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In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.
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Atmospheric fine particulate matter (PM2.5) can trigger the production of cytotoxic reactive oxygen species (ROS), which can trigger or exacerbate oxidative stress and pulmonary inflammation. We collected 111 daily (â¼24 h) ambient PM2.5 samples within an urban region of North China during four seasons of 2019-2020. PM2.5 samples were examined for carbonaceous components, water-soluble ions, and elements, together with their oxidative potential (represent ROS-producing ability) by DTT assay. The seasonal peak DTTv was recorded in winter (2.86 ± 1.26 nmol min-1 m-3), whereas the DTTm was the highest in summer (40.6 ± 8.7 pmol min-1 µg-1). WSOC displayed the highest correlation with DTT activity (r = 0.84, p < 0.0001), but the influence of WSOC on the elevation of DTTv was extremely negligible. Combustion source exhibited the most significant and robust correlation with the elevation of DTTv according to the linear mixed-effects model result. Source identification investigation using positive matrix factorization displayed that combustion source (36.2%), traffic source (30.7%), secondary aerosol (15.7%), and dust (14.1%) were driving the DTTv, which were similar to the results from the multiple linear regression (MLR) analysis. Backward trajectory analysis revealed that the major air masses originate from local and regional transportation, but PM2.5 OP was more susceptible to the influence of short-distance transport clusters. Discerning the influence of chemicals on health-pertinent attributes of PM2.5, such as OP, could facilitate a deep understanding of the cause-and-effect relationship between PM2.5 and impacts.
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PM2.5 pollution in China has decreased dramatically, but how its health effects change is not clear. There are 120 old industrial cities in China, where the sources, composition, and health effects of PM2.5 may be significantly different with other cities. Huangshi, an old industrial city in central China, underwent intense green transformations from 2015 to 2018. In this study, we collected ambient PM2.5 samples in 2015 and 2018 at an urban site in Huangshi. The average PM2.5 concentration decreased from 83.44 ± 48.04 µg/m3 in 2015 to 68.03 ± 39.41 µg/m3 in 2018. However, the average volume-normalized dithiothreitol (DTTv) of PM2.5 increased from 1.38 ± 0.45 nmol/min/m3 to 2.14 ± 1.31 nmol/min/m3 and the DTT normalized by particulate mass (DTTm) increased from 20.6 ± 10.1 pmol/min/µg to 40.07 ± 21.9 pmol/min/µg, indicating increased exposure risk and inherent toxicity. The increased toxicity of PM2.5 might be related to the increased trace elements (TEs) concentrations. The positive matrix factorization and multiple linear regression methods were employed to quantify the contributions of emission sources to PM2.5 and DTTv. The results showed that the contribution of coal combustion, industry, and dust to PM2.5 decreased significantly from 2015 to 2018, while that of vehicle emission and secondary sources increased. Despite the decreased fraction of coal combustion and industry sources, their contribution to DTTv increased slightly, which was caused by the increased intrinsic toxicity. The increased intrinsic toxicity was possibly caused by increased TEs, such as Pb, Cu, and V. Besides, the contribution of vehicle emission to DTTv also increased. Overall, these results provide valuable insights into the effectiveness of controlling strategies in reducing particulate health impacts in old industrial cities, and stress the necessity of formulating toxicity-oriented controlling strategies, with special attention to TEs from coal combustion and industry sources as well as vehicle emissions.
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Soybean (Glycine max [L.] Merr.) is a valuable oil crop but is also highly susceptible to environmental stress. Thus, developing approaches to enhance soybean stress resistance is vital to soybean yield improvement. In previous studies, transcription factor Alfin has been shown to serve as an epigenetic regulator of plant growth and development. However, no studies on Alfin have yet been reported in soybean. In this study, the endoplasmic reticulum (ER) stress- and reactive oxygen species (ROS)-related GmAlfin09 was identified. Screening of genes co-expressed with GmAlfin09 unexpectedly led to the identification of soybean peroxidase 6 (GmPRDX6). Further analyses revealed that both GmAlfin09 and GmPRDX6 were responsive to ER stress, with GmPRDX6 localizing to the ER under stress. Promoter binding experiments confirmed the ability of GmAlfin09 to bind to the GmPRDX6 promoter directly. When GmAlfin09 and GmPRDX6 were overexpressed in soybean, enhanced ER stress resistance and decreased ROS levels were observed. Together, these findings suggest that GmAlfin09 promotes the upregulation of GmPRDX6, and GmPRDX6 subsequently localizes to the ER, reduces ROS levels, promotes ER homeostasis, and ensures the normal growth of soybean even under ER stress. This study highlights a vital target gene for future molecular breeding of stress-resistant soybean lines.
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An alternative metric to account for particulate matter (PM) composition-based toxicity is the ability of PM-species to generate reactive oxygen species (ROS) and deplete antioxidants, the so-called oxidative potential (OP). Acellular OP assays are the most used worldwide, mainly those based on ascorbic acid (AA) and dithiothreitol (DTT) depletion; OP values are calculated from AA/DTT concentration over time kinetic curves. Since a great variability in OP-DTT and OP-AA values can be found in the literature, the understanding of those factors affecting the kinetic rate of AA and DTT oxidation in the presence of PM-bound species will improve the interpretation of OP values. In this work, a kinetic study of the oxidation rate of AA and DTT driven by species usually found in PM (transition metals and naphthoquinone (NQ)) was carried out. In particular, the influence of the concentration of Cu(II), Fe(II), Fe(III), Mn(II), Mn(III), and 1,4-NQ, and the type of fluid used in the assay (phosphate buffer (PB), phosphate buffer saline (PBS) and artificial lysosomal fluid (ALF)) is analysed and discussed. The reaction orders with respect to the AA/DTT and the active compound, and the kinetic rate constants were also determined. The results show great variability in OP values among the studied species depending on the fluid used; the OP values were mostly higher in PB0.05 M, followed by PBS1x and ALF. Moreover, different species concentration-responses for OP-DTT/OP-AA were obtained. These differences were explained by the different reaction orders and kinetic rate constants obtained for each active compound in each fluid.
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Ácido Ascórbico , Cobre , Ditiotreitol , Hierro , Manganeso , Naftoquinonas , Oxidación-Reducción , Cinética , Ácido Ascórbico/química , Naftoquinonas/química , Ditiotreitol/química , Hierro/química , Cobre/química , Manganeso/química , Material Particulado/química , Material Particulado/análisis , Antioxidantes/química , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/químicaRESUMEN
This study investigated the impact of large-scale incineration facilities on PM2.5 levels in Seoul during winter. Due to the challenge of obtaining accurate combustion data from external sources, heat supply records were used as a proxy for combustion activity. To assess health risks, dithiothreitol-oxidative potential (DTT-OP) was analyzed to identify potential hazards to human health. By comparing DTT-OP with PM2.5 sources related to combustion, the study aimed to understand the impact of local pollution sources on human health in Seoul. The diurnal analysis showed that oxidative potential (0.19 µM/m3) and the biomass burning factor (5.53 µg/m3) peaked between 4:00 and 8:00 AM, with lower levels observed from 12:00 to 20:00. A significant correlation was found between combustion sources and oxidative potential, with a high correlation coefficient (r2 = 0.92). The presence of terephthalic acid (TPA) in the Cellulose combustion source profile, which is produced by the pyrolysis of plastics like polyester fiber and polyethylene terephthalate (PET), further supported the link to emissions from incineration facilities. These findings suggest that the biomass burning source is strongly correlated with DTT-OP, indicating a significant association with health risks among various local sources of PM2.5 in Seoul.
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Contaminantes Atmosféricos , Biomasa , Monitoreo del Ambiente , Incineración , Material Particulado , Ácidos Ftálicos , Ácidos Ftálicos/análisis , Contaminantes Atmosféricos/análisis , Material Particulado/análisis , Seúl , Oxidación-Reducción , Contaminación del Aire/estadística & datos numéricos , República de CoreaRESUMEN
Certain therapies that target CD markers on some blood cells can affect pretransfusion testing. Key examples are anti-CD38, CD47 monoclonal antibody (mAb) therapies such as daratumumab (DARA) and magrolimab, which have presented a challenge for transfusion medicine laboratories around the globe. Scientists have been faced with not only introducing a protocol to provide safe blood to patients but also investigating the most effective method to remove the pretransfusion pan-agglutinating interference caused. A number of papers in the last 5 years have reported on various methods to remove pretransfusion interference; however, most of these studies have been conducted only in a few countries. Most recent reviews on this topic have focused on techniques and reagents to remove pretransfusion interference, and dithiothreitol is currently the gold standard for removing DARA interference. However, it was clear from this review that while many laboratories have developed processes for addressing interference in pretransfusion testing, and DARA interference may not be a major issue, there are still laboratories around the world, that may not have adequately addressed this issue. In addition, the impact of mAb interference on widely used techniques such as flow cytometry is unclear.
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ADP-Ribosil Ciclasa 1 , Anticuerpos Monoclonales , Humanos , Anticuerpos Monoclonales/uso terapéutico , ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , ADP-Ribosil Ciclasa 1/inmunología , Transfusión Sanguínea , Glicoproteínas de Membrana/inmunología , Antígeno CD47/antagonistas & inhibidores , Citometría de Flujo/métodosRESUMEN
Critically ill pediatric patients supported on ventricular assist devices (VADs) are increasingly being anticoagulated on bivalirudin, but with difficulty monitoring anticoagulation. Activated partial thromboplastin time (aPTT) has recently been shown to poorly correlate with bivalirudin plasma concentrations, while dTT had excellent correlation. However, aPTT is the more common monitoring test and dTT testing is rarely used. In addition, effects of frequent clinical VAD scenarios (such as inflammation) on the accuracy of aPTT and dTT testing remains uncertain. We reviewed the effects of clinical scenarios (infection/inflammation, chylothorax, and steroids administration) on anticoagulation monitoring in 10 pediatric VAD patients less than 3 years at Cincinnati Children's Hospital Medical Center from 10/27/2020 to 5/6/2022 using bivalirudin for anticoagulation. There were 16 inflammation/infection, 3 chylothorax, and 6 steroids events. Correlation between dTT and aPTT was significantly lower after infection/inflammation, with dTT increasing prior to inflammation/infection while aPTT remained unchanged. In addition, steroids are administered to VAD patients to reduce inflammation and thus additionally stabilize anticoagulation. However, this anticoagulation stabilization effect was reflected more accurately by dTT compared to aPTT. In children requiring VAD support utilizing bivalirudin anticoagulation, inflammation/infection is a common occurrence resulting in anticoagulation changes that may be more accurately reflected by dTT as opposed to aPTT.
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Exposure to elevated particulate matter (PM) concentrations in ambient air has become a major health concern over urban areas worldwide. Reactive oxygen species (ROS) generation due to ambient PM (termed as their oxidative potential, OP) is shown to play a major role in PM-induced health effects. In the present study, the OP of the ambient PM2.5 samples, collected during summer 2019 from New Delhi, were measured using the dithiothreitol (DTT) assay. Average volume-normalized OP (OPV) was 2.9 ± 1.1 nmol DTT min-1 m-3, and mass-normalized OP (OPm) was 61 ± 29 pmol DTT min-1 µg-1. The regression statistics of OPv vs chemical species show the maximum slope of OPV with the elemental carbon (EC, r2 = 0.72) followed by water-soluble organic carbon (WSOC, r2 = 0.72), and organic carbon (OC, r2 = 0.64). A strong positive correlation between OPm and secondary inorganic aerosols (SIA, such as NH4+ and NO3- mass fractions) was also observed, indicating that the sources emitting NO2 and NH3, precursors of NO3- and NH4+, also emit DTT-active species. Interestingly, the slope value of OPv vs OC for aged aerosols (OM/OC > 1.7, f44 > 0.12 and f43 < 0.04) was 1.7 times higher than relatively fresh organic aerosols (OA, OM/OC < 1.7, f44 < 0.12, f43 > 0.04). An increase in OPv and OPoc with f44 indicates the formation of more DTT active species with the ageing of OA. A linear increase in OPoc with increasing Nitrogen/Carbon (N/C) ratio suggests that nitrogenous OA have higher OP.
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In the context of air quality research, the collection and analysis of fine particulate matter (PM2.5, with a diameter less than 2.5 µm) and volatile organic compound (VOCs) play a pivotal role in understanding and addressing environmental issues across the Korean Peninsula. PM2.5 and VOCs were collected over 4-hr intervals from October 17 to November 26, 2021 during the 2021 Satellite Integrated Joint Monitoring of Air Quality campaign at Olympic Park in the Republic of Korea to understand the factors controlling air quality over the Seoul Metropolitan Area. Source apportionment was performed using the positive matrix factorization (PMF) model incorporating PM2.5 and VOCs. The factor identified by chlorinated VOCs as a major component was presumed to be due to transboundary influx and was referred to as the long-range transport factor. The long-range transport factor of PM2.5 was composed of NO3-, SO42-, NH4+, and di-carboxylic acids. Back trajectory analysis showed that the airflows originated from China and passed through the west coast of Korea to the Korean Peninsula. In the PMF results using PM2.5 and VOCs, long-range transport factors were identified in both analyses, and the high correlation observed between these factors confirms that they were transported from abroad. The dithiothreitol oxidation potential normalized to quinine showed the highest oxidation potential during the same period as the long-range transport factors increased. In conclusion, PM2.5 from external sources significantly contribute to elevated levels of dithiothreitol assay-oxidative potential (DTT-OP) in Korea. The toxic concentration, expressed as the mean ± standard deviation, was determined to be 0.29 ± 0.05 µM/m³, peaking at 0.39 µM/m³. This level is 1.8 times higher than that observed outside the event period. A notable increase in secondary pollutants was observed during these periods. These pollutants are known to enhance oxidative potential, thereby potentially impacting human health.
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Contaminantes Atmosféricos , Contaminación del Aire , Compuestos Orgánicos Volátiles , Humanos , Contaminantes Atmosféricos/análisis , Contaminación del Aire/análisis , China , Ditiotreitol , Monitoreo del Ambiente/métodos , Estrés Oxidativo , Material Particulado/análisis , Emisiones de Vehículos/análisis , Compuestos Orgánicos Volátiles/análisisRESUMEN
Biofilms are multicellular aggregates of bacteria immersed in an extracellular matrix that forms on various surfaces, including biological tissues and artificial surfaces. However, more and more reports point out the fact that even biological fluids and semifluid, such as synovial liquid, blood, urine, or mucus and feces, harbor "non-attached" biofilm aggregates of bacteria, which represent a significant phenomenon with critical clinical implications that remain to be fully investigated. In particular, biofilm aggregates in biological fluid samples have been shown to play a relevant role in bacterial count and in the overall accuracy of microbiological diagnosis. In line with these observations, the introduction in the clinical setting of fluid sample pretreatment with an antibiofilm chemical compound called dithiothreitol (DTT), which is able to dislodge microorganisms from their intercellular matrix without killing them, would effectively improve the microbiological yield and increase the sensitivity of cultural examination, compared to the current microbiological techniques. While other ongoing research continues to unveil the complexity of biofilm formation in biological fluids and its impact on infection pathogenesis and diagnosis, we here hypothesize that the routine use of a chemical antibiofilm pretreatment of fluid and semi-solid samples may lead to a paradigm shift in the microbiological approach to the diagnosis of biofilm-related infections and should be further investigated and eventually implemented in the clinical setting.
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A positive flow cytometry crossmatch (FCXM) due to donor specific antibodies (DSA) constitutes a risk for kidney transplantation; such a finding may indicates an unacceptable donor for this patient. However, positive FCXM in the absence of DSA is considered discordant and need further investigations. During COVID-19 pandemic, we observed 22% discordant results out of 445 FCXM performed during eight months period in our laboratory and another 7% were invalid due to high background negative control (NC). No study has addressed the impact of COVID-19 pandemic on FCXM and the overall pre-kidney transplant workups or described a solution to deal with these non-specific reactivities. Herein, we analyzed all FCXM results in SARS-CoV-2 seropositive patients and addressed how this pandemic affected significantly the pre-kidney transplant workups, highlighting both technical and financial implications. We also shared our modified FCXM procedures using dithiotheritol (DTT) sera treatment or blocking donor cells with negative control human serum (NCS) which we found to be successful to abrogate 98% of all discordant FCXM results and to validate all invalid results due to high background NC. In conclusion, COVID-19 pandemic has affected our HLA laboratory significantly by creating many false positive or invalid crossmatch results. Transplant laboratories must consider this before test interpretations and immune risk assessments. We recommend the use of DTT serum treatment to remove nonspecific bindings in the sera of kidney transplant candidates and the use of NCS-blocked donor cells to correct high background when performing FCXM in transplant candidates or donors with recent history of SARS-CoV-2 immunization respectively.
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【Objective】 To explore the feasibility of blood transfusion compatibility testing for multiple myeloma(MM) patients treated with anti-CD38 monoclonal antibody daratumumab (DARA) after DARA-Fab fragment blocking, and to evaluate the transfusion efficacy by comparing with dithiothreitol(DTT) method. 【Methods】 After DARA was prepared into DARA-Fab fragments using PierceFab preparation kit, the neutralization effects of different volumes (5, 10, 15, 30 μL) on screening cells and panel cells were confirmed. DARA-Fab fragments and screening cells with specific antigens and corresponding monoclonal antibody reagents were used as the experimental group and the control group with the same volume of saline for incubating and centrifugin.Twenty MM patients treated with DARA were selected for cross-matching with DARA-Fab and DTT respectively, and the laboratory indexes before and after transfusion were statistically analyzed, and the two blood matching methods were compared. 【Results】 After incubating and centrifuging, the results of DARA-Fab fragments(15, 30 μL) with screening cells and serum mixed with DARA were negative, while those of DARA-Fab(5, 10 μL) were positive. 15μL DARA-Fab treated antibody identification cells (2, 3, 4, 5, 7, 9, 11) were negative, antibody identification cells (1, 6, 8, 10, 12) were negative after 30 μL DARA-Fab fragments treatment; the results of MNS, Duffy, Kidd, Kell, Lewis, Rh blood group system of the experimental group were consistent with those of the control group; the hemoglobin (Hb) (g/L) of 20 patients after infusion of RBC (73.90±1.90) was significantly higher than that before transfusion (63.60±1.58), P0.05).And no statistical difference was noticed in Hb (10.75±1.04 vs 10.30±0.98), TBil (3.31±1.47 vs 3.31±0.55), DBIL(2.76±1.24 vs 2.60±0.83), and I-Bil(1.97±0.40 vs 2.82±0.53) between the DTT treatment method and the DARA Fab fragment treatment before and after transfusion(P>0.05). 【Conclusion】 DARA-Fab can remove the interference of RBC on cross matching by blocking CD38 antigen. This method has no effect on the antigens of common RBC blood group systems, and shows significant blood transfusion efficacy as that of DTT method.
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This study reports size-resolved dithiothreitol (DTT)-based oxidative potential (OP: total and water-soluble) in rural kitchens using liquefied petroleum gas (LPG), firewood (FW), and mixed biomass (MB) fuels in northeastern (NE) India. In comparison to LPG, volume-normalized total OP (OPtotal(v)DTT) was enhanced by a factor of â¼5 in biomass-using kitchens (74 ± 35 to 78 ± 42 nmol min-1 m-3); however, mass-normalized total OP (OPtotal(m)DTT) was similar between LPG and FW users and higher by a factor of 2 in MB-using kitchens. The water-insoluble OP (OPwi(v, m)DTT) fraction in OPtotal(v, m)DTT was greater than 50% across kitchens. Size distributions across kitchens and OPDTT categories ranged from unimodal to trimodal. OPws(v)DTT was driven by metals as well as organics across size fractions while OPwi(v)DTT was majorly constrained by metals with an increasing importance of organics in fine particles of biomass-using kitchens. Multiple linear regression analysis revealed that Cu and Ba explained 71% of the OPtotal(v)DTT variability in LPG-using kitchens, while water-soluble organic carbon (WSOC) and Ba were responsible for 44% variability in FW-using kitchens. Finally, the high internal dose of OPtotal(v)DTT (28-31 nmol min-1 m-3) in biomass-using kitchens established the severity of oxidative stress on the exposed population.
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Contaminantes Atmosféricos , Contaminación del Aire Interior , Petróleo , Material Particulado/análisis , Contaminantes Atmosféricos/análisis , India , Aerosoles , Estrés Oxidativo , Ditiotreitol , Agua , Monitoreo del Ambiente , Contaminación del Aire Interior/análisisRESUMEN
The patch clamp method is a widely applied electrophysiological technique used to understand ion channel activity and cellular excitation. The formation of a high resistance giga-ohm seal is required to obtain high-quality recordings but can be challenging due to variables including operator experience and cell preparation. Therefore, the identification of methods to promote the formation and longevity of giga-ohm seals may be beneficial. In this report, we describe our observation that the application of reducing agents (DTT and TCEP) to the external bath solution during whole-cell patch clamp recordings of heterologous cells (HEK and LM) and cultured primary cells (DRG neurons) enhanced the success of giga-ohm seal formation. Reducing agents also maintained the integrity of the seal for longer periods of time at strong hyperpolarizing voltages, whereas an oxidizing agent (H2O2) appeared to have the opposite effect. In summary, we report a useful tool to improve the quality of patch clamp recordings that may be helpful in certain experimental contexts.
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Peróxido de Hidrógeno , Sustancias Reductoras , Células CultivadasRESUMEN
BACKGROUND: Hemolytic disease of the newborn (HDN) results in the decreased lifespan of the red cells. HDN related to ABO incompatibility is mostly unnoticed because routine screening is not being done. This study was done to assess the prevalence of ABO-HDN and to compare different immunohematological tests. Methods-In this study 213 O group mothers and the 122 ABO-incompatible newborns born to them were included. Quantifying the maternal IgG anti-A/anti-B antibody titer was done by Conventional Tube Technique (CTT) using Dithiothreitol (DTT) pretreated maternal serum. Hemolysin test was performed on the mothers having titer > 256. These cases were followed up and, after delivery, were monitored for ABO HDN, along with direct antiglobulin testing and elution studies. The prevalence of ABO-HDN was calculated, and the different diagnostic parameters of the tests were calculated. Results- The prevalence of ABO-HDN in our population was estimated to be 1.7%, 6.1% & 10.6% in our population, O group mothers, and O group mothers with ABOincompatible newborns, respectively. Maternal titer≥ 512 strongly correlated with ABOHDN. DAT positivity is a good predictor of ABO-HDN, especially using sensitive techniques. Maternal IgG titers have the highest sensitivity & Negative Predictive Value, while DAT has the highest specificity & Positive Predictive Value. Conclusion - Maternal ABO antibody titration may be advocated in the centers to identify high-risk groups. It can advocate institutional delivery and dedicated follow-up of newborns with ABO-HDN. Blood grouping & DAT may be performed in all newborns born to O blood group to identify high-risk cases.
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Eritroblastosis Fetal , Recién Nacido , Humanos , Femenino , Embarazo , Prevalencia , Centros de Atención Terciaria , Eritroblastosis Fetal/diagnóstico , Eritroblastosis Fetal/epidemiología , Incompatibilidad de Grupos Sanguíneos , Sistema del Grupo Sanguíneo ABO , Inmunoglobulina G , Pruebas Diagnósticas de Rutina , Prueba de CoombsRESUMEN
Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.
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Actinas , Estrellas de Mar , Animales , Femenino , Masculino , Ditiotreitol/farmacología , Ditiotreitol/metabolismo , Actinas/metabolismo , Semen/metabolismo , Oocitos/metabolismo , FertilizaciónRESUMEN
Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.
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Luz , Espectrometría de Masas en Tándem , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes/químicaRESUMEN
The toxicity associated with the fine particulate matter (PM2.5) has not been well studied, particularly in relation to the emissions from on-road vehicles and other sources in low- and middle-income countries such as India. Thus, a study was conducted to examine the oxidative potential (OP) of PM2.5 at a roadside (RS) site with heavy vehicular traffic and an urban background (BG) site in Mumbai using the dithiothreitol (DTT) assay. Simultaneous gravimetric PM2.5 was measured at both sites and characterized for carbonaceous constituents and water-soluble trace elements and metals. Results depicted higher PM2.5, elemental carbon (EC), and organic carbon (OC) concentrations on the RS than BG (by a factor of 1.7, 4.6, and 1.2, respectively), while BG had higher water-soluble organic carbon (WSOC) levels (by a factor of 1.4) and a higher WSOC to OC ratio (86%), likely due to the dominance of secondary aerosol formation. In contrast, the measured OPDTTv at RS (8.9 ± 5.5 nmol/min/m3) and BG (8.1 ± 6.4 nmol/min/m3) sites were similar. However, OPDTTv at BG was higher during the afternoon, suggesting the influence of photochemical transformation on measured OPDTTv at BG. At RS, OC and redox-active metals (Cu, Zn, Mn, and Fe) were significantly associated with measured OP (p < 0.05), while at BG, WSOC was most strongly associated (p < 0.05). The coefficient of divergence (COD) for PM2.5, its chemical species, and OPDTTv was >0.2, indicating spatial heterogeneity between the sites, and differences in emission sources and toxicity. The estimated hazard index (HI) was not associated with OPDTTv, indicating that current PM2.5 mass regulations may not adequately capture the health effects of PM2.5. The study highlights the need for further studies examining PM2.5 toxicity and developing toxicity-based air quality regulations.
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Contaminantes Atmosféricos , Material Particulado , Material Particulado/análisis , Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente/métodos , Oxidación-Reducción , Aerosoles/análisis , Carbono , Metales , Agua , Estrés Oxidativo , Emisiones de Vehículos/análisisRESUMEN
Oxidative stress is a mechanism that might raise the toxicity of mineral dust aerosols. We evaluated the oxidative potential (OP) of four reference materials (RMs) of mineral dusts using dithiothreitol assay. The OP of the water-soluble fraction of the dust RMs accounts for 40%-70% of the OP of the total fraction. The values of total and water-soluble OP normalized by the surface area of insoluble particles showed agreement among the different dust RMs. The surface area of insoluble dust particles was therefore inferred as an important factor affecting the OP of mineral dust. Using the relation between total OP and the surface area of insoluble particles of the dust RMs, we estimated the total OPs of fine and coarse atmospheric mineral dust aerosols assuming a typical particle size distribution of Asian dust aerosols observed in Japan. Mass-normalized total OPs were estimated at 44 and 23 pmol min-1 µg-1 for fine and coarse atmospheric mineral dust particles. They closely approximate the values observed for urban aerosols in Japan, which suggests that mineral dust plume advection can lead to a marked increase in human exposure to redox-active aerosols, even far downwind from mineral dust source regions.