Serum Samples Research Articles

Serum proteomics for the identification of biomarkers to flag predilection of COVID19 patients to various organ morbidities

BackgroundCOVID19 is a pandemic that has affected millions around the world since March 2020. While many patients recovered completely with mild illness, many patients succumbed to various organ morbidities. This heterogeneity in the clinical presentation of COVID19 infection has posed a challenge to clinicians around the world. It is therefore crucial to identify specific organ-related morbidity for effective treatment and better patient outcomes. We have carried out serum-based proteomic experiments to identify protein biomarkers that can flag organ dysfunctions in COVID19 patients.MethodsCOVID19 patients were screened and tested at various hospitals across New Delhi, India. 114 serum samples from these patients, with and without organ morbidities were collected and annotated based on clinical presentation and treatment history. Of these, 29 samples comprising of heart, lung, kidney, gastrointestinal, liver, and neurological morbidities were considered for the discovery phase of the experiment. Proteins were isolated, quantified, trypsin digested, and the peptides were subjected to liquid chromatography assisted tandem mass spectrometry analysis. Data analysis was carried out using Proteome Discoverer software. Fold change analysis was carried out on MetaboAnalyst. KEGG, Reactome, and Wiki Pathway analysis of differentially expressed proteins were carried out using the STRING database. Potential biomarker candidates for various organ morbidities were validated using ELISA.Results254 unique proteins were identified from all the samples with a subset of 12–31 differentially expressed proteins in each of the clinical phenotypes. These proteins establish complement and coagulation cascade pathways in the pathogenesis of the organ morbidities. Validation experiments along with their diagnostic parameters confirm Secreted Protein Acidic and Rich in Cysteine, Cystatin C, and Catalase as potential biomarker candidates that can flag cardiovascular disease, renal disease, and respiratory disease, respectively.ConclusionsLabel free serum proteomics shows differential protein expression in COVID19 patients with morbidity as compared to those without morbidity. Identified biomarker candidates hold promise to flag organ morbidities in COVID19 for efficient patient care.

Detection of C-reactive protein using a label-free NIR fluorescent aptasensor with a large Stokes shift based on an AIEE anthracene derivative

BackgroundC-reactive protein (CRP), one of the classic biomarkers of inflammation, is closely related to infectious inflammation, cardiovascular disease, cancer, and other diseases. Therefore, timely and accurate detection of CRP in human blood is crucial for the discovery, diagnosis, and treatment of the aforementioned diseases. Herein, a novel label-free NIR fluorescence aptasensor with a large Stokes shift based on an AIEE anthracene derivative B and a molybdenum disulfide (MoS2) platform was developed and used for the high sensitivity and specificity detection of CRP. ResultsCompound B could emit near-infrared (NIR) fluorescence with a large Stokes shift (190 nm). Notably, this compound could bind with the aptamer of CRP (CRP-Apt) through electrostatic attraction to form a B/CRP-Apt complex, generating an aggregation-induced emission enhancement effect and enhancing the fluorescent intensity of B. B/CRP-Apt could be adsorbed on the surface of MoS2 with the addition of MoS2 to its solution, and the fluorescence of Compound B was quenched. CRP was then added to the above solution. CRP-Apt had a substantially higher affinity for CRP than MoS2. Therefore, B/CRP-Apt detached from the surface of MoS2 and bound to CRP, thereby restoring the fluorescence of B. Experimental results showed a good linear relationship between the fluorescent recovery intensity of B and the concentration of CRP in the concentration range of 0.3–70 ng mL−1, with a limit of detection as low as 0.1 ng mL−1. Significance and NoveltyThe aptasensor integrates the advantages of high sensitivity of NIR fluorescence, high specificity of aptamers, good water-solubility and AIEE effect of Compound B. And it could be applied to the determination of CRP in human serum samples, while most of the reported methods can only determine CRP in spiked human serum samples.

The association between N-nitrosamines exposure and lipid metabolism in the high incidence area of esophageal cancer: A case-control analysis

Environmental carcinogens such as N-nitrosamines are high-risk factors for the development of esophageal cancer (EC). However, the association between nitrosamines exposure and lipid metabolism disorders in human EC remained largely obscure. Therefore, we conducted a population-based case-control study established with esophageal inflammation (BCH), esophageal heterotrophic hyperplasia (DYS), patients with primary EC and matched controls in high prevalence area of EC in China. Our prospective work investigated the joint and independent effects of N-nitrosamines co-exposure on the risk of EC development. Lipidomics analysis was employed to screen differential lipid biomarkers in serum, and the mediating effects of key lipid metabolites in the association between nitrosamines exposure and EC were evaluated. After adjustment for confounders, N-nitrosodimethylamine (NDMA, 1.79 (95% CI: 1.35, 2.39)), N-nitrosodi-n-propylamine (NDPA, 1.55 (95%CI: 1.15, 2.09)), N-nitrosodiethylamine (NDEA, 1.82 (95%CI: 1.36, 2.45)), N-nitrosodibutylamine (NDBA, 1.60 (95%CI: 1.20, 2.13)), N-nitrosomethylethylamine (NMEA, 1.81 (95%CI: 1.36, 2.41)) and N-nitrosomorpholine (NMOR, 1.84 (95%CI: 1.38, 2.45)) exposure all elevated the risks of EC development. The Bayesian kernel machine regression (BKMR) with hierarchical variable selection showed significant positive joint associations of urinary NDMA, NDPA, NMOR, NDEA and EC development, when all nitrosamines were at the 55th percentiles or above, compared with the median. Lipidomic screening of serum samples from the stages of BCH, DYS and EC suggested the perturbation in the biosynthesis of unsaturated fatty acid metabolism pathway, of which myristic acid (FFA 14:0), palmitoleic acid (FFA 16:1), docosahexaenoic acid (FFA 22:6) exerted remarkable mediation effects in the association between N-nitrosamines exposure and EC development. These findings provided new sights for screening early lipid biomarkers and intervention targets in human EC.

An optimized ROP6 mRNA construct successfully expressed immunogenic Toxoplasma gondii ROP6 protein in cell culture

Toxoplasma gondii is an apicomplexan parasite infecting all mammals including humans and causes toxoplasmosis. There is no vaccine available for humans and thus vaccine development efforts continue using novel antigens and/or vaccine platforms. Since our previous microarray screening study showed that ROP6 is a suitable antigen to be used in vaccine studies, in this study, we aimed to design an optimized mRNA construct encoding the ROP6 protein and then demonstrate its efficiency and immunogenicity using in vitro methods. For this, we constructed a pT7CFE1-Chis/ROP6 vector encoding optimized ROP6 mRNA containing EMCV 5′UTR with IRES and a 20 nucleotides fragment from alpha globin 3′ UTR. Then, we generated the optimized ROP6 mRNAs with anti-reverse cap analogue (ARCA) and approximately 150 nucleotide long poly-A tail. Next, HEK293T cells were transfected with the optimized ROP6 mRNAs to show recombinant ROP6 protein expression capability. Moreover, we expressed in vitro recombinant ROP6 protein in HeLa cell lysate using the pT7CFE1-Chis/ROP6 vector to reveal the immunogenicity of recombinant ROP6 protein using sera samples collected from mice infected with PRU strain of T. gondii. The IFA and Western blot results showed that the optimized ROP6 mRNAs successfully expressed the recombinant ROP6 protein in HEK293T cells. Moreover the recombinant ROP6 protein expressed in HeLa cell lysate strongly reacted with sera samples collected from mice. The absorbance difference detected among positive and negative mice serum samples analyzed was statistically significant, indicating that the recombinant ROP6 protein was immunogenic (P = 0.0003). In conclusion, this study demonstrated that the optimized ROP6 mRNAs can be used in the development of mRNA vaccines against toxoplasmosis.

Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Based on a Recombinant Matrix Protein for the Serological Study of Porcine Deltacoronavirus in Mexican Pigs.

Porcine deltacoronavirus (PDCoV) is an infectious disease that causes diarrhoea in pigs of different ages; however, piglets are more susceptible. PDCoV was first reported in 2012 in China and Hong Kong. Later, it was first reported in the USA in 2014 and in Mexico in 2019. Several studies have shown that M protein is highly conserved and, therefore, suitable for diagnostic systems. In this study, for the first time, an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant M protein (rM-PDCoV) was developed to evaluate the seroprevalence of PDCoV in four states in Mexico. High sensitivity (83%) and specificity (100%) were observed for the iELISA. The kappa index calculated a nearly perfect agreement (0.8831) compared to the Western blot (gold standard test), suggesting acceptable statistical value support. In this study, 50.38% of the serum samples from backyard pigs were PDCoV-positive. The serological comparison showed that PDCoV/PEDV coinfections occurred in 31.98% of the analysed sera. These results can enrich our understanding of how this virus spreads and enable the evaluation of PDCoV infections. Moreover, it highlights the importance of continually investigating the seroprevalence of PDCoV in Mexico because there is also no information about the current prevalence of the disease.

Serological analyses against endemic human coronaviruses and SARS-CoV-2 in children and adults using samples collected before the COVID-19 pandemic

ObjectivesFour endemic human coronaviruses (HCoVs), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, infect humans during childhood and cause the common cold. Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, leads to mild symptoms in children, possibly owing to the protection conferred by immunity developed during HCoV infection. This study analyzed the seroreactivity of four endemic HCoVs and SARS-CoV-2 in children and adults. MethodsA total of 747 serum samples (from individuals aged 6 months to 69 years) were collected from 2015 to 2019 before the COVID-19 pandemic in Japan. The samples were tested for IgG antibodies against the four endemic HCoVs and SARS-CoV-2 wild-type spike ectodomain proteins using enzyme-linked immunosorbent assay. ResultsThe seroprevalence of endemic HCoVs (except HCoV-229E) showed 90% positivity by 3–4 years old, whereas HCoV-229E seroprevalence was observed at 8 years old. Approximately 35% of the samples showed reactivity to SARS-CoV-2 and did not change with age. However, the children's group presented higher antibody levels than the adult group. Sample reactivity against SARS-CoV-2 did not confirm neutralization capability. ConclusionsReactive samples against SARS-CoV-2 showed varying antibody levels among different age groups. These findings may contribute to a deeper understanding of the clinical symptoms of COVID-19 and coronavirus diseases.

Tunable Multi-Enzyme Activities of Platinum Nanoclusters for Enhanced Specificity and Sensitivity in Biosensing

Nanozymes have gained prominence for their utility in biosensing and disease diagnostics. However, challenges arise from complex sample matrices and nonspecific enzyme activities that contribute to false signals. This study introduces multifunctional platinum nanoclusters (Pt NCs) exhibiting peroxidase-like (POD-like), oxidase-like (OXD-like), and laccase-like activities tailored for enhanced biosensing capabilities. By adjusting pH, we optimized the conditions to achieve distinct POD-like and OXD-like responses, thereby reducing background signals and improving detection accuracy. The addition of ATP further amplified the POD-like activity while minimizing interference from OXD-like activity. This combined strategy substantially enhanced biomarker detection selectivity, demonstrated through glucose detection in human serum samples. Moreover, thiol inhibition of laccase-like activity in Pt NCs was leveraged for thiol-based antioxidant assessment, revealing their application in quantifying total antioxidant capacity (TAC) in human liver cancer cells, accounting for 44% of TAC. The Pt NCs demonstrated robust sensitivity and reusability, offering a novel multi-enzyme nanomaterial with potential for precise and interference-free biosensing applications. These findings contribute to the development of advanced nanozyme-based biosensors, addressing specificity regulation challenges and expanding their practical application in biosensing and disease diagnostics.

Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis.

In this retrospective longitudinal study, we aimed at exploring the role of (a) MuSK-immunoglobulin G (IgG) levels, (b) predominant MuSK-IgG subclasses, and (c) antibody affinity as candidate biomarkers of severity and outcomes in MuSK-MG, using and comparing different antibody testing techniques. Total MuSK-IgGs were quantified with radioimmunoassay (RIA), ELISA, flow cytometry, and cell-based assay (CBA) serial dilutions using HEK293 cells transfected with MuSK-eGFP. MuSK-IgG subclasses were measured by flow cytometry. SAffCon assay was used for determining MuSK-IgG affinity. Forty-three serum samples were obtained at different time points from 20 patients with MuSK-MG (median age at onset: 48 years, interquartile range = 27.5-72.5; women, 16/20), with 9 of 20 (45%) treated with rituximab. A strong correlation between MuSK-IgG levels measured by flow cytometry and RIA titers was found (rs = 0.74, 95% CI 0.41-0.89, p = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (rs = 0.47, 95% CI 0.01-0.77, p = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (rs = 0.39, 95% CI 0.06-0.64, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 0.032). In individual patients, clinical improvement was associated with decrease in MuSK-IgG levels, as measured by either flow cytometry or CBA end-point titration. In all samples, MuSK-IgG4 was the most frequent isotype (mean ± SD: 90.95% ± 13.89). A significant reduction of MuSK-IgG4 and, to a lesser extent, of MuSK-IgG2, was seen in patients with favorable clinical outcomes. A similar trend was confirmed in the subgroup of rituximab-treated patients. In a single patient, MuSK-IgG affinity increased during symptom exacerbation (KD values: 62 nM vs 0.6 nM) while total MuSK-IgG and IgG4 levels remained stable, suggesting that affinity maturation may be a driver of clinical worsening. Our data support the quantification of MuSK antibodies by flow cytometry. Through a multimodal investigational approach, we showed that total MuSK-IgG levels, MuSK-IgG4 and MuSK-IgG2 levels, and MuSK-IgG affinity may represent promising biomarkers of disease outcomes in MuSK-MG.

De novo granulocyte–macrophage colony-stimulating factor antibody production has been linked to acute graft-versus-host disease following hematopoietic stem cell transplantation from an HLA-matched, unrelated donor

The role of non-HLA antibodies in hematopoietic stem cell transplantation (HSCT) and acute graft-versus-host disease (aGVHD) is not established. Serum samples collected from 58 adult patients before and after HSCT were examined for non-HLA antibodies. Following HSCT, 47 out of 58 patients (81.0 %) had various antibody patterns, with 23 of them (39.7 %) producing denovo antibodies. The most prevalent antibodies were directed against granulocyte–macrophage colony-stimulating factor (GM-CSF). The Fisher exact test revealed a statistically significant correlation between the incidence of acute graft-versus-host disease and denovo production of GM-CSF antibodies in patients fully HLA-matched with their donors (p = 0.001). There were no cases of denovo GM-CSF antibody production seen in non-permissively HLA-mismatched patients. Consequently, we hypothesize that the development of aGVHD after HLA-matched HSCT may differ from that following HLA-mismatched HSCT.

Determining factor of enzyme conjugates, bridge heterology and analytical variables of immunogens in prednisolone ELISA

In this study, adipic acid dihydrazide (ADH), carbohydrazide (CH), ethylenediamine (EDA), and urea (U) were used as spacer molecules, covalently linking prednisolone (PSL) to carrier proteins for immunogen preparation, and PSL to enzymes for enzyme conjugate preparation using N-hydroxysuccinimide (NHS)-mediated carbodiimide reactions. The resulting immunogens were used to generate antiserum in New Zealand white rabbits. Antibodies produced against immunogens with various spacers were tested for immunoreactivity with enzyme conjugates, both with and without spacers, in a total of twenty different combinations. All combinations demonstrated binding and were subsequently evaluated through displacement studies. Sensitivity and specificity tests revealed that the combination of PSL-21-HS-U-BSA-antibody with PSL-21-HS-HRP enzyme conjugate exhibited the best sensitivity (0.032 ng/mL) and limited cross-reactivity with other steroids. This combination was further examined for analytical parameters, showing recovery rates of 93.87–101.19 % for PSL from spiked human serum samples, with intra- and inter-assay CVs of <8.88 %. The serum PSL values obtained by this method showed strong correlation with a commercially available ELISA kit (r2 = 0.97, n = 78).

Interrogating post-transplant donor HLA-specific antibody characteristics and effector functions using clinical bead assays

Single antigen bead (SAB) assays are the most common and sensitive method used to detect and monitor post-transplant donor specific HLA antibodies (DSA). However, a direct comparison across traditional and modified SAB assays to improve routine DSA monitoring using pre-treated IgG sera to eliminate interference has not been performed. We performed a technical comparison of 251 post-transplant DSA from n = 91 serum samples tested neat (pre-treated, undiluted), at a single 1:16 dilution, in the C1q bead assay, and for IgG subclasses (IgG1, IgG2, IgG3, IgG4) with IgG-enriched sera. We found that DSAs that are detectable by 1:16 dilution and/or C1q are associated with higher IgG MFI values and results could be predicted by testing neat sera. DSA detected at 1:16 dilution correlated with >7000 IgG MFI in neat sera and identified DSA that exceeded the SAB linear range for semiquantitative measurements. C1q positive DSA correlated with >15,000 IgG MFI in neat sera. C1q binding correlated most strongly with total IgG MFI (Spearman r = 0.82, p = 0.002) and not specific subclasses, demonstrating that DSA C1q binding capacity in this cohort is driven by HLA-specific IgG concentration. Evaluation of engineered pan-HLA class I-specific human IgG1 and IgG2 subclass monoclonal antibodies by SAB C1q and C3d assays revealed that IgG2 antibodies can bind complement at higher concentrations. The strengths and limitations of modified SAB assays must be considered to optimize efficient testing and accurate clinical interpretation.

Seroprevalence and associated risk factors for Fasciola hepatica in sheep in Nile Delta of Egypt

Fascioliasis is a globally distributed zoonotic parasitic disease that affects ruminants, including sheep. This study conducted from January to December 2023, aimed to determine the prevalence of Fasciola hepatica in sheep across three governorates in Egypt's Nile Delta and to assess associated risk factors. A total of 455 serum samples were analyzed using a commercial ELISA kit, revealing antibodies against F. hepatica in 22.2 % of the tested sheep. There was no significant association between locality or sex and the seroprevalence of F. hepatica in sheep; however, the highest prevalence was observed in Kafr Sheikh and in female sheep.Concerning risk factors, poor conditioned sheep aged between 1 and 2 years showed 2.1 and 3.8 times higher of infection probability than others. In addition, the risk of F. hepatica infection in sheep increased significantly in winter season (OR = 6.6, 95 %CI: 2.6–16.8), in absence of prophylactic treatment (OR = 2.2, 95 %CI: 1.3–3.3) and in presence of snail (OR = 3, 95 %CI: 3–5.4). The existence of antibodies against F. hepatica in examined sheep raising in Nile Delta indicating that the disease is reported in the studied areas and needs to be managed on farms through control and preventative measures.

Seroprevalence and Associated Risk Factors of Infectious Bovine Rhinotracheitis (IBR) and Animal Owners' Knowledge, Attitude and Practice (KAP) Towards the Disease in Selected Districts of East Wollega Zone, Oromia Regional State, Ethiopia.

Infectious bovine rhinotracheitis (IBR) is one of the most important diseases affecting production and productivity. Cross-sectional study was aimed at to determine the seroprevalence of IBR and associated risk factors, and animal owners' knowledge, attitude and practice towards the disease from April 2021 to June 2022. Accordingly, a total of 384 serum samples were collected from both crossbreed (70) and local breed (314) cattle from purposively selected districts of East Wollega zone of Western Ethiopia. Competitive enzyme-linked immunosorbent assay (ELISA) was used for testing glycoprotein antibodies (anti-gB) for bovine herpes virus-1 (BoHV-1) virus in collected serum, and the obtained data were analysed by multiple logistic regressions by using R software 3.62 version. However, questionnaire data were analysed for descriptive statistics by SPSS version 20.0 (IBM. Corp, 2011). The total prevalence of IBR in the study area was found to be 70.54% at herd and 80.47% at individual cattle level. The significant association (p ) was found for breed, age, body condition and herd size but not for district and sex as risk factors. The BoHV-1 virus seropositivity in adult animals increased significantly, with an odds ratio of 1.65 (95% CI 0.705-3.85) compared to young. Local breed cattle were 2.055 times more likely to test positive for IBR with an odds ratio of 0.77 (95% CI 0.23-2.22) compared to crossbreed cattle. The chances of cattle in medium herds testing positive for the BoHV-1 virus with an odds ratio of (1.78 95% CI 1.303-7.50) are greater than the chances of cattle in smaller herds testing positive. The survey results showed that 70% of animal owners identified IBR as a major challenge in animal production, whereas 35% mentioned long calving intervals. However, 92% of the participants were not informed about the level of knowledge and attitude regarding particular diseases such as IBR. This study showed that there is a high prevalence of IBR in cattle in the study area, and that owners have low awareness of the disease. Therefore, it is necessary to develop an immediate control system and conduct additional research on molecular detection to evaluate its effects on reproductive performance.

Target-promoted catalytic DNA molecular circuit network coupled with endonuclease amplification for sensitive and label-free electrochemical aptamer antibiotic assay

Sarafloxacin (SAR) is a common antibiotic that accumulates in animal tissues, which potentially poses health risks to humans via ingestion. Sensitive detection of SAR in animal-derived foods is therefore critical. Traditional SAR detection methods based on HPLC or antibody immunoassays encounter the limitations of cost, sensitivity and operational simplicity. In this study, with the design of a new DNAzyme/catalytic hairpin assembly (CHA) molecular circuit network coupled with endonuclease amplification, we developed an electrochemical aptamer (AP) biosensor with high sensitivity for label-free SAR detection in milk. The sensor functions by specific binding of SAR to APs, which release DNAzyme sequences to initiate the catalytic DNAzyme/CHA molecular circuit network amplification for forming many ssDNAs and dsDNA duplexes. These sequences further hybridize with hairpin signal probes on sensor electrode to create favorable nicking sites for endonuclease, which cleaves hairpin signal probes to liberate lots of G-quadruplex strands to complex with and capture hemin. Subsequent hemin reduction by electrochemistry thus results in dramatically enhanced currents to achieve label-free detection of SAR in linear range between 10 pM and 80 nM with detection limit of 1.62 pM. Our sensor also shows satisfactory reproducibility, repeatability and stability, demonstrates high selectivity for the target SAR against other interfering antibiotics, e.g., norfloxacin, penicillin and gentamicin and can realize detection of low concentrations of SAR in diluted complex milk and human serum samples, thereby underscoring its promises for convenient and sensitive assay of different trace antibiotics.

Lanthanide coordination polymers as luminescent laccase mimics for ratiometric sensing of dopamine

Some metal ions, with inner enzyme-like catalytic activity, could be doped into lanthanide coordination polymers (Ln CPs) through coordination, which has been proved as a facile strategy to prepare the luminescent nanozymes. In this study, Cu-doped Ln CPs with laccase-mimic activity and double luminescence were rationally designed and synthesized by self-assembly of guanine monophosphate (GMP), 2-aminoterephthalic acid (ATA), Cu2+ and Tb3+ in buffer solution at room temperature. The obtained probes Tb/Cu-GMP/ATA CPs not only emitted green fluorescence of Tb3+ and blue fluorescence of ATA simultaneously under irradiation at the same wavelength, but also processed enhanced laccase-like activity for catalyzing the oxidation of phenolic substrates. Upon dopamine (DA), the probes catalyzed the oxidation of DA to polydopamine (PDA), which effectively quenched the fluorescence of Tb3+ due to the internal filtration effect. Based on this, a ratiometric fluorescent sensor for DA was constructed accordingly, and the corresponding fluorescence intensity ratio of Tb3+to ATA (F547/F427) was linearly correlated with the DA concentration in the range of 1 to 400 μM, with a detection limit of 0.44 μM. Besides, this sensor could be used to detect DA in human serum samples with good recovery, which results were highly consistent with that of HPLC method. The constituent and luminescence tunability, as well as the extraordinarily facile synthesis, made Ln CPs a potential platform for designing and preparing the integrated multifunctional probe for special target in sensing applications.

Mediterranean Diet Pattern: Potential Impact on the Different Altered Pathways Related to Cardiovascular Risk in Advanced Chronic Kidney Disease

Background: Cardiovascular disease (CVD) remains the most common cause of mortality in chronic kidney disease (CKD) patients. Several studies suggest that the Mediterranean diet reduces the risk of CVD due to its influence on endothelial function, inflammation, lipid profile, and blood pressure. Integrating metabolomic and proteomic analyses of CKD could provide insights into the pathways involved in uremia-induced CVD and those pathways modifiable by the Mediterranean diet. Methods: We performed metabolomic and proteomic analyses on serum samples from 19 patients with advanced CKD (aCKD) and 27 healthy volunteers. The metabolites were quantified using four different approaches, based on their properties. Proteomic analysis was performed after depletion of seven abundant serum proteins (Albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen). Integrative analysis was performed using MetaboAnalyst 4.0 and STRING 11.0 software to identify the dysregulated pathways and biomarkers. Results: A total of 135 metabolites and 75 proteins were differentially expressed in aCKD patients, compared to the controls. Pathway enrichment analysis showed significant alterations in the innate immune system pathways, including complement, coagulation, and neutrophil degranulation, along with disrupted linoleic acid and cholesterol metabolism. Additionally, certain key metabolites and proteins were altered in aCKD patients, such as glutathione peroxidase 3, carnitine, homocitrulline, 3-methylhistidine, and several amino acids and derivatives. Conclusions: Our findings reveal significant dysregulation of the serum metabolome and proteome in aCKD, particularly in those pathways associated with endothelial dysfunction and CVD. These results suggest that CVD prevention in CKD may benefit from a multifaceted approach, including dietary interventions such as the Mediterranean diet.

Laboratory support for seromonitoring of anti-pertussis post-vaccination immunity

Monitoring of humoral immunity in 3–4 year-old anti-pertussis vaccinated children is one of the approaches for pertussis epidemiological surveillance. It’s a predictive indicator for comprehensive assessment of morbidity in the region. The agglutination reaction (RA) used earlier has been displaced with enzyme immunoassay (ELISA). So, two questions arose: regarding i) a relevance of using ELISA in whole-cell vaccinated individuals, and ii) correctness of large-scale data interpretation without applying a unified IgG reference value. Study aims were to assess humoral anti-pertussis population immunity by ELISA, to determine features of humoral protection in vaccinated subjects after using various vaccine types, to identify age features, to make recommendations for evaluating seromonitoring data. Blood serum samples from anti-pertussis vaccinated children in Samara region (n = 1729) were examined in 2016–2020; children were divided into 4 age groups ( 3, 3–4, 5–7, ≥ 7.1 years) and into 3 type vaccines groups: cellular, acellular and combined. We used two ELISA kits, which are designed to assess post-vaccination immunity, according to the manufacturer’s instructions, quantifying IgG againstB. pertussis, which differ in units and reference values. Statistical analysis was performed using StatTech program v.2.6.2. The largest and smallest percentage of seronegative children was in the cellular-vaccinated and acellular-vaccinated group, respectively. Seronegative percentage in the “indicator group” reaches 55.2% being significantly higher than acceptable 10.0%-level, and differs from the literature data (30–40%), which were obtained mainly with RA. This may suggest about an incorrect interpretation of post-vaccination immunity data evaluation strictly according to manufacturers’ reference criteria, especially for whole-cell vaccinated subjects. A method for calculating the seronegative percentage is proposed that relies on the prognostic negative data level as a correction factor. The required values were obtained during comparison studies of random serum samples from 3–4-year-old children (n = 70) using two ELISA kits (“RIDASCREEN®Bordetella”, “SeroPertussis™ IgG”) and intra-laboratory comparison panel. It should be given preference for sets with a greater prognostic negative data level, if population seronegative percentage is assessed.

Early Diagnosis of Triple-Negative Breast Cancer Based on Dual microRNA Detection Using a Well-Defined DNA Crown-Carbon Dots Structure as an Electrochemiluminescence Sensing Platform.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Thus, early detection and accurate diagnosis of this cancer are crucial for improving the survival rate of patients. Specific microRNAs (miRNAs) have been implicated in the occurrence, proliferation, and metastasis of TNBC. Addressing this need, our study developed a biosensor platform for early and accurate TNBC diagnosis by integrating electrochemiluminescence (ECL) technology with a DNA sensing strategy. Specifically, synthesized positively charged carbon dots (CDs) were used to neutralize the electrostatic repulsion between DNA strands and facilitate the assembly of DNA triangular prisms (DNA TP-CDs). Hairpins were then incorporated into the DNA TP-CDs to form the final DNA crown structure. The early TNBC biomarker, microRNA-93-3p (miR-93-3p), allowed for the binding between the DNA Crown and the DNA track on the electrode and initiated the ECL signal. Subsequently, microRNA-210 (miR-210) unlocked the DNA tripedal walker, and its movement on the DNA Crown eventually quenched the ECL signal, enabling accurate TNBC diagnosis and tumor stage assessment. Our proposed biosensor had satisfactory sensing efficiency due to the ordered DNA track and rapid-moving DNA walker. The data revealed a good linear relationship between the ECL signals and the logarithm of miRNA concentrations, with miR-93-3p having a detection limit of 31.04 aM and miR-210 having a detection limit of 7.69 aM. The biosensor also showed satisfactory performance in serum samples and cells. Taken together, this study hopes to provide ideas and applications for clinical diagnosis as well as the personalized treatment of TNBC.

Serum Lipoprotein Profiling by NMR Spectroscopy Reveals Alterations in HDL-1 and HDL-2 Apo-A2 Subfractions in Alzheimer’s Disease

Identifying biomarkers for Alzheimer’s disease (AD) is crucial, due to its complex pathology, which involves dysfunction in lipid transport, contributing to neuroinflammation, synaptic loss, and impaired amyloid-β clearance. Nuclear magnetic resonance (NMR) is able to quantify and stratify lipoproteins. The study investigated lipoproteins in blood from AD patients, aiming to evaluate their diagnostic potential. Serum and plasma were collected from AD patients (n = 25) and healthy individuals (n = 25). We conducted a comprehensive lipoprotein profiling on serum samples using NMR spectroscopy, analysing 112 lipoprotein subfractions. In plasma, we measured unspecific markers of neuronal damage and AD hallmark proteins using single molecule array technology. Additionally, clinical data and cerebrospinal fluid biomarker levels were also collected to enrich our data. Our findings, after adjusting for age and sex differences, highlight significant alterations in two specific lipoproteins; high-density lipoprotein (HDL)-1 Apo-A2 (H1A2) and HDL-2 Apo-A2 (H2A2), both with area under the curve (AUC) values of 0.67, 95% confidence interval (CI) = 0.52–0.82). These results indicate that these lipoprotein subfractions may have potential as indicators of AD-related metabolic changes.

Evidence for stability of cardiac troponin T concentrations measured with a high sensitivity TnT test in serum and lithium heparin plasma after six-year storage at-80 °C and multiple freeze-thaw cycles.

As high-sensitivity cardiac troponin T (hs-cTnT) is making the transition from diagnostic to prognostic use, a long-term stability study of 5th generation hs-cTnT according to EFLM CRESS recommendations was set up for investigation of frozen clinical specimens (two matrices). Study samples collected in serum tubes and lithium heparin tubes with gel from patients admitted for suspected minor myocardial damage were measured directly after completion of the study (0 years), and after 3-year and 6-year storage at-80 °C, and recovery of hs-cTnT concentrations after long-term storage (%hs-cTnT concentration compared to 0-year) was calculated. Hs-cTnT changes were also compared to decisive delta changes, such as the ones proposed in the ESC NSTEMI 0 h/1 h algorithm (<3 or >5 ng/L for ruling out and ruling in suspected NSTEMI patients). Eighty-six patients were included in the study, whereof 28 both lithium heparin plasma and serum samples were collected simultaneously, in others only serum (n=30) or plasma (n=28). Multiple aliquots per patient were made, so that 479 serum and 473 plasma samples were available for analysis. Across the overall hs-cTnT measuring range, median recovery after 6 years was 105.4 % and 106.2 % for serum and plasma, respectively. Based on these decisive delta changes, serum showed consistent results upon long term storage (max 0.8 % of samples above delta threshold of >5 ng/L) as compared to heparin plasma (up to 19.2 % of samples above threshold). Over 6 years of storage at-80 °C, recovery of hs-cTnT in serum and heparin plasma was similar and within common lot-to-lot variation. Yet, when evaluating absolute delta increments around hs-cTnT clinical decision points, long-term stored sera displayed better clinical performance compared to heparin plasma samples.