial loads. However, it is encouraging that the hydrogen subsystem can reach 35 – 40 % when the fuel cell and the electrolyser are allowed to run at nominal power levels, in addition to optimal arrangement of the hydrogen purification unit. These energy efficiencies are higher than efficiencies achieved with diesel-fuelled generators. Besides, stand-alone power systems often resides in remote areas where transportation of diesel is costly, thus local production of the fuel by means of electrolyser and excess renewable energy can be profitable. Regarding the difficulty of measuring the true amount of hydrogen present in the metal hydride, and because this system parameter is important in the control strategy, a pressurised vessel is recommended instead of the air-cooled metal hydride. Furthermore, it is recommended to use DC/DC converters in the hydrogen power system in order to ensure power quality within specifications and robust operation.dr.ing.dr.ing

Stability characteristics and applications of native and chemically-modified horseradish peroxidases

Due to its inherent stability, ease of handling and availability, horseradish peroxidase (HRP) will continue to be used more extensively in analytical and industrial situations. It is often used as a model for other peroxidases. Developments in the fields of protein stabilisation and biosensor construction are discussed in Chapter one; also, various enzymatic methods for treating phenolic effluents are reviewed. The effects of chemical modifiers on native HRP were investigated (Chapter two). Homobifunctional crosslinkers specific for lysine residues were employed. No loss of enzyme activity occured on reaction with such Nhydroxysuccinimide (NHS) compounds. Derivative forms of HRP displayed greater thermostability and a greater tolerance of water-miscible organic solvents. Enhanced resistance towards dénaturants was noted. Structural changes in the vicinity of the heme of HRP derivatives were studied by UV/Visible spectrophotometry and fluorimetry. The extent of modification on HRP’s six lysines has been determined. The NHS derivatives of HRP have also been employed in the removal of phenols from aqueous solution (Chapter three). HRP catalyses the oxidation of toxic aromatic compounds in the presence of hydrogen peroxide. Reaction products polymerise to form high molecular weight materials which can be easily separated from aqueous solution. Modified peroxidases displayed greater removal efficiencies of phenols compared to the native enzyme over a wide range of reaction conditions, including high temperatures. For some pollutants, the efficiency of removal is high. Native HRP has also been used in the development of a biosensor for the selective determination of uric acid (Chapter four). The sensor was found to function efficiently without the necessity for an electron transfer mediator. The mechanism of the sensor’s response was thought to be due to direct electron transfer from the electrode to HRP. A monomer, o-aminophenol, which was electrodeposited at the working surface of the electrode, was found to protect the biocomponents from interferences and fouling. The sensor was incorporated into a flow injection system for the quantification of uric acid in human serum. Recoveries compared favourably with a standard spectrophotometric method

Study on the Application of Lesson Study in Increasing the Effectiveness of Learning Practices at SDN 15 Mataram

In essence, pupils have a right to education. However, a sizable portion of the class still doesn't study. The learning outcomes are not ideal since many pupils do not learn. It is stated that learning is ineffective since a large number of pupils' learning results are subpar. This is why lesson study, a teacher forum, is necessary to enhance learning and make it more efficient. In light of this backdrop, the research's goals are to: a) assess the degree of lesson study implementation at SDN 15 Mataram. b) to evaluate if lesson study has a positive impact on SDN 15 Mataram's instructional methods. There is descriptive qualitative research in this study. Using percentages and values, the information gathered through observation and interviews was examined. The use of lesson study at SDN 15 Mataram can improve learning practices, according to the findings and analysis of the research data, which show that: a) the implementation of lesson study there is adequate

En deskriptiv studie av innmeldte legemiddelhåndteringsavvik i hjemmetjenesten

Bakgrunn og hensikt: Legemiddelbruk kan utgjøre en stor risiko for pasientsikkerheten. Uforsvarlig legemiddelhåndtering er årsaken bak en betydelig andel pasientskader, til tross for at slike avvik i mange tilfeller er mulige å forebygge. Når hjemmetjenesten har ansvaret for håndtering av pasienters legemidler, er både de ansatte og virksomheten ansvarlig for at legemiddelhåndteringen skjer forsvarlig. Hjemmetjenesten skiller seg fra andre deler av helsetjenesten, og pasientene de bistår blir stadig eldre og sykere. Det er gjort lite forskning på legemiddelhåndteringsavvik i kommunehelsetjenesten. Formålet med denne studien var derfor å kategorisere og beskrive legemiddelhåndteringsavvik innmeldt av ansatte i hjemmetjenesten i Trondheim kommune over en bestemt tidsperiode. Det var ikke studiens hensikt å fremskaffe eksakte tall på avviksforekomst, men se på omfanget av de innmeldte avvikene. Videre hensikt var å vurdere riktigheten av registrert legemiddelhåndteringsprosess i avviksmeldingene, samt fordele avvik innen utdeling på bestemte underkategorier. Metode: En deskriptiv, retrospektiv studie ved gjennomgang av innmeldte legemiddelhåndteringsavvik over en toårsperiode (01.01.2022 - 31.12.2023). Det ble brukt en kombinasjon av kvalitativ og kvantitativ metode for å samle inn, analysere og kategorisere avviksmeldingene. En kvalitativ deduktiv dokumentanalyse ble benyttet for å vurdere innholdet i avviksmeldingene opp mot bestemte kriterier, i flere omganger. Data ble videre analysert kvantitativt ved bruk av deskriptiv statistikk i Excel for å kvantifisere og sammenfatte de ulike kategoriene. Resultater: Gjennomgangen av de innmeldte avvikene (n=1099) resulterte i at 11 % ble ekskludert som følge av de ikke var reelle avvik, inneholdt mangelfull informasjon eller var samhandlingsavvik. Omtrent 40 % av de inkluderte avvikene for 2023 var feilregistrert med hensyn til legemiddelhåndteringsprosess. Flere avviksmeldinger var dermed enten mangelfulle eller feilregistrerte. Utdelingsavvik utgjorde 65 % av de inkluderte avvikene for 2023, hvor 7 av 10 av disse omhandlet at legemiddel ikke var gitt. Legemiddel ikke gitt utgjorde nesten halvparten av alle de inkluderte avvikene for 2023. Konklusjon: For å forebygge avvik og sikre pasientsikkerheten i framtiden, er det viktig at hjemmetjenestens ansatte har gode kunnskaper om legemiddelhåndtering og avviksrapportering, og forståelse for ansvaret og viktigheten av dette. Avviksrapportering av god kvalitet er viktig for at hjemmetjenesten kan lære av feil og jobbe videre med forbedrings- og kvalitetsarbeid i virksomheten. Forbedring av opplæring og prosedyrer rundt avviksrapportering og utdeling, virker å være spesielt viktig.Background and aim: The use of medication can pose a significant risk to patient safety, and despite them being preventable in many cases, a substantial portion of patient injuries can be attributed to improper medication management. In situations where home care services are responsible for managing medications for patients, both the staff and the organization in question are accountable for ensuring that the management of medication is carried out safely. In home care services, patients can often present with multimorbidity, and are thus prescribed multiple medications. This could potentially present greater challenges in ensuring patient safety, but despite this fact, limited research is conducted exploring medication management errors in the municipal health care service. The purpose of the study was therefore to categorize and describe medication management errors reported by employees in the home care service in Trondheim muncipality over a specific timeperiod, aiming for an examination of the extent of the reported errors. In addition, a further purpose was to map the actual distribution of errors in the medication management prosess, demonstrating how administration errors were distributed among predefined subcategories. Method: A descriptive, retrospective study was conducted by reviewing reported medication management errors over the timeperiod 01.01.2022 - 31.12.2023, utilizing both qualitative and quantitative methods to collect, analyze and categorize the error reports. To analyze the error reports, a qualitative deductive document analysis was used to evaluate the text against several predefined criteria. The collected data were analyzed quantitatively by the use of descriptive statistics in Excel. Results: Out of the total reported errors (n=1099), 11 % were excluded due to them not being actual errors by not meeting inclusion requirements for the present study. Several of the reported errors contained incomplete information and were incorrectly recorded in terms of medication management process. Accounting for 65 % of the included errors for 2023, the largest proportion of errors was attributed to the process of medication administration. Comprising 7 out of 10 of the errors related to administration and almost half of all included errors for 2023, the most frequent type of error was medication not given. Conclusion: To prevent medication errors and thus ensuring patient safety in the future, it is important that home care staff have sufficient capability and competance regarding medication management and incident reporting. It is important that reported errors maintan a certain quality, enabling them to sufficiently contribute to quality improvement work within the organization. Enhancing training and procedures related to reporting errors and administrating medication, appears to be especially important

Accuracy of Death Certificate Data in Reporting Suicide in the United States

Suicide is one of the leading causes of death in the United States. Death certificates are currently being used by public health professionals and policy makers as a main source of public health surveillance data on suicide. The accuracy of death certificates in tracking and reporting suicides has not been well quantified or evaluated in the United States. Death certificates from other countries have been found to under-report suicide. The purpose of this study was to evaluate the accuracy of death certificates in reporting suicides in the United States. The National Violence Death Reporting System (NVDRS) collects information on all violent deaths from multiple sources including medical records, law enforcement reports, and vital records. Suicide cases recorded in the NVDRS data from 2003 through 2017 were used as a reference standard to evaluate the accuracy of death certificates in reporting suicide in the United States (n = 201,912). Using a multifactorial conceptual framework and a quantitative cross-sectional design, several risk factors were analyzed to determine if they influenced the accurate reporting of suicide on the death certificate. Using a binomial logistic regression model, 13.4% (Nagelkerke R2 = .134) of the variation in accuracy can be attributed to age, race, marital status, education, method of suicide, substance abuse status, year, and state. This study establishes that the death certificate is highly accurate (99.57%) at reporting suicide deaths overall. Therefore, death certificate data can be used as an accurate data source upon which to base public health decisions, interventions, and tracking. It is important that policy and intervention decisions be based on accurate data to effectively and efficiently influence social change