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Protein A (PA) is a bacterial cell wall component of Staphylococcus aureus whose function is to bind to Immunoglobulin G (IgG). Given its ability to bind IgG as well as its stability and resistance to harsh acidic and basic cleaning conditions, it is commonly used in the affinity chromotography purification of biotherapeutics. This use can result in levels of PA being present in a drug product and subsequent patient exposure. Interestingly, PA was previously evaluated in clinical trials as well as supporting nonclinical studies, resulting in a database that enables the derivation of a health-based exposure limit (HBEL). Given the widespread use of PA in the pharmaceutical industry, the IQ DruSafe Impurities Working Group (WG) evaluated the available information with the purpose of establishing a harmonized parenteral HBEL for PA. Based on this thorough, collaborative evaluation of nonclinical and clinical data available for PA, a parenteral HBEL of 1.2 µg/kg/dose (60 µg/dose for a 50 kg individual) is expected to be health protective for patients when it is present as an impurity in a biotherapeutic.
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Antibiotics have been a vital component in the fight against microbial diseases for over 75 years, saving countless lives. However, the global rise of multi-drug-resistance (MDR) bacterial infections is pushing us closer to a post-antibiotic era where common infections may once again become lethal. To combat MDR Acinetobacter baumannii, we investigated chiral phthalimides and used molecular docking to identify potential targets. Outer membrane protein A (OmpA) is crucial for A. baumannii resistant to antibiotics, making it a pathogen of great concern due to its high mortality rate and limited treatment options. In this study, we evaluated three distinct compounds against the OmpA protein: FIA (2-(1,3-dioxoindolin-2yl)-3-phenylpropanoic acid), FIC (2-(1,3-dioxoindolin-2yl)-4-(methylthio) butanoic acid), and FII (3-(1,3-dioxoindolin-2yl)-3-phenylpropanoic acid). Molecular docking results showed that these three compounds exhibited strong interactions with the OmpA protein. Molecular dynamics (MD) simulation analysis further confirmed the stability and binding efficacy of these compounds with OmpA. Their antimicrobial activities were assessed using the agar well diffusion method, revealing that FIA had an optimal zone of inhibition of 24 mm. Additionally, the minimum inhibitory concentrations (MIC) of these compounds were determined, demonstrating their bactericidal properties against A. baumannii, with MICs of 11 µg/µL for FIA, 46 µg/µL for FIC, and 375 µg/µL for FII. In vitro cytotoxicity data indicated that none of the three compounds were hemolytic when exposed to human red blood cells. This finding is particularly significant as it highlights the superior efficacy of FIA against A. baumannii compared to the other compounds. With thorough pharmacokinetic validations, these chiral phthalimides are promising alternative therapeutic options for treating infections caused by A. baumannii, offering new hope in the face of rising antibiotic resistance.
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Proliferation of renal tubular epithelial cells (TEC) is essential for restoring tubular integrity and thereby to support renal functional recovery from kidney ischemia/reperfusion (KI/R) injury. Activation of transcriptional factor c-Myc promotes TEC proliferation following KI/R; however, the mechanism regarding c-Myc activation in TEC is incompletely known. Heat shock protein A12A (HSPA12A) is an atypic member of HSP70 family. In this study, we found that KI/R decreased HSPA12A expression in mouse kidneys and TEC, while ablation of HSPA12A in mice impaired TEC proliferation and renal functional recovery following KI/R. Gain-of-functional studies demonstrated that HSPA12A promoted TEC proliferation upon hypoxia/reoxygenation (H/R) through directly interacting with c-Myc and enhancing its nuclear localization to upregulate expression of its target genes related to TEC proliferation. Notably, c-Myc was lactylated in TEC after H/R, and this lactylation was enhanced by HSPA12A overexpression. Importantly, inhibition of c-Myc lactylation attenuated the HSPA12A-induced increases of c-Myc nuclear localization, proliferation-related gene expression, and TEC proliferation. Further experiments revealed that HSPA12A promoted c-Myc lactylation via increasing the glycolysis-derived lactate generation in a Hif1α-dependent manner. The results unraveled a role of HSPA12A in promoting TEC proliferation and facilitating renal recovery following KI/R, and this role of HSPA12A was achieved through increasing lactylation-mediated c-Myc activation. Therefore, targeting HSPA12A in TEC might be a viable strategy to promote renal functional recovery from KI/R injury in patients.
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Proliferación Celular , Células Epiteliales , Proteínas HSP70 de Choque Térmico , Túbulos Renales , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myc , Daño por Reperfusión , Animales , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratones , Células Epiteliales/metabolismo , Células Epiteliales/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Humanos , Riñón/metabolismo , Riñón/patologíaRESUMEN
BACKGROUND: Maternal gestational diabetes (GDM), small (SGA) and large (LGA) for gestational age neonates are associated with increased morbidity in both mother and child. We studied how different levels of first trimester pregnancy associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotropin (fß-hCG) were associated with SGA and LGA in GDM pregnancies and controls. METHODS: Altogether 23 482 women with singleton pregnancies participated in first trimester combined screening and delivered between 2014 and 2018 in Northern Finland and were included in this retrospective case-control study. Women with GDM (n = 4697) and controls without GDM (n = 18 492) were divided into groups below 5th and 10th or above 90th and 95th percentile (pc) PAPP-A and fß-hCG MoM levels. SGA was defined as a birthweight more than two standard deviations (SD) below and LGA more than two SDs above the sex-specific and gestational age-specific reference mean. Odds ratios were adjusted (aOR) for maternal age, BMI, ethnicity, IVF/ICSI, parity and smoking. RESULTS: In pregnancies with GDM the proportion of SGA was 2.6% and LGA 4.5%, compared to 3.3% (p = 0.011) and 1.8% (p < 0.001) in the control group, respectively. In ≤ 5th and ≤ 10th pc PAPP-A groups, aORs for SGA were 2.7 (95% CI 1.5-4.7) and 2.2 (95% CI 1.4-3.5) in the GDM group and 3.8 (95% CI 3.0-4.9) and 2.8 (95% CI 2.3-3.5) in the reference group, respectively. When considering LGA, there was no difference in aORs in any high PAPP-A groups. In the low ≤ 5 percentile fß-hCG MoM group, aORs for SGA was 2.3 (95% CI 1.8-3.1) in the control group. In fß-hCG groups with GDM there was no association with SGA and the only significant difference was ≥ 90 percentile group, aOR 1.6 (95% CI 1.1-2.5) for LGA. CONCLUSION: Association with low PAPP-A and SGA seems to be present despite GDM status. High PAPP-A levels are not associated with increased LGA risk in women with or without GDM. Low fß-hCG levels are associated with SGA only in non-GDM pregnancies.
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Gonadotropina Coriónica Humana de Subunidad beta , Diabetes Gestacional , Macrosomía Fetal , Recién Nacido Pequeño para la Edad Gestacional , Primer Trimestre del Embarazo , Proteína Plasmática A Asociada al Embarazo , Humanos , Femenino , Embarazo , Proteína Plasmática A Asociada al Embarazo/análisis , Proteína Plasmática A Asociada al Embarazo/metabolismo , Gonadotropina Coriónica Humana de Subunidad beta/sangre , Primer Trimestre del Embarazo/sangre , Adulto , Estudios de Casos y Controles , Estudios Retrospectivos , Diabetes Gestacional/sangre , Diabetes Gestacional/epidemiología , Recién Nacido , Macrosomía Fetal/sangre , Macrosomía Fetal/epidemiología , Finlandia/epidemiología , Factores de Riesgo , Peso al NacerRESUMEN
Staphylococcus aureus is a major human pathogen, yet the immune factors that protect against infection remain elusive. High titers of opsonic IgG antibodies, achieved in preclinical animal immunization studies, have consistently failed to provide protection in humans. Here, we investigate antibody responses to the conserved S. aureus surface glycan wall teichoic acid (WTA) and detect the presence of WTA-specific IgM and IgG antibodies in the plasma of healthy individuals. Functionally, WTA-specific IgM outperforms IgG in opsonophagocytic killing of S. aureus and protects against disseminated S. aureus bacteremia through passive immunization. In a clinical setting, patients with S. aureus bacteremia have significantly lower WTA-specific IgM but similar IgG levels compared to healthy controls. Importantly, low WTA-IgM levels correlate with disease mortality and impaired bacterial opsonization. Our findings may guide risk stratification of hospitalized patients and inform future design of antibody-based therapies and vaccines against serious S. aureus infection.
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Anticuerpos Antibacterianos , Inmunoglobulina G , Inmunoglobulina M , Polisacáridos , Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/inmunología , Inmunoglobulina M/inmunología , Inmunoglobulina M/sangre , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Polisacáridos/inmunología , Ácidos Teicoicos/inmunología , Animales , Femenino , Masculino , Fagocitosis/inmunología , Bacteriemia/inmunología , Bacteriemia/microbiología , Ratones , Adulto , Persona de Mediana Edad , Opsonización/inmunologíaRESUMEN
Many cell wall anchored surface proteins of Gram-positive bacteria harbor a highly conserved YSIRK/G-S signal peptide (SPYSIRK+), which deposits surface protein precursors at the cell division septum where they are subsequently anchored to septal peptidoglycan. Previously we identified that LtaS-mediated lipoteichoic acid (LTA) synthesis regulates septal trafficking of YSIRK+ proteins in S. aureus. Interestingly, both LtaS and SPYSIRK+ are cleaved by the signal peptidase SpsB, but the biological implications remain unclear. Here we show that SpsB is required for cleaving SPSpA(YSIRK+) of staphylococcal surface protein A (SpA). Depletion of spsB not only diminished SPSpA processing but also abolished SpA septal localization. The mis-localization is attributed to the cleavage activity of SpsB, as an A37P mutation of SPSpA that disrupted SpsB cleavage also abrogated SpA septal localization. Strikingly, depletion of spsB led to aberrant cell morphology, cell cycle arrest and daughter cell separation defects. Localization studies showed that SpsB predominantly localized at the septum of dividing staphylococcal cells. Finally, we show that SpsB spatially regulates LtaS as spsB depletion enriched LtaS at the septum. Collectively, the data suggest a new dual-mechanism model mediated by SpsB: the abundant YSIRK+ proteins are efficiently processed by septal localized SpsB; SpsB cleaves LtaS at the septum, which spatially regulates LtaS activity contributing to YSIRK+ proteins septal trafficking. The study identifies SpsB as a novel and key regulator orchestrating protein secretion, cell cycle and cell envelope biogenesis.
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Polysorbates (PS) are commonly used as stabilizers of biopharmaceuticals such as monoclonal antibodies (mAbs). However, they are prone to chemical and enzymatic degradation. The latter can be caused by residual host cell proteins (HCPs) in the drug substance. Degradation affects the functionality of the PS surfactant which can lead to formation of particles. An increasing number of publications describe enzymatic PS degradation. Significant efforts have been made to characterize HCP removal during Downstream Processing (DSP) of mAbs and to develop mitigation strategies. Here we describe the use of glycine buffer for acidic elution in Protein A affinity chromatography compared to acetate buffer, which is more commonly used in the biopharmaceutical industry. Increased turbidity was observed during pH re-adjustment after low pH virus inactivation when using glycine buffer. Analytical data suggests that this turbidity is caused by the formation of precipitates which include HCP and DNA impurities. Additionally, as a zwitterion, glycine does not contribute to conductivity; this further enhances HCP removal during anion-exchange flow-through chromatography. Although glycine is well known as a possible elution buffer for Protein A affinity chromatography, its positive impact on HCP removal and PS stability have not yet been described in literature.
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The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1ß, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.
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Leaf senescence is a developmental program regulated by both endogenous and environmental cues. Abiotic stresses such as nutrient deprivation can induce premature leaf senescence, which profoundly impacts plant growth and crop yield. However, the molecular mechanisms underlying stress-induced senescence are not fully understood. In this work, employing a carbon deprivation (C-deprivation)-induced senescence assay in Arabidopsis seedlings, we identified PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, as a negative regulator of C-deprivation-induced senescence. Furthermore, we demonstrated that PRL1 directly interacts with the RPA2A subunit of the single-stranded DNA-binding Replication Protein A (RPA) complex. Consistently, the loss of RPA2A leads to premature senescence, while increased expression of RPA2A inhibits senescence. Moreover, overexpression of RPA2A reverses the accelerated senescence in prl1 mutants, and the interaction with PRL1 stabilizes RPA2A under C-deprivation. In summary, our findings reveal the involvement of the PRL1-RPA2A functional module in C-deprivation-induced plant senescence.
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Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils and function in antiviral innate immunity, RNA editing, and can cause hypermutation in chromosomes. The resulting uracils can be directly replicated, resulting in C to T mutations, or uracil-DNA glycosylase can convert the uracils to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depends on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long-persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed uracils in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of uracils by replicative DNA polymerases. Unexpectedly, we also found that for uracils shielded from Ung1 by wild-type RPA the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.
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Anti-glomerular basement membrane (GBM) nephritis is a rare autoimmune disorder characterized by acute and rapidly progressive glomerulonephritis. In this report, we present the case of a 52-year-old woman with anti-GBM nephritis who was treated with Staphylococcus Protein A immunoadsorption in combination with glucocorticoids and cyclophosphamide. After 8 cycles of immunoadsorption, the patient's anti-GBM antibodies decreased from 363 AU/mL to less than 20 AU/mL, accompanied by a dropped immunoglobin G level, although renal impairment persisted. We reviewed the therapeutic options for anti-GBM nephritis and compared plasma exchange, double filtration plasmapheresis, and immunoadsorption with regard to plasma consumption, allergic events, and plasma components loss. Protein A immunoadsorption appears to be a promising treatment modality for anti-GBM nephritis.
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BACKGROUND: Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain. OBJECTIVE: This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain. METHODS: The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain. RESULTS: When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species. CONCLUSION: Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.
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OBJECTIVE: To compare the differential impact of recombinant protein A immunoadsorption (PAIA) or therapeutic plasma exchange (TPE) on neurological functional improvement and quality of life in patients afflicted with severe acute neuroimmune diseases, including Guillain-Barré syndrome (GBS), myasthenia gravis (MG), neuromyelitis optica spectrum disorder (NMOSD), and anti-NMDA receptor encephalitis (NMDARE). METHODS: The retrospective study included 29 patients with moderate to severe disability (modified Rankin scale, mRS≥3) due to acute neuroimmune diseases at the second Xiangya hospital from January 2021 to January 2023. The clinical efficacy of PAIA and TPE in improving neurological function (ΔmRS≥1) and the difference in favorable functional outcomes (mRS 0-2) at three months were evaluated. The impact of both treatments on patients' health-related quality of life (HRQoL) was assessed using a visual analog scale (EQ-VAS) score ranging from 0 to 100. RESULTS: The findings revealed that the PAIA group exhibited a significantly higher rate of improvement in modified Rankin scale (mRS) scores (ΔmRS≥1) at the three-month follow-up compared to the TPE group (94.4 % vs. 54.5 %, p = 0.018). However, no statistically significant difference was observed between the two treatment modalities in terms of favorable neurological functional outcomes at the three-month mark. Furthermore, the PAIA group demonstrated a significantly higher EQ-VAS score at 14 days post-treatment compared to the TPE group (60.0 vs. 47.7, p = 0.017). CONCLUSION: In the short-term management of severe acute neuroimmune diseases, PAIA may present a greater probability of improving neurological function and facilitating an earlier enhancement of quality of life compared to TPE.
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Intercambio Plasmático , Calidad de Vida , Humanos , Intercambio Plasmático/métodos , Femenino , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto , Técnicas de Inmunoadsorción , Recuperación de la Función , Resultado del Tratamiento , Síndrome de Guillain-Barré/terapia , Síndrome de Guillain-Barré/inmunología , Anciano , Miastenia Gravis/terapia , Miastenia Gravis/inmunología , Adulto JovenRESUMEN
Background: Due to the incomplete standardization of the etiology and diagnostic criteria for fetal growth restriction (FGR), there has been uncertainty in the early prediction of FGR. The comprehensive estimation of FGR was mainly based on various factors, such as maternal characteristics and medical history, nuchal translucency (NT), and serum biochemical markers [pregnancy-associated plasma protein-A (PAPP-A) and free beta human chorionic gonadotropin (free ß-hCG)]. Herein, we performed a retrospective cohort study to investigate the correlation and diagnostic value of maternal markers such as PAPP-A, free ß-hCG, and NT in the first trimester with maternal characteristics, so as to provide theoretical basis for perinatal care and the application of low-dose aspirin. Methods: A retrospective cohort study was conducted to analyze the data of an FGR group and a non-FGR group. Chi-square test and Mann-Whitney U test were used for univariate analysis of qualitative or quantitative data, respectively. Modified Poisson regression calculated the relative risk (RR) and 95% confidence interval (CI) of perinatal variables; P<0.05 was considered statistically significant. Results: The multiple of median (MoM) of PAPP-A level and NT in the FGR group were lower than those of the non-FGR group [0.63 (0.12-2.08) vs. 1.01 (0.28-2.41) MoM, 1.30 (0.80-2.07) vs. 1.40 (0.80-2.20) cm, P<0.05]. The weight, score, and length of newborns in the FGR group were lower than those in the non-FGR group (all P<0.001). Modified Poisson regression analysis showed that gestational hypertension (GH) [RR =1.836 (95% CI: 1.188-2.836)], oligohydramnios [1.420 (95% CI: 1.022-1.973)], premature rupture of membranes (PROM) [0.641 (95% CI: 0.425-0.969)], female infant [1.539 (95% CI: 1.098-2.157)], low infant length [5.700 (95% CI: 3.416-9.509)], low birth weight [1.609 (95% CI: 1.012-2.559), and increased PAPP-A MoM [0.533 (95% CI: 0.369-0.769)] were associated with FGR. The cut-off value of PAPP-A + free ß-hCG + NT for predicting FGR was 0.190, with a sensitivity of 0.547 and a specificity of 0.778. Conclusions: Early screening markers combined with perinatal characteristics have better diagnostic value in predicting FGR and provide a scientific basis for the clinical use of low-dose aspirin to prevent FGR.
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Objective: This study aimed to investigate the effects of the presence of subchorionic hematoma (SH) in early pregnancies with threatened miscarriage (TM) on levels of first-trimester maternal serum markers, pregnancy-associated plasma protein-A (PAPP-A), and free ß-human chorionic gonadotropin (ß-hCG) levels. Methods: The data of TM cases with SH in the first trimester between 2015 and 2021 were evaluated retrospectively. The data of age and gestational age-matched TM cases without SH were also assessed to constitute a control group. Demographic characteristics, obstetric histories, ultrasonographic findings, and free ß-hCG and PAPP-A levels of the groups were compared. Results: There were 119 cases in the study group and 153 cases in the control group. The median vertical and longitudinal lengths of the SH were 31 mm and 16 mm. The median age of both groups was similar (p=0.422). The MoM value of PAPP-A was 0.088 (.93) in the study group and 0.9 (0.63) in the control group (p=0.519). Similarly, the MoM value of free ß-hCG was 1.04 (0.78) in the study group and 0.99 (0.86) in the control group (p=0.66). No significant relationship was found in the multivariate analysis between free ß-hCG MoM, PAPP-A MoM, age, gravida, and vertical and longitudinal lengths of the hematoma (p>0.05). Conclusion: The level of PAPP-A and free ß-hCG were not affected by the SH. Therefore, these markers can be used reliably in TM cases with SH for the first-trimester fetal aneuploidy screening test.
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Gonadotropina Coriónica Humana de Subunidad beta , Hematoma , Primer Trimestre del Embarazo , Proteína Plasmática A Asociada al Embarazo , Humanos , Femenino , Embarazo , Proteína Plasmática A Asociada al Embarazo/análisis , Primer Trimestre del Embarazo/sangre , Gonadotropina Coriónica Humana de Subunidad beta/sangre , Hematoma/sangre , Hematoma/diagnóstico por imagen , Adulto , Estudios Retrospectivos , Biomarcadores/sangre , Estudios de Casos y Controles , Amenaza de Aborto/sangre , Corion/diagnóstico por imagenRESUMEN
BACKGROUND: The serum markers Krebs von den Lungen-6 (KL-6), surfactant protein A (SP-A), and surfactant protein D (SP-D) have been used for the diagnosis, differential diagnosis, and prognosis prediction of interstitial pneumonia. However, the significance of measuring the serum and bronchoalveolar lavage fluid (BALF) KL-6, SP-D, and SP-A levels in predicting the prognosis of chronic fibrosing interstitial pneumonia (CFIP), idiopathic pulmonary fibrosis, and idiopathic nonspecific interstitial pneumonia remains unclear. We aimed to clarify the significance of measuring the serum and BALF KL-6, SP-A, and SP-D levels in predicting the prognosis of patients with CFIP. METHODS: Among 173 patients who were diagnosed with CFIP between September 2008 and February 2021, 39 who underwent bronchoalveolar lavage were included in this study. Among these, patients experiencing an annual decrease in forced vital capacity (FVC) of ≥10% or those facing challenges in undergoing follow-up pulmonary function tests owing to significant deterioration in pulmonary function were categorized as the rapidly progress group. Conversely, individuals with an annual decrease in the FVC of <10% were classified into the slowly progress group. The serum and BALF KL-6, SP-D, and SP-A levels, as well as BALF/serum SP-D and SP-A ratios were compared between the two groups. RESULTS: Among the patients with CFIP, the BALF SP-D level (p=0.0111), BALF SP-A level (p<0.0010), BALF/serum SP-D ratio (p=0.0051), and BALF/serum SP-A ratio (p<0.0010) were significantly lower in the rapidly than in the slowly progress group (p<0.0010). The receiver operating characteristics analysis results demonstrated excellent performance for diagnosing patients with CFIP, with the BALF SP-D level (area under the curve [AUC], 0.7424), BALF SP-A level (AUC, 0.8842), BALF/serum SP-D ratio (AUC, 0.7673), and BALF/serum SP-A ratio (AUC, 0.8556). Moreover, the BALF SP-A level showed a notably superior CFIP diagnostic capability. Survival analysis using the Kaplan-Meier method revealed that patients with a BALF SP-A level of <1500 ng/mL and BALF/serum SP-A ratio of <15.0 had poor prognoses. CONCLUSIONS: Our results suggest that BALF SP-A measurement may be useful for predicting the prognosis in patients with CFIP.
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Biomarcadores , Líquido del Lavado Bronquioalveolar , Mucina-1 , Proteína A Asociada a Surfactante Pulmonar , Proteína D Asociada a Surfactante Pulmonar , Humanos , Proteína D Asociada a Surfactante Pulmonar/sangre , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Líquido del Lavado Bronquioalveolar/química , Mucina-1/sangre , Mucina-1/análisis , Femenino , Masculino , Estudios Retrospectivos , Proteína A Asociada a Surfactante Pulmonar/sangre , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Proteína A Asociada a Surfactante Pulmonar/análisis , Anciano , Persona de Mediana Edad , Pronóstico , Biomarcadores/sangre , Biomarcadores/análisis , Fibrosis Pulmonar Idiopática/sangre , Fibrosis Pulmonar Idiopática/diagnóstico , Fibrosis Pulmonar Idiopática/metabolismo , Enfermedades Pulmonares Intersticiales/sangre , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/metabolismo , Curva ROC , Capacidad Vital , Enfermedad CrónicaRESUMEN
Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.
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Unión Proteica , Proteína Recombinante y Reparadora de ADN Rad52 , Proteína de Replicación A , Dispersión del Ángulo Pequeño , Humanos , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/química , Proteína de Replicación A/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/genética , Recombinasa Rad51/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/genética , Difracción de Rayos X/métodos , Reparación del ADN , Modelos Moleculares , Dominios ProteicosRESUMEN
Diagnostic assays that are able to detect foot-and-mouth disease (FMD) virus infection in the vaccinated population are essential tools in the progressive control pathway for the FMD. However, testing of serum samples using a single diagnostic assay may not completely substantiate freedom from the virus infection. Therefore, viral non-structural proteins (NSPs)-based various serological assays have been developed for the detection of FMD infection. Nevertheless, the NSPs-based ELISAs have been developed in the indirect-ELISA format, thereby necessitating the use of species-specific conjugated secondary-antibodies for the detection of anti-NSP antibodies in various FMD-susceptible species. Therefore, this study presents a novel recombinant 2B-NSP-based indirect ELISA, employing HRP-conjugated protein-A/G detection system which can detect anti-NSPs antibodies from multiple FMD-susceptible species in a single ELISA platform. Recombinant 2B (r2B) protein was expressed as His-SUMO tagged protein in the E. Coli cells and purified using NI-NTA affinity column chromatography. Using the r2B protein and HRP-conjugated protein A/G, an indirect ELISA was developed and validated for the detection of anti-2B antibodies in serum samples collected from multiple FMD-susceptible animal species with known FMD status. Further, a resampling based statistical technique has been reported for determination of optimal cut-off value for the diagnostic assay. Through this technique, the optimal cut-off of 44 percentage of positivity value was determined for the assay. At this optimal cut-off value, the developed diagnostic assay provided diagnostic sensitivity, specificity, and accuracy, positive and negative predictive values (PPV and NPV) of 92.35 %, 98.41 %, 95.21 %, 98.58 %, and 91.67 %, respectively. The assay was validated further by analyzing random serum samples collected across multi-locations in India. The assay can be used as a single platform for testing serum samples from different species of FMDV-susceptible animals and will be useful for NSP-based serosurveillance of FMDV.
Asunto(s)
Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Virus de la Fiebre Aftosa , Fiebre Aftosa , Proteínas no Estructurales Virales , Virus de la Fiebre Aftosa/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/inmunología , Fiebre Aftosa/sangre , Fiebre Aftosa/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Proteínas no Estructurales Virales/inmunología , Bovinos , Proteínas Recombinantes/inmunología , Porcinos , Especificidad de la EspecieRESUMEN
A label-free electrochemical immunosensor was developed to rapidly detect tilmicosin (TMC) residues in pork and milk. The immunosensor was constructed by immobilizing a high-affinity monoclonal antibody against TMC on an rGO-PEI-Ag nanocomposite-modified electrode. The rGO-PEI-Ag nanocomposites were prepared by mixing polyethyleneimine (PEI) modified reduced graphene oxide (rGO) with AgNO3 solution. The prepared rGO-PEI-Ag nanocomposites showed good redox activity and conductivity, as characterized by ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), and X-ray diffraction (XRD). During the preparation process, staphylococcal protein A (SPA) was added to targetedly bind the Fc segment of the monoclonal antibody. The immunosensor showed a low detection limit (LOD) of 0.0013 ng/mL, a linear range of 0.01-100 ng/mL, and recoveries ranging from 92.77 to 100.02% in pork and 92.26-101.23% in milk. Furthermore, the immunosensor exhibited good stability, reproducibility, and specificity in detecting TMC in pork and milk real samples.
Asunto(s)
Técnicas Electroquímicas , Contaminación de Alimentos , Grafito , Límite de Detección , Leche , Nanocompuestos , Plata , Tilosina , Grafito/química , Nanocompuestos/química , Animales , Leche/química , Plata/química , Contaminación de Alimentos/análisis , Porcinos , Tilosina/análogos & derivados , Tilosina/análisis , Tilosina/química , Polietileneimina/química , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Técnicas Biosensibles , Antibacterianos/análisis , Antibacterianos/químicaRESUMEN
Icariside II, a flavonoid glycoside, is the main component found invivo after the administration of Herba epimedii and has shown some pharmacological effects, such as prevention of osteoporosis and enhancement of immunity. Increased levels of marrow adipose tissue are associated with osteoporosis. S100 calcium-binding protein A16 (S100A16) promotes the differentiation of bone marrow mesenchymal stem cells (BMSCs) into adipocytes. This study aimed to confirm the anti-lipidogenesis effect of Icariside II in the bone marrow by inhibiting S100A16 expression. We used ovariectomy (OVX) and BMSC models. The results showed that Icariside II reduced bone marrow fat content and inhibited BMSCs adipogenic differentiation and S100A16 expression, which correlated with lipogenesis. Overexpression of S100A16 eliminated the inhibitory effect of Icariside II on lipid formation. ß-catenin participated in the regulation adipogenesis mediated by Icariside II/S100A16 in the bone. In conclusion, Icariside II protects against OVX-induced bone marrow adipogenesis by downregulating S100A16, in which ß-catenin might also be involved.