Molecular Characterization and Functional Analysis of a Schistosoma mansoni Serine Protease Inhibitor, Smserpin-p46.

Serine protease inhibitors are a superfamily of proteins that regulate various physiological processes including fibrinolysis, inflammation and immune responses. In parasite systems, serpins are believed to play important roles in parasite colonization, inhibition of host immune serine proteases and penetration of defensive barriers. However, serpins are less well characterized in schistosomes. In this study, a Schistosoma mansoni serpin (Smserpin-p46) containing a 1360 base pair open reading frame, was cloned, expressed and functionally characterized. Bioinformatics analysis revealed that Smserpin-p46 contains the key residues, structural domains and motifs characteristic of inhibitory serpins. Gene expression profiling demonstrated stage-specific expression of Smserpin-p46 with the highest expression in adult male worms. Recombinant Smserpin-p46 (rSmserpin-p46) inhibited both human neutrophil cathepsin G and elastase, key serine proteases involved in NETosis, a program for the formation of neutrophil extracellular traps. Using specific rabbit antiserum, Smserpin-p46 was detected in soluble worm antigen preparation and was localized to the adult worm tegument. Cumulatively, the expression of Smserpin-p46 on the parasite tegument and its ability to inhibit proteases involved in NETosis highlights the importance of this serpin in parasite-host interactions and encourages its further investigation as a candidate vaccine antigen for the control of schistosomiasis.

Cost-effectiveness analysis of baricitinib versus dupilumab for moderate to severe atopic dermatitis: an Italian healthcare system perspective.

AIMS: To assess, within the Italian healthcare system, the cost-effectiveness of baricitinib versus dupilumab, both in combination with topical corticosteroids (TCS), in adults with moderate to severe atopic dermatitis (AD) who are eligible for but have failed, have contraindications to, or cannot tolerate ciclosporin. MATERIALS AND METHODS: Using the perspective of the Italian healthcare payer, direct medical costs associated with each intervention were estimated over a lifetime horizon. A Markov cohort model utilized the proportions of patients with ≥75% improvement Eczema Area and Severity Index obtained from clinical trials. Health outcomes were evaluated in quality-adjusted life years (QALYs) to assess the cost effectiveness of baricitinib against a willingness-to-pay threshold of €35,000 per QALY gained. RESULTS: In the base case, with secondary censoring applied, patients treated with dupilumab or baricitinib, in combination with TCS, accumulated total costs of €135,780 or €129,586, and total QALYs of 18.172 or 18.133, respectively. The incremental cost-effectiveness ratio of dupilumab versus baricitinib was estimated at €160,905/QALY. LIMITATIONS: Core assumptions were needed to extrapolate available short-term clinical trial data to lifelong data, adding uncertainty. Benefits of baricitinib seen in clinical trials and not assessed in dupilumab clinical trials were not included. Discontinuation rates for each treatment were derived from different sources potentially introducing bias. Results may not be generalizable to other populations. CONCLUSIONS: This cost-effectiveness analysis shows that, from the Italian healthcare payer perspective, in the treatment of patients with moderate to severe AD who have experienced failure on, are intolerant to, or have contraindication to ciclosporin, dupilumab cannot be considered cost-effective when compared with baricitinib. Given its oral administration, favorable risk/benefit profile and lower acquisition cost compared with dupilumab, baricitinib may offer a valuable, cost-effective treatment option-after failure on conventional systemic agents-for patients with moderate to severe AD in Italy.

Baricitinib is the first oral systemic treatment for patients with moderate to severe atopic dermatitis (AD). The drug was effective for treating patients with AD in clinical trials, producing improvements in skin inflammation, itch, sleep disturbances due to itch and skin pain, as well as the quality of life of patients. However, it is important to ensure that healthcare funds are well spent. We therefore compared the cost-effectiveness of baricitinib, with another new systemic treatment for AD, dupilumab, (both in combination with topical corticosteroids) in patients with moderate to severe AD who are eligible for but have failed or are unable to take ciclosporin, in Italy. We found that using dupilumab to treat these patients with AD cost more than using baricitinib, although dupilumab was more effective. Combining these considerations showed that the cost of obtaining the additional benefit from dupilumab over baricitinib was not cost-effective for the Italian healthcare system. Baricitinib may be a better treatment option because it is given orally, has a favorable balance between the risks and benefits of treatment, and costs less than dupilumab.

Burden of illness associated with overweight and obesity in patients hospitalized with COVID-19 in the United States: analysis of the premier healthcare database from April 1, 2020 to October 31, 2020.

BACKGROUND: SARS-CoV-2 (COVID-19) continues to be a major public health issue. Obesity is a major risk factor for disease severity and mortality associated with COVID-19. OBJECTIVE: This study sought to estimate the healthcare resource use and cost outcomes in patients hospitalized with COVID-19 in the United States (US) according to body mass index (BMI) class. METHODS: Retrospective cross-sectional study analyzing data from the Premier Healthcare COVID-19 database for hospital length-of-stay (LOS), intensive care unit (ICU) admission, ICU LOS, invasive mechanical ventilator use, invasive mechanical ventilator use duration, in-hospital mortality, and total hospital costs from hospital charge data. RESULTS: After adjustment for patient age, gender, and race, patients with COVID-19 and overweight or obesity had longer durations for mean hospital LOS (normal BMI = 7.4 days, class 3 obesity = 9.4 days, p < .0001) and ICU LOS (normal BMI = 6.1 days, class 3 obesity = 9.5 days, p < .0001) than patients with normal weight. Patients with normal BMI had fewer days on invasive mechanical ventilation compared to patients with overweight and obesity classes 1-3 (6.7 days vs. 7.8, 10.1, 11.5, and 12.4, respectively, p < .0001). The predicted probability of in-hospital mortality was nearly twice that of patients with class 3 obesity compared to patients with normal BMI (15.0 vs 8.1%, p < .0001). Mean (standard deviation) total hospital costs for a patient with class 3 obesity is estimated at $26,545 ($24,433-$28,839), 1.5 times greater than the mean for a patient with a normal BMI at $17,588 ($16,298-$18,981). CONCLUSIONS: Increasing levels of BMI class, from overweight to obesity class 3, are significantly associated with higher levels of healthcare resource utilization and costs in adult patients hospitalized with COVID-19 in the US. Effective treatment of overweight and obesity are needed to reduce the burden of illness associated with COVID-19.

The COVID-19 pandemic has caused many people to be seriously ill. People who are overweight are more likely to get sicker from COVID-19 infection and to require hospitalization.In our study, we compared patients who have normal weight to people who have overweight or obesity to understand how excess weight affects their experiences with COVID-19. We looked at: (1) how overweight and obesity is related to how long patients with COVID-19 stay in the hospital, (2) if they stayed in the intensive care unit (ICU) and how long they spent there, (3) whether they needed help breathing with the use of a ventilator and how long they needed a ventilator, (4) if they died during their hospital stay, and (5) how much their hospital stay cost.We found that people who have overweight or obesity stayed in the hospital longer, were more likely to need to stay in the ICU, and were in the ICU longer. They were also more likely to need help breathing with the use of a ventilator and needed that help for a longer time. People who have overweight or obesity died during their hospital stay more often than people with a normal BMI. The costs associated with people who have overweight or obesity were higher than people who have a normal BMI.Overall, this study shows that having overweight or obesity is a significant risk factor for poor outcomes from COVID-19 infection. Treatment for obesity and overweight is needed to help improve outcomes from future pandemics.

A discrete choice experiment investigating HIV testing preferences in South Africa.

BACKGROUND: South Africa (SA) has the world's highest burden of HIV infection, with an estimated 13.7% of the population living with HIV (PLWH/Persons Living With HIV). The early identification of PLWH and rapid engagement of them in HIV treatment are indispensable tools in the fight against HIV transmission. Understanding client preferences for HIV testing may help improve uptake. This study aimed to elicit client preferences for key characteristics of HIV testing options. METHODS: A discrete-choice experiment (DCE) was conducted among individuals presenting for HIV testing at two public primary healthcare facilities in Cape Town, South Africa. Participants were asked to make nine choices between two unlabeled alternatives that differed in five attributes, in line with previous DCEs conducted in Tanzania and Colombia: testing availability, distance from the testing center, method for obtaining the sample, medication availability at testing centers, and confidentiality. Data were analyzed using a random parameter logit model. RESULTS: A total of 206 participants agreed to participate in the study, of whom 199 fully completed the choice tasks. The mean age of the participants was 33.6 years, and most participants were female (83%). Confidentiality was the most important attribute, followed by distance from the testing center and the method of obtaining a sample. Patients preferred finger prick to venipuncture as a method for obtaining the sample. Medication availability at the testing site was also preferred over a referral to an HIV treatment center for a positive HIV test. There were significant variations in preferences among respondents. CONCLUSION: In addition to accentuating the importance of confidentiality, the method for obtaining the sample and the location of sites for collection of medication should be considered in the testing strategy. The variations in preferences within target populations should be considered in identifying optimal testing strategies.

The improved survival rate and cost-effectiveness of a 7-day continuous subcutaneous insulin infusion set.

AIMS: The purpose of this article is to compare the insulin cost-savings of the Medtronic Extended Infusion Set (or EIS, a.k.a. Extended Wear Infusion Set) designed and labeled for up to 7-day use with rapid-acting insulins to the current standard of care, 2- to 3-day infusion sets. METHODS: There are three major improvements (reducing insulin waste, plastic waste, and adverse events) with the extended duration of infusion set wear. This analysis focuses on cost savings from reduced insulin wastage during set changes. Studies published on insulin infusion set survival and EIS clinical trial data (NCT04113694) were used to estimate device lifetime performance using a Markov chain Monte Carlo model, including the assessment of adverse effects and device failure. Total costs associated with infusion set change or failure were systematically found in published literature or estimated based on physical usage, and the direct impact on insulin costs was calculated. RESULTS: Based on the model and clinical data, EIS users can expect to change their infusion sets about 75 fewer times than standard set users each year. The costs related to unrecoverable insulin during an infusion set and reservoir change in the US were estimated to range from $19.79 to $22.48, resulting in approximately $1324 to $1677 in annual cost-savings for the typical user from minimizing insulin wastage. LIMITATIONS: The study only assessed devices used within a monitored setting, that is, clinical trials. In addition, the variability associated with healthcare standards and costs and individual treatment variability including insulin dosages, contribute to the uncertainties with the calculations. CONCLUSIONS: Our analysis demonstrates that by extending the duration of infusion set wear, there may be substantial cost savings by reducing insulin wastage.

Setting and maintaining standards for patient-reported outcome measures: can we rely on the COSMIN checklists?

As test-developers we have often been troubled by published reviews of patient-reported outcome measures (PROMs). Too often minor issues are judged important while other reviews exclude the best measures available. Perhaps this led several groups to make recommendations for evaluating the quality of PROMs. The COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist is the latest set of recommendations. While reviewing the COSMIN literature and reviews conducted using their recommendations several concerns became apparent. The checklist is not evidence-based, relying on the opinion of researchers experienced in health-related quality of life. PROMs measuring other types of outcomes are inadequately covered by the checklist. COSMIN choose to focus on Classical Test Theory and the checklists are not appropriate for use with PROMs developed using modern measurement. Such an approach only obstructs progress in the field of outcome measurement. The retrospective nature of the evaluations also penalizes new PROMs. While the checklists imply that composite, ordinal level measurement is acceptable, crucial aspects of instrument development and quality are excluded. Reviews based on the COSMIN checklist produce contradictory conclusions and fail to provide evidence to support the recommendations. These problems suggest that the checklists themselves lack reliability and validity. It is also clear that several reviewers lack the expertise to apply the checklists. Researchers require a good grounding in instrument development and psychometrics to produce quality reviews. The science of modern PROM development is still in an early phase. Few available PROMs have sufficient quality, limiting the need for complex reviews. Standards need to be agreed for high quality outcome measurement. The issue is who should set these standards? Most published reviews merely scratch the surface and lack essential detail. All reviews of PROMs should be treated with caution, irrespective of whether the COSMIN checklist was employed.

Src homolog and collagen homolog1 isoforms in acute and chronic liver injuries.

Src homolog and collagen homolog (SHC) proteins are adaptor proteins bound to cell surface receptors that play an important role in signal transduction and related diseases. As an important member of the SHC protein family, SHC1 regulates cell proliferation and apoptosis, reactive oxygen species (ROS) production, and oxidative stress. Three isomeric proteins namely, p46shc, p52shc, and p66shc, are produced from the same SHC1 gene locus. All the three proteins are found in the liver, and are widely expressed in various hepatic cells. SHC1 has been proven to be associated with acute and chronic liver injuries of different etiologies, and plays important roles in liver fibrosis and hepatocellular carcinoma (HCC). Therefore, this review summarizes recent studies that discuss and explore the role of SHC1 in the occurrence and progression of liver diseases. We also provide a theoretical basis for future studies.

Willingness to pay for primary health care at public facilities in the Western Cape Province, Cape Town, South Africa.

BACKGROUND AND OBJECTIVES: As facilities are being prepared for the implementation of National Health Insurance (NHI) in South Africa, there is a pressing need to understand how the public equates the provision of health services at Primary Health Care (PHC) centres with monetary value. Accordingly, this exploratory study was designed to ascertain the willingness to pay (WTP) for public primary healthcare services in South Africa and to identify factors that influence the WTP. METHODS: The study was conducted in Cape Town, South Africa, among 453 persons presenting at two public primary health care centres, namely Bothasig Community Day Centre (CDC) and Goodwood CDC. The study used the contingent valuation range methodology. Descriptive statistics, multiple logistic and tobit regression analyses were conducted to assess demographics, socio-economic, and health access factors that influence WTP. RESULTS: Overall, 60% of participants were willing to pay for services offered at the PHC facilities. The average willingness to pay for all participants was 49.44 ZAR, with a median of 25 ZAR. The multiple logistic regression for grouped facilities showed unemployment, public transport, and the facility attended to be significant while public transport, facility visits, and facility attended were the only significant variables in the tobit model. There was less willingness to pay for those unemployed in comparison with students, those using public transport rather than walking, those frequenting the facilities more than first-time visitors and those attending Goodwood facility in comparison with Bothasig. CONCLUSION: This study revealed factors related to the participants' WTP and to their willingness to contribute towards the health service, though at very low amounts. Understanding the economic value placed upon a service provided in a facility is essential in decision-making for quality care improvements, especially as the South African health system is making the facilities ready for NHI.

Structure of P46, an immunodominant surface protein from Mycoplasma hyopneumoniae: interaction with a monoclonal antibody.

Mycoplasma hyopneumoniae is a prokaryotic pathogen that colonizes the respiratory ciliated epithelial cells in swine. Infected animals suffer respiratory lesions, causing major economic losses in the porcine industry. Characterization of the immunodominant membrane-associated proteins from M. hyopneumoniae may be instrumental in the development of new therapeutic approaches. Here, the crystal structure of P46, one of the main surface-antigen proteins, from M. hyopneumoniae is presented and shows N- and C-terminal α/ß domains connected by a hinge. The structures solved in this work include a ligand-free open form of P46 (3.1 Šresolution) and two ligand-bound structures of P46 with maltose (2.5 Šresolution) and xylose (3.5 Šresolution) in open and closed conformations, respectively. The ligand-binding site is buried in the cleft between the domains at the hinge region. The two domains of P46 can rotate with respect to each other, giving open or closed alternative conformations. In agreement with this structural information, sequence analyses show similarities to substrate-binding members of the ABC transporter superfamily, with P46 facing the extracellular side as a functional subunit. In the structure with xylose, P46 was also bound to a high-affinity (Kd = 29 nM) Fab fragment from a monoclonal antibody, allowing the characterization of a structural epitope in P46 that exclusively involves residues from the C-terminal domain. The Fab structure in the complex with P46 shows only small conformational rearrangements in the six complementarity-determining regions (CDRs) with respect to the unbound Fab (the structure of which is also determined in this work at 1.95 Šresolution). The structural information that is now available should contribute to a better understanding of sugar nutrient intake by M. hyopneumoniae. This information will also allow the design of protocols and strategies for the generation of new vaccines against this important swine pathogen.

Mucosal and systemic immune responses induced by intranasal immunization of recombinant Bacillus subtilis expressing the P97R1, P46 antigens of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-γ in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae.

Guidelines-based treatment associated with improved economic outcomes in nontuberculous mycobacterial lung disease.

Background: The prevalence of nontuberculous mycobacterial lung disease (NTMLD) in the US has increased; however, data characterizing the associated healthcare utilization and expenditure at the national level are limited. Objective: To examine associations between economic outcomes and the use of anti-Mycobacterium avium complex (MAC) guidelines-based treatment (GBT) for newly-diagnosed NTMLD in a US national managed care claims database (Optum® Clinformatics® Data Mart). Methods: NTMLD was defined as having ≥2 claims for NTMLD (ICD-9 031.0; ICD-10 A31.0) on separate occasions ≥30 days apart (between 2007 and 2016). The cohort included patients insured continuously over a period of at least 36 months (12 months before initial NTMLD diagnostic claim and for the subsequent 24 months). Treatment was classified as GBT (consistent with American Thoracic Society/Infectious Diseases Society of America guidelines), non-GBT, or untreated. All-cause hospitalization rates and total healthcare expenditures at Year 2 were assessed as outcomes of the treatment prescribed in Year 1 after NTMLD diagnosis. Results: A total of 1,039 patients met study criteria for NTMLD (GBT, n = 294; non-GBT, n = 298; untreated, n = 447). After adjustment for baseline characteristics, GBT was associated with a significantly lower all-cause hospitalization risk vs non-GBT (odds ratio [OR] = 0.53; 95% CI = 0.33-0.85, p = 0.008), and vs being untreated (OR = 0.57; 95% CI = 0.35-0.91, p = 0.020). Adjusted total healthcare expenditure in Year 2 with GBT ($69,691) was lower than that with non-GBT ($77,624) with a difference of -$7,933 (95% CI = -$14,968 to -$899; p = 0.03). Conclusions: Patients with NTMLD in a US managed care claims database who were prescribed GBT had lower hospitalization risk than those who were prescribed non-GBT or were untreated. GBT was associated with lower total healthcare expenditure compared with non-GBT.

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association.

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines.

PURPOSE: Pax-6 is a master regulator for eye and brain development. Previous studies including ours have shown that Pax-6 exists in 4 major isoforms. According to their sizes, they are named p48, p46, p43 and p32 with the corresponding molecular weight of 48, 46, 43 and 32 kd, respectively. While p48 and p46 is derived from alternative splicing, p32 Pax-6 is generated through an internal translation initiation site. As for 43 kd Pax-6, two resources have been reported. In bird, it was found that an alternative splicing can generate a p43 Pax-6. In human and mouse, we reported that the p43 kd Pax-6 is derived from sumoylation: addition of a 11 kd polypeptide SUMO1 into the p32 Pax-6 at the K91 residue. Whether other Pax-6 isoforms can be sumoylated or not remains to be explored. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: Both non-sumoylated and sumoylated isoforms of Pax-6 exist in 6 major types of ocular cells among which five are lens epithelial cells, and one is retinal pigment epithelial cell. Our results revealed that the most abundant isoforms of Pax-6 are the p32 and p46 Pax-6. These two major isoforms can be sumoylated to generate p43 (mono-sumoylated p32 Pax-6), p57 and p68 Pax-6 (mono- and di-sumoylated p46 Pax-6). In addition, the splicing-generated p48 Pax-6 is also readily detected. CONCLUSION: Our results for the first time, have determined the relative isoform abundance and also the sumoylation patterns of pax-6 in 6 major ocular cell lines.

Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients.

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria.

Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo.

Geographical sequence variation in the Leishmania major virulence factor P46.

Cutaneous leishmaniasis as caused by Leishmania major is a zoonotic infection with wide epidemiological impact. The L. major P46 virulence gene was shown to boost the parasite's virulence and extends its range of experimental hosts. Here we show that P46 is subject to significant geographical sequence variations that may reflect the adaption to different reservoir hosts. This view is supported by the results of passage experiments using P46 variants in different experimental hosts. Conversely, loss of P46 expression leads to attenuation both in vitro and in BALB/c mice. Although part of the L. major exosomal protein payload, P46 is not required for exosome-mediated immune modulation.

Contribuição ao estudo polarográfico de naftilazoderivados / Contribution to the polarographic study of naphthylazoderivatives

Análise polarográfica de naftilazo derivados de sulfamidas com núcleo pirimidínico é descrita (AU).