Protein Antibodies Research Articles

Can SARS-CoV-2 Infection Status in Preschool Children be Predicted by Pandemic-related Attitudes and Concerns of their Parents? Data from the Würzburg Daycare Centre Studies (Wü-KiTa-CoV, 2020-2022)

As part of the Würzburg KiTa-CoV study series, SARS-CoV-2 pandemic-related attitudes/concerns of parents of preschool children attending day care centres were surveyed over a 21-month period. We expected associations between these parental attitudes/concerns and their change over the course of the pandemic, on the one hand, and the SARS-CoV-2 infection status of the children, on the other. Parents of children from nine day-care centres who completed a survey on pandemic-related attitudes/concerns in October 2020, July 2021 and July 2022, were included in the analysis. In July 2022, the children's infection status was determined by measuring nucleocapsid protein antibodies (N-AK) and, in the case of N-AK negative, unvaccinated children, the measurement results of spike protein antibodies (S-AK) against SARS-CoV-2 as indicators of previous infection were also taken into account. Changes in parental attitudes/concerns were analysed using trend tests, and their significance in predicting the child's infection status was analysed using multiple binomial regression analyses. Data were available from parents of 159 children (mean age 3.2 years). The overall group of parents showed an increasingly relaxed attitude towards the pandemic over the study period, although the parents' concerns towards a possible infection of their own increased. The infection status of the children could not be predicted from the initial attitudes and concerns of the parents. However, the subgroup of parents with an uninfected child tended to have a higher level of concerns over the course of the pandemic than the parents with an infected child and did not show the same increasing composure towards the pandemic event. The parents' increasing composure is understandable in the context of the reduced virulence of the virus variants prevailing over time, the increasing availability of testing strategies and vaccinations, and the withdrawal of pandemic-related restrictions. In addition, the mildness of most children's infections may have contributed to their parents' sense of security. The seemingly contradictory increase in parental concerns about infection may be related to the strong increase in the incidence due to omicron variant infections in the population since January 2022. It is possible that the higher level of concerns among parents of uninfected children had a protective effect behaviourally.

Enhancing De Novo Protein Sequencing through the C-Terminal Labeling Strategy: Resolving Isobaric Ambiguities by Electron-Transfer/Higher Energy Collision Dissociation (EThcD).

De novo protein sequencing via a bottom-up approach requires various proteases to produce overlapping peptides. However, peptides generated by proteases other than trypsin, LysC, and ArgC often yield C-terminal fragments with suboptimal ionization in positive mode mass spectrometry (MS). This study introduces a novel peptide labeling strategy that involves modifying peptides at the C-terminal and at the carboxyl groups of Aspartic and Glutamic acid with arginine methyl ester (R-met) to improve peptide fragmentation and resolve isobaric ambiguities encountered during sequencing. An amidation reaction is used with coupling reagents to conjugate R-met to the peptide's C-terminal end, introducing a functional group that enhances the detectability of C-terminal peptide fragment ions by mass spectrometry. Subsequently, selecting a charge state of +2 or higher can facilitate optimal fragmentation of the derivatized peptides using electron-transfer/higher energy collision dissociation (EThcD), thereby generating essential w-ions to resolve common isobaric ambiguities. Demonstrating this strategy across diverse protein types, including albumin and antibodies and using different proteases for digestion, highlights the unique characteristics of combining the proposed amidation reaction with the specific proteases tested.

Production of mRNA lipid nanoparticles using advanced crossflow micromixing.

Lipid nanoparticles (LNPs) play a crucial role in RNA-based therapies, and their production is generally based on nanoprecipitation and coalescence of lipids around an RNA core. This study investigated crossflow micromixing to prepare LNPs across various mixing ratios and production speeds. A range of LNPs were prepared using crossflow micromixing across production speeds of 10-500ml/min, and their physico-chemical characteristics (size, polydispersity index (PDI), zeta potential, and mRNA encapsulation), in vitro mRNA expression and in vitro efficacy (protein expression and antibody and cytokine responses). Our results demonstrate the reproducible production of mRNA-LNPs with controlled critical quality attributes, including high mRNA encapsulation from the initial screening scale through to GMP-scale production, where the same mixing ratio can be adopted across all product speeds from 30 to 500ml/min used. We confirm the applicability of stainless-steel crossflow membrane micromixing for the entire spectrum of mRNA-LNP production, ranging from initial discovery volumes to GMP-production scale.

8234 Composite Pheochromocytoma-Ganglioneuroma: Two Clinical Cases Presenting To St. Elizabeth’s Medical Center

Abstract Disclosure: M. El Najjar: None. E. Naous: None. C. Blake: None. A.T. Sweeney: None. Introduction: Pheochromocytomas (PCCS) are rare catecholamine producing tumors that originate from the chromaffin cells of the adrenal medulla. In 3-9% of cases, they may be accompanied by other tumors. Ganglioneuromas are the most encountered tumor type that occur with PCC and are termed composite PCC-GN. The entity is very rare with < 35 cases reported. Herein, we present 2 cases of composite PCC-GN. Case Presentation: Patient 1 (Pt1) & Patient 2 (Pt2): were 35- & 54-year-old obese females. Pt1 had controlled hypertension and symptoms of anxiety along with palpitations, diaphoresis and flushing. Pt2 had type 2 diabetes and complained of abdominal pain. Metabolic studies showed elevated total plasma fractionated metanephrines(MNs). MNs in Pt1 and Pt2 were: 76 and 61pg/ml respectively (normal <57 pg/ml) and normetaneprines levels were 161 and 116pg/ml respectively (normal <148 pg/ml). Abdominal computed tomography (CT) scans demonstrated right adrenal masses in both cases with Pt1 having a 2.3 x 2.3 x 2.6 cm mass (Figure1) with smooth margins measuring 36 Hounsfield units (HU) on non-contrast(NC) CT imaging with 0% washout on delayed imaging after IV contrast. Pt2 on NCCT had a 4.5 x 3.5 cm right adrenal mass measuring 73 HU and a subsequent abdominal MRI(Figure2) demonstrated the mass to have heterogenous signal intensity. Both patients were prepared with pre-operative a and b blockade and underwent laparoscopic robotic adrenalectomies without complications. Pathological analyses in both cases revealed composite PCC-GN. Pt2 underwent genetic testing which did not reveal any known mutations. Surveillance in both patients demonstrated no recurrence. Clinical lesson: The clinical presentation of PCC-GNs is indistinguishable from isolated PCCs. The definitive diagnosis is through histopathological analysis. Histologically the composite tumor is composed of two distinct patterns with PCC showing angular cells with granular, basophilic cytoplasm, arranged in nests, while GNs show ganglion cells in fibrous stroma. Immunohistochemistry show the PCCs staining for chromogranin A and synaptophysin while GNs stain for S-100 protein and neurofilament antibody. Most cases occur in patients between 40 - 60 years old. Most PCC-GNs are functional, with increased MNs. PCC-GN composite tumors may occur sporadically but have also been described as part of syndromes such as MEN2A and NF1. The definitive treatment for PCC-GN is complete surgical excision which is associated with an excellent prognosis. The probability of recurrence is low though distant metastases have rarely been reported. Therefore, lifetime surveillance is critical. Presentation: 6/3/2024

Flour Treatments Affect Gluten Protein Extractability, Secondary Structure, and Antibody Reactivity

Commercial Brazilian wheat flour was subjected to extrusion, oven, and microwave treatments. The solubility, monomeric and polymeric proteins, and the glutenin and gliadin profiles of the gluten were analyzed. In addition, in vitro digestibility and response against potential celiac disease immune-stimulatory epitopes were investigated. All treatments resulted in low solubility of the polymeric and monomeric proteins. The amounts of insoluble proteins increased from 5.6% in control flour to approximately 10% for all (treatments), whereas soluble proteins decreased from 6.5% to less than 0.5% post treatment. In addition, the treatments affected glutenin and gliadin profiles. The amount of α/β-gliadin extracted decreased after all treatments, while that of γ-gliadin was unaffected. Finally, the potential celiac disease immune stimulatory epitopes decreased in oven and microwave treatment using the G12 ELISA, but no change was observed using the R5 antibody. However, the alteration of the gluten structure and complexity was not sufficient to render a product safe for consumption for individuals with celiac disease; the number of potential celiac disease immune-stimulatory epitopes remained high.

The effect of Plasmodium falciparum exposure and maternal anti-circumsporozoite protein antibodies on responses to RTS,S/AS01E vaccination in infants and children: an ancillary observational immunological study to a phase 3, randomised clinical trial

The RTS,S/AS01E malaria vaccine showed lower antibody response and protective efficacy in infants aged 6-12 weeks compared with children aged 5-17 months (for whom this vaccine is recommended). We aimed to study the effect of previous Plasmodium falciparum exposure on the antibody responses to RTS,S/AS01E vaccination in infants and children, and the mediating effect of baseline (including maternal) anti-circumsporozoite protein (CSP) antibodies. In this observational study, we included children and infants from six African countries (Burkina Faso, Gabon, Ghana, Kenya, Mozambique, and Tanzania) enrolled in the MAL067 immunology ancillary study of the RTS,S/AS01E phase 3 clinical trial from March 27, 2009, to Jan 21, 2011. We used comparator-vaccinated infants and children to identify antibody-based signatures of previous P falciparum exposure, which were later applied to RTS,S/AS01E-vaccinated infants and children. In these participants, we explored the relationship between vaccine antibody immunoglobulin G (IgG) responses measured by ELISA and pre-vaccination serological markers of malaria exposure by assessing the IgG levels against 1000 P falciparum antigens using partial proteome microarrays. We included 718 comparator-vaccinated infants (348 [48%]) and children (370 [52%]) and 606 RTS,S/AS01E-vaccinated infants (329 [54%]) and children (277 [46%]). Anti-CSP IgG responses to primary vaccination did not correlate with a baseline signature of previous exposure in children, suggesting that prior P falciparum exposure does not significantly affect antibody immunogenicity in children (Pearson's r=-0·02 [95% CI -0·13 to 0·10]). By contrast, high P falciparum exposure signature levels at the time of vaccination in infants, presumably driven by maternally transferred antibodies and declining within the initial 6-12 months of life, correlated with reduced RTS,S/AS01E responses (r=-0·17 [-0·27 to -0·06]). This negative correlation was stronger for anti-CSP IgG than for the exposure signature or any other more immunogenic blood stage P falciparum antigens (r=-0·42 [-0·50 to -0·33]), persisted after adjustment by baseline levels of the exposure signature (semi-partial correlation r=-0·44 [-0·55 to -0·33]), and involved antibodies to the central NANP region (r=-0·39 [-0·49 to -0·28]) but not the C-terminal region (r=0·02 [-0·10 to 0·15]) of CSP. The negative effect of maternal anti-CSP IgG in infants did not appear to be confounded by other malaria transmission-dependent variables. Interference between passive immunity and vaccine response is clinically significant and might affect the implementation of next-generation CSP-based vaccines for young infants and mothers as well as passive immunisation with human monoclonal antibodies. US National Institutes of Health, National Institute of Allergy and Infectious Diseases; PATH-Malaria Vaccine Initiative; Spanish Ministerio de Economía y Competitividad (Instituto de Salud Carlos III), European Regional Development Fund and European Social Fund; Fundación Ramón Areces; Spanish Ministry of Science and Innovation; and Generalitat de Catalunya (CERCA Program).

Preparation of bovine coronavirus virus-like particles and its immunogenicity in mice and cattle

The widespread prevalence of bovine coronavirus (BCoV) disease worldwide has impacted the livestock industry economically. No effective vaccine is available in China. In this study, we produced BCoV virus-like particles (VLPs) containing E, M, N, S, and hemagglutinin-esterase (HE) proteins using a baculovirus expression system. Five recombinant baculoviruses were co-infected with Sf9 cells, and the VLPs were assembled and characterized. Mice and cattle were immunized by VLPs mixed with MF59 and CpG 55.2 adjuvants. Two doses of the VLPs/MF59/CpG vaccine were administered in mice and cattle. The immune effect of the VLPs/MF59/CpG vaccine was measured using indirect ELISA and neutralization assays. After immunization, the serum IgG-specific antibody titer against S protein and neutralization antibody titer increased to 1:1.28 × 104 (p < 0.01) and 1:128 (p < 0.01) in mice, respectively. Interestingly, the high IgG antibody and neutralizing antibody titers were maintained for seven days in mice. In addition, the serum IgG-specific antibody titer against S proteins and neutralization antibody titer increased to 1:1.024 × 105 and 1:512 (p < 0.05) in cattle, respectively. The high IgG antibody and neutralizing antibody titers were maintained for 21 days in cattle. Notably, BCoV VLPs group interferon-gamma (IFN-γ) lymphocytes in spleens were significantly increased (p < 0.01). These findings suggest that BCoV VLPs induced strong cellular and humoral immune responses in mice and cattle. These findings suggest that BCoV VLPs could serve as a potent immunogen for vaccine development.

A Case of the Anti-aquaporine-4 Antibody-negative Neuromyelitis Optica Spectrum Disorders Associated with Atopic Disease with High Concentration of Anti-IgE Autoantibody and HyperIgEemia

We report a 70-year-old male patient with the sero-negative neuromyelitis optica spectrum disorders (NMOSD) associated with atopic disease (AD). He was diagnosed with allergic rhinitis at the age of 20. When he was 61 years old, he subacutely developed orthostatic hypotension, bilateral optic neuritis, quadriparesis, urinary retention, and constipation. The laboratory results revealed allergen-specific IgE positivity for cryptomeria japonica and hinoki, hyperIgEemia, and Th (helper T cell) 1 dominance. The serological tests for autoantibodies revealed negative anti-aquaporine 4 antibody, and high concentration of anti-IgE autoantibody (anti-IgE AAb). Cerebrospinal fluid was negative for anti-myelin-oligodendrocyte glycoprotein antibody and glial fibrillary acidic protein antibody. Fluid-attenuated inversion recovery on brain magnetic resonance imaging (MRI) showed high signal intensities in bilateral cerebral deep white matter. T2 weighted image on spine MRI showed longitudinally extensive high signal intensities in the spinal cord, specifically involving C1 vertebral level to conus medullaris. Intravenous methylprednisolone (IVMP) and plasma exchange resulted in partial improvement. Following the onset of NMOSD, he had relapse of NMOSD four times. In each episode, IVMP was to be partially effective with anti-IgE AAb reduction. Anti-IgE AAb may be a reasonable clinical indicator of increased disease activity in the sero-negative NMOSD associated with AD.

Managing RS3PE: Diagnostic approach, initial steroid response, and long-term management with DMARDs

Background. RS3PE, or Remitting Seronegative Symmetrical Synovitis with Pitting Edema, is a unique and relatively rare form of inflammatory arthritis that predominantly affects older adults. It is distinguished by its hallmark features: symmetrical swelling of the joints, significant pitting edema in the extremities, and a negative serological profile for common markers associated with rheumatoid arthritis, such as rheumatoid factor (RA factor) and anti-citrullinated protein antibodies (anti-CCP). This condition is often characterized by a remitting course, meaning that symptoms may improve or resolve over time, particularly with appropriate treatment. The condition usually affects older adults and can sometimes be associated with other underlying health issues, such as malignancies or infections. The “remitting” aspect of RS3PE refers to the fact that the symptoms often improve or resolve over time, particularly with appropriate treatment, which usually involves corticosteroids or other anti-inflammatory medications. Case report. This case study details the clinical presentation, diagnosis, and management of a 56-year-old female patient with known hypertension who presented with bilateral dorsal hand swelling, pain, and early morning stiffness over a 15-day period. The patient’s initial examination revealed bilateral pitting edema and active arthritis in the proximal interphalangeal joints, with significant warmth and tenderness. Laboratory tests showed elevated ESR and CRP, normocytic normochromic anemia, and negative rheumatoid factor and anti-CCP antibodies. Imaging studies, including X-ray, ultrasound, and mammogram, showed no erosive changes or malignancy, while a Pap smear and serum CA-125 levels were within normal limits. The clinical picture, combined with the exclusion of other conditions, supported the diagnosis of RS3PE. Treatment commenced with intravenous low-dose dexamethasone, which led to significant symptomatic improvement. This was followed by a transition to oral prednisolone, and the patient initially showed complete resolution of symptoms. However, recurrence of symptoms occurred upon discharge, prompting the introduction of steroid-sparing agents: methotrexate and hydroxychloroquine, alongside continued oral steroid treatment. Over a two-month follow-up period, the patient experienced no signs of relapse. This case fits the clinical and investigative criteria for RS3PE. The patient showed symptomatic improvement after treatment with low-dose steroids.

Quantum Dot-Based Nanosensors for In Vitro Detection of Mycobacterium tuberculosis.

Despite the existing effective treatment methods, tuberculosis (TB) is the second most deadly infectious disease, its carriers in the latent and active phases accounting for more than 20% of the world population. An effective method for controlling TB and reducing TB mortality is regular population screening aimed at diagnosing the latent form of TB and taking preventive and curative measures. Numerous methods allow diagnosing TB by directly detecting Mycobacterium tuberculosis (M.tb) biomarkers, including M.tb DNA, proteins, and specific metabolites or antibodies produced by the host immune system in response to M.tb. PCR, ELISA, immunofluorescence and immunochemical analyses, flow cytometry, and other methods allow the detection of M.tb biomarkers or the host immune response to M.tb by recording the optical signal from fluorescent or colorimetric dyes that are components of the diagnostic systems. Current research in biosensors is aimed at increasing the sensitivity of detection, a promising approach being the use of fluorescent quantum dots as brighter and more photostable optical tags. Here, we review current methods for the detection of M.tb biomarkers using quantum dot-based nanosensors and summarize data on the M.tb biomarkers whose detection can be made considerably more sensitive by using these sensors.

Analysis of Beyfortus® (Nirsevimab) Immunization Campaign: Effectiveness, Biases, and ADE Risks in RSV Prevention.

Respiratory infections with respiratory syncytial virus (RSV) account for an important part of hospital admissions for acute respiratory infections. Nirsevimab has been developed to reduce the hospital burden of RSV infections. Compared with the product previously used, it has a stronger binding capacity to RSV F protein and a high affinity for FcRn (neonatal receptor for the Fc fragment of IgG), which extends its lifespan. Nirsevimab has been shown to be highly effective in reducing hospitalization rates of RSV infections but a large or unknown number of treated subjects have been excluded in clinical and post-marketing studies. However, analysis of these studies cannot exclude that, in rare cases, nirsevimab facilitates and worsens RSV infection (or other respiratory infections). This could be attributable to antibody-dependent enhancement (ADE) which has been observed with RSV F protein antibodies in inactivated vaccine trials. This risk has been incompletely assessed in pre-clinical and clinical trials (incomplete exploration of nirsevimab effector functions and pharmacokinetics). ADE by disruption of the immune system (not studied and due to FcRn binding) could explain why there is no reduction in all-cause hospital admissions in treated age groups. Given the high price of nirsevimab, the cost-effectiveness of mass immunization campaigns may therefore be debated from an economic as well as a scientific point of view.

Real-world treatment patterns and outcomes of patients with advanced melanoma treated with nivolumab plus relatlimab

Abstract The use of programmed death-1 (PD-1) inhibitors has been a pivotal advancement in treating advanced melanoma, yet their efficacy is limited. The approval of relatlimab (RELA), a lymphocyte activation gene 3 protein (LAG-3) antibody, in combination with nivolumab (NIVO), a PD-1 inhibitor, marked a significant stride toward enhancing treatment efficacy for metastatic and unresectable stage 3 and 4 melanoma. This combination has been shown to synergistically improve antitumor activity and effector T-cell activity in the tumor microenvironment, despite limited data on real-world outcomes. Our retrospective review at a tertiary cancer center of patients with stage 3 and 4 melanoma treated with NIVO-RELA revealed an overall response rate (ORR) of 39%, with notable improvements in median PFS and ORR, especially in first-line treated patients. Our study highlights the superior efficacy of NIVO-RELA over previous reports, demonstrating its significant potential in the treatment landscape of advanced melanoma.

A guide to selecting high-performing antibodies for Rab10 (UniProt ID: P61026) for use in western blot, immunoprecipitation, and immunofluorescence

Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson’s disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Nectin1 is a pivotal host factor involved in attachment and entry of red-spotted grouper nervous necrosis virus in the early stages of the viral life cycle.

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV.

A guide to selecting high-performing antibodies for CSNK1A1 (UniProt ID: P48729) for use in western blot, immunoprecipitation, and immunofluorescence

CSNK1A1 is a key regulator of various signalling pathways, including the Wnt/β-catenin pathway. Playing a central role in cellular function and disease pathology, CSNK1A1 has emerged as an attractive protein target for therapeutic development. In this study we characterize ten CSNK1A1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Enhanced Association of Novel Cardiovascular Biomarkers Fetuin-A and Catestatin with Serological and Inflammatory Markers in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with increased cardiovascular disease (CVD) risk and mortality. This work aimed to evaluate the serum levels of the novel CV biomarkers fetuin-A (fet-A), Dickkopf-1 (DKK-1), galectin-3 (Gal-3), interleukin-32 (IL-32), and catestatin (CST) in RA patients and their associations with RA parameters and CVD markers. A cohort of 199 RA patients was assessed for traditional CVD risk factors, RA disease activity, and biomarker levels. Carotid ultrasound was used to measure carotid intima-media thickness (cIMT) and carotid plaque presence (cPP). Multivariate analyses examined correlations between biomarkers and RA parameters, serological markers, and CVD markers. Adjusted models showed that elevated CST expression levels were associated with rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) positivity (OR = 2.45, p = 0.0001 and OR = 1.48, p = 0.04, respectively) in the overall cohort and for RF in men and women, respectively. In addition, fet-A concentration was inversely associated with the erythrocyte sedimentation rate (ESR) in the overall cohort (β = -0.15, p = 0.038) and in women (β = -0.25, p = 0.004). Fet-A levels were also negatively correlated with disease activity (DAS28-ESR) scores (β = -0.29, p = 0.01) and fibrinogen concentration (β = -0.22, p = 0.01) in women. No adjusted associations were observed for Gal-3, DKK-1 or IL32 concentration. The study revealed no significant associations between the biomarkers and cIMT or cPP. The measurement of CST and fet-A levels could enhance RA patient management and prognosis. However, the utility of biomarkers for evaluating CV risk via traditional surrogate markers is limited, highlighting the need for continued investigations into their roles in RA.

Immunogenicity and safety of different dose levels of Ad26.RSV.preF/RSV preF protein vaccine in adults aged 60 years and older: A randomized, double-blind, placebo-controlled, phase 2a study

BackgroundRespiratory syncytial virus (RSV) can cause severe illness in older adults. A combination vaccine containing Ad26.RSV.preF and purified recombinant RSV preF protein has previously demonstrated efficacy and tolerability in older adults. We report results of a dose-ranging study to determine immunogenicity and safety of different doses of the Ad26.RSV.preF component in the combined Ad26.RSV.preF/RSV preF protein vaccine to support Ad26.RSV.preF drug product release and stability specifications. MethodsIn this randomized, double-blind, placebo-controlled, phase 2a study, adults aged ≥60 years in good or stable health were randomly assigned within 1 of 3 cohorts to receive either placebo or Ad26.RSV.preF/RSV preF protein, composed of different doses of Ad26.RSV.preF with a fixed dose of RSV preF protein (150 μg). Ad26.RSV.preF doses in Cohort 1 (4 dose-down groups) ranged from 3.7 × 109 to 1.0 × 1011 viral particles (vp). Doses in Cohorts 2 and 3 (2 dose-up groups, each) ranged from 1.0 to 1.6 × 1011 vp. Primary endpoints were immunogenicity (RSV preF protein antibody titers) for Cohort 1 and safety (solicited local and systemic adverse events [AEs] and unsolicited AEs) for Cohorts 2 and 3. Immunogenicity analyses (RSV preF protein antibody titers, RSV A2 neutralizing antibodies, and RSV-F–specific interferon-γ enzyme-linked immunosorbent spot) were performed on the day of vaccination and 14 days, 3 months, and 6 months postvaccination. Safety was monitored from vaccination until study end. ResultsOverall, 454 participants were enrolled and received 1 dose of study vaccine or placebo (Cohort 1, n = 226; Cohort 2, n = 124; Cohort 3, n = 104). No substantial differences in measured immune responses were observed between lower or higher Ad26.RSV.preF doses compared with Ad26.RSV.preF 1.0 × 1011 vp across all postvaccination time points. All Ad26.RSV.preF doses between 3.7 × 109 vp and 1.6 × 1011 vp were well tolerated, with no safety issues identified. ConclusionsResults of this dose-ranging study may be used to inform the refinement of Ad26.RSV.preF drug product release and stability specifications.Clinical Trial Registration:ClinicalTrials.gov Identifier: NCT04453202

Immobilization coupling with aptamer assisted dual cycle amplification for sensitive sEVs isolation and analysis.

Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

Plug-and-play protein biosensors using aptamer-regulated in vitro transcription

Molecular biosensors that accurately measure protein concentrations without external equipment are critical for solving numerous problems in diagnostics and therapeutics. Modularly transducing the binding of protein antibodies, protein switches or aptamers into a useful output remains challenging. Here, we develop a biosensing platform based on aptamer-regulated transcription in which aptamers integrated into transcription templates serve as inputs to molecular circuits that can be programmed to a produce a variety of responses. We modularly design molecular biosensors using this platform by swapping aptamer domains for specific proteins and downstream domains that encode different RNA transcripts. By coupling aptamer-regulated transcription with diverse transduction circuits, we rapidly construct analog protein biosensors and digital protein biosensors with detection ranges that can be tuned over two orders of magnitude and can exceed the binding affinity of the aptamer. Aptamer-regulated transcription is a straightforward and inexpensive approach for constructing programmable protein biosensors that could have diverse applications in research and biotechnology.

Expression of goat poxvirus P32 protein and monoclonal antibody preparation.

P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method.