RESUMEN
The aim of this study was to evaluate transcriptional changes in sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis was removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis was obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time QPCR and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of pro-inflammatory transcripts interleukin 1 α (IL1A), interleukin1 ß (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), and caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA) and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.
RESUMEN
Podermatitis aseptica hemorrhagica circumscripta is associated with metalloproteinase 2 weakening of distal phalangeal suspensory structures and sinkage of the distal phalanx in the claw capsule. Pressure from the tuberculum flexorium on the sole epidermis and dermis produces hemorrhagic tissue injury and defective horn production appearing as yellow-red, softened claw horn in region 4 of the sole. A model of the MAPK/ERK signal cascade orchestrating epidermal-dermal homeostasis was employed to determine if sterile inflammatory responses are linked to disturbed signal transduction for epidermal homeostasis in sole epidermis and dermis. The objective was to assess shifts in target genes of inflammation, up- and downstream MAPK/ERK signal elements, and targeted genes supporting epidermal proliferation and differentiation. Sole epidermis and dermis was removed from lateral claws bearing lesions of podermatitis aseptica hemorrhagica circumscripta, medial claws from the same limb and lateral claws from completely normal limbs of multiparous, lactating Holstein cows. The abundance levels of targeted transcripts were evaluated by real-time QPCR. Lesion effects were assessed by ANOVA, and mean comparisons were performed with t-tests to assess variations between mean expression in ulcer-bearing or medial claw dermis and epidermis and completely normal lateral claw dermis and epidermis or between ulcer-bearing dermis and epidermis and medial claw dermis and epidermis. The lesions were sterile and showed losses across multiple growth factors, their receptors, several downstream AP1 transcription components, CMYC, multiple cell cycle and terminal differentiation elements conducted by MAPK/ERK signals and ß 4, α 6 and collagen 17A hemidesmosome components. These losses coincided with increased cytokeratin 6, ß 1 integrin, proinflammatory metalloproteinases 2 and 9, IL1B and physiologic inhibitors of IL1B, the decoy receptor and receptor antagonist. Medial claw epidermis and dermis from limbs with lateral claws bearing podermatitis aseptica hemorrhagica circumscripta showed reductions in upstream MAPK/ERK signal elements and downstream targets that paralleled those in hemorrhagic lesions. Inhibitors of IL1B increased in the absence of real increases in inflammatory targets in the medial claw dermis and epidermis. Losses across multiple signal path elements and downstream targets were associated with negative effects on targeted transcripts supporting claw horn production and wound repair across lesion-bearing lateral claws and lesion-free medial claw dermis and epidermis. It was unclear if the sterile inflammation was causative or a consequence of these perturbations.
RESUMEN
Hypothermia during obstetric spinal anaesthesia is a common and important problem, yet temperature monitoring is often not performed due to the lack of a suitable, cost-effective monitor. This study aimed to compare a noninvasive core temperature monitor with two readily available peripheral temperature monitors during obstetric spinal anaesthesia. We undertook a prospective observational study including elective and emergency caesarean deliveries, to determine the agreement between affordable reusable surface temperature monitors (Welch Allyn SureTemp® Plus oral thermometer and the Braun 3-in-1 No Touch infrared thermometer) and the Dräger T-core© (using dual-sensor heat flux technology), in detecting thermoregulatory changes during obstetric spinal anaesthesia. Predetermined clinically relevant limits of agreement (LOA) were set at ± 0.5 °C. We included 166 patients in our analysis. Hypothermia (heat flux temperature < 36 °C) occurred in 67% (95% CI 49 to 78%). There was poor agreement between devices. In the Bland-Altman analysis, LOA for the heat flux monitor vs. oral thermometer were 1.8 °C (CI 1.7 to 2.0 °C; bias 0.5 °C), for heat flux monitor vs. infrared thermometer LOA were 2.3 °C (CI 2.1 to 2.4 °C; bias 0.4 °C) and for infrared vs. oral thermometer, LOA were 2.0 °C (CI 1.9 to 2.2 °C; bias 0.1 °C). Error grid analysis highlighted a large amount of clinical disagreement between methods. While monitoring of core temperature during obstetric spinal anaesthesia is clinically important, agreement between monitors was below clinically acceptable limits. Future research with gold-standard temperature monitors and exploration of causes of sensor divergence is needed.
RESUMEN
South Africa is classified as a low- and middle-income country, with a complex mixture of resource-rich and resource-limited settings. In the major referral hospitals, the necessary skill level exists for the management of complex challenges. However, this contrasts with the frequently-inadequate skill levels of anaesthesia practitioners in resource-limited environments. In Japan, obstetricians administer anaesthesia for 40% of caesarean deliveries and 80% of labour analgesia. Centralisation of delivery facilities is now occurring and it is expected that obstetric anaesthesiologists will be available 24â¯h a day in centralised facilities in the future. In China, improvements in women's reproductive, maternal, neonatal, child, and adolescent health are critical government policies. Obstetric anaesthesia, especially labour analgesia, has received unprecedented attention. Chinese obstetric anaesthesiologists are passionate about clinical research, focusing on efficacy, safety, and topical issues. The Latin-American region has different landscapes, people, languages, and cultures, and is one of the world's regions with the most inequality. There are large gaps in research, knowledge, and health services, and the World Federation of Societies of Anaesthesiologists is committed to working with governmental and non-governmental organisations to improve patient care and access to safe anaesthesia. Anaesthesia workforce challenges, exacerbated by coronavirus disease 2019, beset North American healthcare. Pre-existing struggles by governments and decision-makers to improve health care access remain, partly due to unfamiliarity with the role of the anaesthesiologist. In addition to weaknesses in work environments and dated standards of work culture, the work-life balance demanded by new generations of anaesthesiologists must be acknowledged.
Asunto(s)
Anestesia Obstétrica , COVID-19 , Embarazo , Adolescente , Recién Nacido , Niño , Humanos , Femenino , América Latina , Japón , Sudáfrica , China , América del NorteRESUMEN
Dihydrofolate reductase (DHFR) is a well-studied, clinically relevant enzyme known for being highly dynamic over the course of its catalytic cycle. However, the role dynamic motions play in the explicit hydride transfer from the nicotinamide cofactor to the dihydrofolate substrate remains unclear because reaction initiation and direct spectroscopic examination on the appropriate time scale for such femtosecond to picosecond motions is challenging. Here, we employ pre-steady-state kinetics to observe the hydride transfer as directly as possible in two different species of DHFR: Escherichia coli and Homo sapiens. While the hydride transfer has been well-characterized in DHFR from E. coli, improvements in time resolution now allow for sub-millisecond dead times for stopped-flow spectroscopy, which reveals that the maximum rate is indeed faster than previously recorded. The rate in the human enzyme, previously only estimated, is also able to be directly observed using cutting-edge stopped-flow instrumentation. In addition to the pH dependence of the hydride transfer rates for both enzymes, we examine the primary H/D kinetic isotope effect to reveal a temperature dependence in the human enzyme that is absent from the E. coli counterpart. This dependence, which appears above a temperature of 15 °C is a shared feature among other hydride transfer enzymes and is also consistent with computational work suggesting the presence of a fast promoting-vibration that provides donor-acceptor compression on the time scale of catalysis to facilitate the chemistry step.
Asunto(s)
Escherichia coli , Tetrahidrofolato Deshidrogenasa , Catálisis , Escherichia coli/metabolismo , Humanos , Cinética , Modelos Moleculares , Niacinamida , Conformación Proteica , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
The solar conversion of CO2 to low carbon fuels has been heralded as a potential solution to combat the rise in greenhouse gas emissions. Here we report the first light-driven activation of [NiFe] CODH II from Carboxydothermus hydrogenoformans for the reduction of CO2 to CO. To accomplish this, a hybrid photosystem composed of CODH II and CdSe/CdS dot-in-rod nanocrystals was developed. By incorporating a low-potential redox mediator to assist electron transfer, quantum yields up to 19% and turnover frequencies of 9 s-1 were achieved. These results represent a new standard in efficient CO2 reduction by an enzyme-based photocatalytic systems. Furthermore, successful photoactivation of CODH II allows for future exploration into the enzyme's not fully understood mechanism.
Asunto(s)
Compuestos de Cadmio , Nanotubos , Compuestos de Selenio , Aldehído Oxidorreductasas , Dióxido de Carbono , Monóxido de Carbono/química , Complejos MultienzimáticosRESUMEN
[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H2 binding site and a diiron cluster ligated by CO, CN-, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S]H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.
Asunto(s)
Chlamydomonas reinhardtii , Hidrogenasas , Chlamydomonas reinhardtii/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Hidrógeno/química , Concentración de Iones de Hidrógeno , Hidrogenasas/química , Ligandos , Protones , Análisis EspectralRESUMEN
In single-molecule force spectroscopy (SMFS), a tethered molecule is stretched using a specialized instrument to study how macromolecules extend under force. One problem in SMFS is the serial and slow nature of the measurements, performed one molecule at a time. To address this long-standing challenge, we report on the origami polymer force clamp (OPFC) which enables parallelized manipulation of the mechanical forces experienced by molecules without the need for dedicated SMFS instruments or surface tethering. The OPFC positions target molecules between a rigid nanoscale DNA origami beam and a responsive polymer particle that shrinks on demand. As a proof-of-concept, we record the steady state and time-resolved mechanical unfolding dynamics of DNA hairpins using the fluorescence signal from ensembles of molecules and confirm our conclusion using modeling.
Asunto(s)
ADN/química , Polímeros/química , Imagen Individual de Molécula , Temperatura , Fenómenos Ópticos , Tamaño de la PartículaRESUMEN
Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre- to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Internalización del Virus , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/patología , Gripe Humana/virología , Conformación Proteica , Dominios Proteicos , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump-probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116-132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.
Asunto(s)
Nanopartículas del Metal/química , Modelos Moleculares , Tetrahidrofolato Deshidrogenasa/química , Catálisis , Dominio Catalítico/efectos de la radiación , Oro/química , Calefacción/efectos adversos , Cinética , Nanopartículas del Metal/efectos de la radiación , Conformación Proteica/efectos de la radiación , Tetrahidrofolato Deshidrogenasa/ultraestructuraRESUMEN
BACKGROUND: In South Africa, hypertensive disorders of pregnancy are the leading cause of maternal mortality. More than 50% of anaesthesia-related maternal deaths are attributed to complications of airway management. We compared the prevalence and risk factors for hypoxaemia during induction of general anaesthesia in parturients with and without hypertensive disorders of pregnancy. We hypothesised that hypertensive disorders of pregnancy are associated with desaturation during tracheal intubation. METHODS: Data from 402 cases in a multicentre obstetric airway management registry were analysed. The prevalence of peri-induction hypoxaemia (SpO2 <90%) was compared in patients with and without hypertensive disorders of pregnancy. Quantile regression of SpO2 nadir was performed to identify confounding variables associated with, and mediators of, hypoxaemia. RESULTS: In the cohort of 402 cases, hypoxaemia occurred in 19% with and 9% without hypertension (estimated risk difference, 10%; 95% CI 2% to 17%; P=0.005). Quantile regression demonstrated a lower SpO2 nadir associated with hypertensive disorders of pregnancy as body mass index increased. Room-air oxygen saturation, Mallampati grade, and number of intubation attempts were associated with the relationship. CONCLUSIONS: Clinically significant oxygen desaturation during airway management occurred twice as often in patients with hypertensive disorders of pregnancy, compounded by increasing body mass index. Intermediary factors in the pathway from hypertension to hypoxaemia were also identified.
Asunto(s)
Hipertensión Inducida en el Embarazo , Manejo de la Vía Aérea , Femenino , Humanos , Hipertensión Inducida en el Embarazo/epidemiología , Hipoxia/epidemiología , Intubación Intratraqueal , Saturación de Oxígeno , Embarazo , Sistema de RegistrosRESUMEN
The fabrication of dynamic, transformable biomaterials that respond to environmental cues represents a significant step forward in the development of synthetic materials that rival their highly functional, natural counterparts. Here, we describe the design and synthesis of crystalline supramolecular architectures from charge-complementary heteromeric pairs of collagen-mimetic peptides (CMPs). Under appropriate conditions, CMP pairs spontaneously assemble into either 1D ultraporous (pore diameter >100 nm) tubes or 2D bilayer nanosheets due to the structural asymmetry that arises from heteromeric self-association. Crystalline collagen tubes represent a heretofore unobserved morphology of this common biomaterial. In-depth structural characterization from a suite of biophysical methods, including TEM, AFM, high-resolution cryo-EM, and SAXS/WAXS measurements, reveals that the sheet and tube assemblies possess a similar underlying lattice structure. The experimental evidence suggests that the tubular structures are a consequence of the self-scrolling of incipient 2D layers of collagen triple helices and that the scrolling direction determines the formation of two distinct structural isoforms. Furthermore, we show that nanosheets and tubes can spontaneously interconvert through manipulation of the assembly pH and systematic adjustment of the CMP sequence. Altogether, we establish initial guidelines for the construction of dynamically responsive 1D and 2D assemblies that undergo a structurally programmed morphological transition.
Asunto(s)
Colágeno/química , Nanoestructuras/química , Péptidos/química , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Nanotubos/química , PorosidadRESUMEN
Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediator-catalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core-shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from -550 to -670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.
Asunto(s)
Hidrogenasas , Proteínas Hierro-Azufre , Transporte de Electrón , Electrones , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Rayos Láser , Oxidación-Reducción , OxidorreductasasRESUMEN
BACKGROUND AND PURPOSE: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is associated with stroke. The role of sex on stroke outcome has not been investigated. To objective of this paper is to describe the characteristics of a diverse cohort of acute stroke patients with COVID-19 disease and determine the role of sex on outcome. METHODS: This is a retrospective study of patients with acute stroke and SARS-CoV-2 infection admitted between March 15 to May 15, 2020 to one of the six participating comprehensive stroke centers. Baseline characteristics, stroke subtype, workup, treatment and outcome are presented as total number and percentage or median and interquartile range. Outcome at discharge was determined by the modified Rankin Scale Score (mRS). Variables and outcomes were compared for males and females using univariate and multivariate analysis. RESULTS: The study included 83 patients, 47% of which were Black, 28% Hispanics/Latinos, and 16% whites. Median age was 64 years. Approximately 89% had at least one preexisting vascular risk factor (VRF). The most common complications were respiratory failure (59%) and septic shock (34%). Compared with females, a higher proportion of males experienced severe SARS-CoV-2 symptoms requiring ICU hospitalization (73% vs. 49%; pâ¯=â¯0.04). When divided by stroke subtype, there were 77% ischemic, 19% intracerebral hemorrhage and 3% subarachnoid hemorrhage. The most common ischemic stroke etiologies were cryptogenic (39%) and cardioembolic (27%). Compared with females, males had higher mortality (38% vs. 13%; pâ¯=â¯0.02) and were less likely to be discharged home (12% vs. 33%; pâ¯=â¯0.04). After adjustment for age, race/ethnicity, and number of VRFs, mRS was higher in males than in females (ORâ¯=â¯1.47, 95% CIâ¯=â¯1.03-2.09). CONCLUSION: In this cohort of SARS-CoV-2 stroke patients, most had clinical evidence of coronavirus infection on admission and preexisting VRFs. Severe in-hospital complications and worse outcomes after ischemic strokes were higher in males, than females.
Asunto(s)
Isquemia Encefálica/epidemiología , Infecciones por Coronavirus/epidemiología , Disparidades en el Estado de Salud , Hemorragias Intracraneales/epidemiología , Neumonía Viral/epidemiología , Accidente Cerebrovascular/epidemiología , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/terapia , COVID-19 , Chicago/epidemiología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/terapia , Femenino , Humanos , Hemorragias Intracraneales/diagnóstico , Hemorragias Intracraneales/terapia , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/terapia , Pronóstico , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/terapia , Factores de TiempoRESUMEN
BACKGROUND: Postoperative nausea and vomiting is one of the most common anaesthetic complications of caesarean section. This study examined the association between hyperemesis gravidarum during pregnancy and nausea and vomiting after caesarean section. METHODS: A single-centre, retrospective cohort study, using electronic databases of patients with and without hyperemesis gravidarum, undergoing caesarean section from 2015 to 2019. The incidence and severity of postoperative nausea and vomiting were established by a review of the documentation of administration of postoperative anti-emetics within the 24-h period after surgery, and examined using univariable, multivariable binary and ordered logistic regression models. RESULTS: Data were compared for 76 patients with hyperemesis gravidarum and 315 patients without the condition. The incidence of postoperative nausea and vomiting in the hyperemesis group versus the non-hyperemesis group was 43.4% vs 29.6%, respectively. The odds of experiencing postoperative nausea and vomiting was 1.95 times higher in women with hyperemesis gravidarum than in those without (aOR 1.95, 95% CI 1.13 to 3.36, P=0.016). The odds of having more severe postoperative nausea and vomiting were greater in the hyperemesis gravidarum group (aOR 1.91, 95% CI 1.14 to 3.20, P=0.014). CONCLUSION: Patients with hyperemesis gravidarum are more likely to develop nausea and vomiting after caesarean section, and this is likely to be of greater severity than in those without the condition. This finding should assist the effective provision of intra-operative and postoperative anti-emetics for patients with hyperemesis gravidarum undergoing caesarean section.
Asunto(s)
Cesárea , Hiperemesis Gravídica/epidemiología , Náusea y Vómito Posoperatorios/epidemiología , Adulto , Estudios de Cohortes , Comorbilidad , Femenino , Humanos , Incidencia , Embarazo , Estudios Retrospectivos , Índice de Severidad de la EnfermedadRESUMEN
Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan-tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Tetrahidrofolato Deshidrogenasa/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutación , Conformación Proteica , Espectrometría de Fluorescencia , Tetrahidrofolato Deshidrogenasa/genética , Triptófano/química , Triptófano/genéticaRESUMEN
This study reports how the length of capping ligands on a nanocrystal surface affects its interfacial electron transfer (ET) with surrounding molecular electron acceptors, and consequently, impact the H2 production of a biotic-abiotic hybrid artificial photosynthetic system. Specifically, we study how the H2 production efficiency of a hybrid system, combining CdS nanorods (NRs), [NiFe] hydrogenase, and redox mediators (propyl-bridged 2,2'-bipyridinium, PDQ2+), depends on the alkyl chain length of mercaptocarboxylate ligands on the NR surface. We observe a minor decrease of the quantum yield for H2 production from 54 ± 6 to 43 ± 2% when varying the number of methylene units in the ligands from 2 to 7. In contrast, an abrupt decrease of the yield was observed from 43 ± 2 to 4 ± 1% when further increasing n from 7 to 11. ET studies reveal that the intrinsic ET rates from the NRs to the electron acceptor PDQ2+ are all within 108-109 s-1 regardless of the length of the capping ligands. However, the number of adsorbed PDQ2+ molecules on NR surfaces decreases dramatically when n ≥ 10, with the saturating number changing from 45 ± 5 to 0.3 ± 0.1 for n = 2 and 11, respectively. These results are not consistent with the commonly perceived exponential dependence of ET rates on the ligand length. Instead, they can be explained by the change of the accessibility of NR surfaces to electron acceptors from a disordered "liquid" phase at n < 7 to a more ordered "crystalline" phases at n > â¼7. These results highlight that the order of capping ligands is an important design parameter for further constructing nanocrystal/molecular assemblies in broad nanocrystal-based applications.
Asunto(s)
Anestesia Obstétrica , Anestesia Raquidea , Hipotensión , Cesárea , Femenino , Humanos , EmbarazoRESUMEN
Epidemiological data establish that lameness is second only to mastitis as the dairy industry's most prevalent and costly animal welfare issue. Using an automatic lameness detection (ALD) system in which continuous, accurate detection is coupled with proper treatment, is key for reducing economic losses due to lameness. It is reasonable to assume that the cost of lameness would vary with its severity. Therefore, our first objective was to estimate the cost of different lameness severity levels as a function of milk production, lameness risk, conception probability, and treatment cost using a dynamic programming (DP) model. Our second objective was to conduct a cost benefit analysis for ALD systems which can reduce production losses through early detection and treatment of lameness, when compared to visual-detection (VD; i.e., performed by humans) systems. The default production loss parameters for the VD system used as inputs to the DP model were either sourced from the literature or were estimated based on data from a field trial. The production loss parameters for the ALD system used as inputs to the DP model were based on extrapolations of parameter values used for the VD system. The profit per present cow per year under assumed expenses and revenues decreased from $426.05 (when lameness incidence was assumed to be 0%) to $389.69 when lameness incidence was 19.5 %. Out of the 19.5 % lameness incidence in our default scenario, 9.8 % were moderate cases and 9.7 % were severe cases. Average cost of lameness was $36.36 at 19.5 % incidence. Average cost of lameness increased with increased incidence and was respectively $82.05, $195.05, and $286.87 at the low, medium, and high incidence scenarios. We used an operational framework which compared the lameness costs between the VD and ALD systems with 25 %, 50 % and 75 % net avoided costs (NAC) for the 10 year lifespan of the ALD system, at default, low, medium and high lameness incidence scenarios. The net return per cow per year from using an ALD system over a VD system was $13, at low incidence and 25 % NAC. The net return per cow per year for the ALD system was as high as $99 at high incidence and 75 % NAC. Out of 351 (3 system prices, 3 system efficiencies, 3 levels of lameness incidence and 13 different herd sizes) scenarios tested, 295 resulted in a net profit within the system lifespan of 10 years, thus justifying the investment in ALD systems.