Protein Markers Research Articles

Comparative Assessment of Mitochondria Isolation Buffers for Optimizing Tissue-Specific Yields in Buffalo

Introduction: Mitochondrial studies are crucial for assessing livestock health and performance. While extensive research has been done on cattle and pigs, the influence of mitochondria in Indian buffalo remains unexplored. Therefore, in order to understand functions of mitochondria, their energy-related processes, or any additional mitochondrial traits in buffaloes, it is imperative to isolate high-yield mitochondria with purity and functionality. Mitochondria are extracted by few conventional buffers. These buffers were previously characterized for their effectiveness in isolating mitochondria from rodent and human tissues. Therefore, the present study is to assess the performance of mitochondria isolation buffers specifically in buffalo tissues. Methods: The study involved isolation of mitochondria from four different tissues, i.e., liver, brain, heart and muscles of slaughtered buffalo (n = 3), using: (i) Tris-Mannitol buffer (ii) Tris-Sucrose buffer, and (iii) MOPS-Sucrose buffer. Buffer efficiency in preserving high fidelity during mitochondria isolation was assessed by comparison with Cayman’s MitoCheck® Mitochondrial Isolation Kit (control). Further mitochondrial purity and functionality was assessed through comparative estimation of protein concentration and marker enzyme assays, respectively. Results: Our results revealed insights into the suitability of specific buffer for functional mitochondria isolation from specific type of buffalo tissue. Notably for obtaining high quality functional mitochondria from buffalo, MOPS-Sucrose buffer appeared optimal for soft tissues (liver and brain), while Tris-Mannitol buffer was efficient for hard tissues (muscles and heart). Conclusions: Thus, our research highlights the influence of buffer composition and tissue-specific variations in buffer effectiveness on mitochondrial activity in different tissues, leading to improved mitochondrial isolation in buffalo.

Stretchable wireless optoelectronic synergistic patches for effective wound healing

Physiotherapies play a crucial role in noninvasive tissue engineering for wound healing. However, challenges such as the implementation of complex interventions and unsatisfactory treatment outcomes impede widespread application. Here, we proposed a stretchable and wirelessly-powered optoelectronic synergistic patch with a dual-layer serpentine wireless receiver circuit to drive the optoelectronic modulation component. Optimized structure and impedance matching enable the patch to seamlessly attach to irregular skin surfaces and operate robustly over a 30% tensile strain range. Based on Sprague-Dawley rat wound model. The wound closure rate of the optoelectronic synergistic group significantly outperformed both monointervention and blank control groups. Mechanistically, optoelectronic synergistic intervention enhances the secretion of vascular endothelial marker proteins and growth factors, and stabilizes mitochondrial function during oxidative stress. Overall, the scalable amalgamation of flexible electronics, wireless transmission, and synergistic interventions promise to improve wound care.

LPS-induced systemic inflammation is suppressed by the PDZ motif peptide of ZO-1 via regulation of macrophage M1/M2 polarization.

The gram-negative bacterium lipopolysaccharide (LPS) is frequently administered to generate models of systemic inflammation. However, there are several side effects and no effective treatment for LPS-induced systemic inflammation. PEGylated PDZ peptide based on zonula occludens-1 (ZO-1) was analyzed for its effects on systemic inflammation induced by LPS. PDZ peptide administration led to the restoration of tissue injuries (kidney, liver, and lung) and prevented alterations in biochemical plasma markers. The production of pro-inflammatory cytokines was significantly decreased in the plasma and lung BALF in the PDZ-administered mice. Flow cytometry analysis revealed the PDZ peptide significantly inhibited inflammation, mainly by decreasing the population of M1 macrophages, and neutrophils (immature and mature), and increasing M2 macrophages. Using RNA sequencing analysis, the expression levels of the NF-κB-related proteins were lower in PDZ-treated cells than in LPS-treated cells. In addition, wild-type PDZ peptide significantly increased mitochondrial membrane integrity and decreased LPS-induced mitochondria fission. Interestingly, PDZ peptide dramatically could reduce LPS-induced NF-κB signaling, ROS production, and the expression of M1 macrophage marker proteins, but increased the expression of M2 macrophage marker proteins. These results indicated that PEGylated PDZ peptide inhibits LPS-induced systemic inflammation, reducing tissue injuries and reestablishing homeostasis, and may be a therapeutic candidate against systemic inflammation.

LPS-induced systemic inflammation is suppressed by the PDZ motif peptide of ZO-1 via regulation of macrophage M1/M2 polarization

The gram-negative bacterium lipopolysaccharide (LPS) is frequently administered to generate models of systemic inflammation. However, there are several side effects and no effective treatment for LPS-induced systemic inflammation. PEGylated PDZ peptide based on zonula occludens-1 (ZO-1) was analyzed for its effects on systemic inflammation induced by LPS. PDZ peptide administration led to the restoration of tissue injuries (kidney, liver, and lung) and prevented alterations in biochemical plasma markers. The production of pro-inflammatory cytokines was significantly decreased in the plasma and lung BALF in the PDZ-administered mice. Flow cytometry analysis revealed the PDZ peptide significantly inhibited inflammation, mainly by decreasing the population of M1 macrophages, and neutrophils (immature and mature), and increasing M2 macrophages. Using RNA sequencing analysis, the expression levels of the NF-κB-related proteins were lower in PDZ-treated cells than in LPS-treated cells. In addition, wild-type PDZ peptide significantly increased mitochondrial membrane integrity and decreased LPS-induced mitochondria fission. Interestingly, PDZ peptide dramatically could reduce LPS-induced NF-κB signaling, ROS production, and the expression of M1 macrophage marker proteins, but increased the expression of M2 macrophage marker proteins. These results indicated that PEGylated PDZ peptide inhibits LPS-induced systemic inflammation, reducing tissue injuries and reestablishing homeostasis, and may be a therapeutic candidate against systemic inflammation.

Investigation of the Immunoexpression of SVIP and UPR Pathway Proteins in Ovarian Adenocarcinoma Cell Line OVCAR-3

Ovarian cancer is the deadliest gynecological cancer. The endoplasmic reticulum (ER), a vital cell organelle, is involved in the folding, synthesis, and modification of a wide range of soluble and insoluble proteins. ER stress initiates the unfolded protein response (UPR), an evolutionary conserved cell stress mechanism. The UPR is mediated by three ER transmembrane sensors: IRE1, ATF6, and PERK. An inhibitor of ERAD is a small VCP/p97-interacting protein (SVIP). The study aimed to investigate the relationship between SVIP and the ER stress protein markers in the human ovarian cancer cell line OVCAR-3. The SVIP and GRP78, PERK, ATF4 immunoexpression levels were analyzed. Furthermore, employing immunofluorescence, the colocalization of three ER sensors and SVIP was ascertained. The immunoexpression of SVIP and GRP78, ATF4, and PERK were shown in the OVCAR-3 cell line. Additionally, immunofluorescence results showed the colocalization of SVIP and UPR-related proteins in the cytoplasm of OVCAR-3 cells. In conclusion, we demonstrated the cellular localization of SVIP and the proteins involved in the UPR pathway. However, further studies are needed to determine the relation between SVIP and these proteins in cancer cells.

Epidemiology and clinical characteristics of acute viral gastroenteritis in 350 paediatric patients hospitalised between 2019 and 2022 in the Military Institute of Medicine – National Research Institute in Warsaw, Poland: a single-centre retrospective analysis

Introduction and objective: Acute gastroenteritis, a common childhood illness worldwide, manifests with symptoms including fever, abdominal pain, vomiting, and diarrhoea. It is highly contagious, with transmission occurring through contaminated water, food, or poor hygiene. Globally, 1.7 billion cases of diarrhoeal diseases are diagnosed annually, causing approximately 525,000 deaths among children under the age of five. Dehydration caused by diarrhoea is a primary cause of hospitalisation, particularly in developing countries. Aim: Analysis of the aetiology, frequency, and seasonal distribution of viral pathogens responsible for acute gastroenteritis in children hospitalised between 2019 and 2022 at the Military Institute of Medicine – National Research Institute in Warsaw, Poland. Materials and methods: Medical records of patients aged 0 to 18 diagnosed with rotavirus, norovirus, and adenovirus-induced acute gastroenteritis were analysed. The pathogens had been identified with reliable immunochromatographic tests. Exclusion criteria encompassed bacterial infections, dietary errors, and inflammatory conditions. Data examined included age, gender, aetiology of gastroenteritis, seasonality, duration of hospitalisation, and clinical symptoms (diarrhoea, vomiting, fever, abdominal pain, and dehydration). Laboratory results such as white blood cell count, C-reactive protein, electrolyte levels, and organ function markers were also evaluated. Results: Data from 350 children hospitalised between 2019 and 2022 were analysed. The highest number of hospitalisations occurred in 2019 and 2022, with fewer cases in 2020–2021, likely reflecting the impact of the COVID-19 pandemic. Most patients were between 6 and 12 months old. Rotavirus infection commonly presented with fever, vomiting, diarrhoea, and dehydration. Adenovirus and mixed infections were associated with slightly higher C-reactive protein levels, while rotavirus infections showed mildly elevated aspartate aminotransferase levels. Haematocrit, blood urea nitrogen, creatinine, and electrolyte levels were similar across all cases. Conclusions: Our analysis showed that rotavirus was the most frequent cause of acute viral diarrhoea in children, followed by norovirus and adenovirus, with mixed infections being the least common. The peak incidence occurred in autumn and winter, except in 2021, reflecting changes in the dynamics of infections related to the coronavirus pandemic. Biochemical findings were not sufficiently characteristic to infer the aetiology of the disease.

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells

ABSTRACT Background Checkpoint kinase 2 (CHEK2) and its regulated tumor protein p53 (TP53) have been correlated with osteogenic differentiation of osteoblast-like cells. Based on bioinformatics predictions, this study aims to investigate the effect of the CHEK2/TP53 axis on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to explore the regulatory mechanism. Methods PDLSCs were isolated from human impacted wisdom teeth, and they were cultured in normal medium (NM) or osteogenic medium (OM). Protein levels of CHEK2 and TP53 were examined using western blot analysis. Osteogenic differentiation ability of PDLSCs was analyzed by measuring marker proteins (RUNX2, OCN, and OSX), ALP activity, and ALP staining. Molecular interaction between NEDD4 like E3 ubiquitin protein ligase (NEDD4L) and CHEK2 was examined by ubiquitination and co-immunoprecipitation assays. Gain- and loss-of function assays of NEDD4L, CHEK2, and TP53 were performed to analyze their function in osteogenic differentiation of PDLSCs. A rat model of mandibular bone defect was generated for in vivo validation. Results NEDD4L was upregulated, while CHEK2 and TP53 were downregulated in PDLSCs cultured in OM. CHEK2 protected TP53 from degradation, while NEDD4L reduced CHEK2 protein level by ubiquitination modification. NEDD4L silencing reduced osteogenic differentiation ability of PDLSCs both in vitro and in vivo, which was restored by CHEK2 silencing. By contrast, CHEK2 overexpression blocked the osteogenic differentiation of PDLSCs in vitro. Conclusion This study demonstrates that NEDD4L affects protein stability of the CHEK2/TP53 axis through ubiquitination modification, thus increasing osteogenic differentiation of PDLSCs.

Exercise-Induced Proteomic Profile Changes in Patients with Advanced Heart Failure.

The pathophysiological background of the processes activated by physical activity in patients with heart failure (HF) is not fully understood. Proteomic studies can help to preliminarily identify new protein markers for unknown or poorly defined physiological processes. We aimed to analyse the changes in the plasma proteomic profile of HF patients after a cardiopulmonary exercise test (CPET) to define pathways involved in the response to exercise. The study prospectively enrolled 20 male patients with advanced HF (aged 53.3 ± 8.3 years). Blood samples were taken from the patients before and immediately after the CPET to obtain plasma proteomic profiles. Two-sample t-tests (paired or non-paired) were performed with and without false discovery rate (FDR) correction for multiple testing. Enrichment analysis was performed to associate biological processes and pathways with the study results. A total of 968 plasma proteins were identified, of which 722 underwent further statistical analysis. Of these, 236 proteins showed differential expression when comparing all plasma samples collected before and after CPT (p < 0.05), and for 86 of these the difference remained statistically significant after FDR correction. Proteins whose expression changed after exercise are mostly involved in immune response and inflammatory processes, coagulation, cell adhesion, regulation of cellular response to stimulus and regulation of programmed cell death. There were no differences in resting proteomics according to HF etiology (ischemic vs. non-ischemic). Changes in the proteomic profile revealed a complexity of exercise-induced processes in patients with HF, suggesting that few major physiological pathways are involved. Further studies focusing on specific pathways are needed.

12349 Increased Total Oxidative Status/Total AntioxidantCapacityandReduced Microparticle Size In Diabetes Kidney Disease

Abstract Disclosure: S.C. Oliveira: None. A. Bridi: None. B.G. Rossato: None. R.N. Moresco: None. J.C. Silveira: None. M.W. Lauria: None. F.V. Comim: None. Diabetic kidney disease (DKD) is an important cause of morbimortality in the adult population with Type 1 Diabetes Mellitus (T1DM). Our cross-sectional study explored the association of abnormalities in extracellular vehicles (EVs) and oxidative stress markers in three distinct groups of clinical outpatients: normal controls, T1DM (without DKD), and DKD. Clinical and biochemical data were obtained from these groups. The isolation and characterization of EVs from serum were obtained through a series of processes, including the ultracentrifugation and characterization of EVs was identified by specific protein markers by western blot morphology checked by Scanning Electron Microscope (FEI Tecnai 20; LAB6 emission; 200 kV). EVs particle size and concentration were determined by NanoSight NS300 instrument (Malvern Panalytical, UK). Total Oxidant Status (TOS), Total Antioxidant Capacity (TAC), Advanced Oxidation of Protein Products (AOPP), (Ferric Reducing Antioxidant Power), C reactive Protein, Uric Acid, Aloxan levels were determined in the serum or the plasma.Overall, 124 patients were evaluated: 61 normal controls, 42 T1DM (without DKD), and 21 DKD defined according the KDIGO guidelines. The three groups were similar regarding age (mean± SD) of 40.3 ± 13.7 years, BMI 25.8 ± 5.1 Kg/m2, and Abdominal Circumference 89.6 ± 12.3 cm. The albumin/creatinine ratio was statistically different among the groups, being its (median [95%CI]) of 4.0 [3.9-5.14] mg/g in Controls, 6.4[5.5-10.0]mg/g in T1DM, and 81.4 [48.5 -1415] mg/g in DKD (p&amp;lt;0.001). The DKD patients exhibited higher SBP and an increased proportion of retinopathy and neuropathy than T1DM, but HBa1c levels were similar between the two diabetes groups. Patients with DKD show an increased TOS/TAC Ratio ( μmol H2O2 Equiv/L /mmol Trolox Equiv/L) (ANOVA p&amp;lt;0.001) superior to T1DM and controls. The mean ± SD was 61.1 ± 31.0 in Controls, 103.0± 68 in T1DM, and 145.0± 62.8 in DKD. Interestingly, T1DM exhibited an increased TOS/TAC Ratio than Controls. No modifications in AOPP or FRAP could be observed among the three groups.There is a gradual decrease in EVs size (but not concentration) in patients with DKD in comparison to T1DM and controls, and T1DM with controls. The mean of the moda size of EVs was (mean + SE) 144.5 ± 3.04 nm in Controls versus 136.14 ± 3.5 nm in T1DM, and 135.4 ± 3.6nm in DKD (ANOVA p=0.04). The concentration was (mean ± SE) 4.57 x109 ± 5.57 x108 particles/mL in Controls, 3.58 x109 ± 2.9 x108 particles/mL in T1DM and 3.94 x109 ± 3.7 x108 particles/mL in DKD (NS).Our results suggest an association of increased oxidative stress status (TOS/TAC ratio) and reduced microvesicles size in the presence of T1DM; both abnormalities progress towards the development of DKD. Presentation: 6/3/2024

Structural diversity inside the mouse subiculum revealed by a new marker protein fibronectin 1.

The subiculum is one of the major output structures of the hippocampal formation and is an important brain region for memory. We have previously reported that the subiculum of rodents can be morphologically divided into its temporal (ventral) two-thirds and the septal (dorsal) third and that the former can be further subdivided into the distal (Sub1) and proximal (Sub2) regions, on a basis of immunohistochemical localizations of several Sub2-specific proteins. However, it remains unclear whether detailed structural organization found in the temporal subiculum is applicable to the septal subiculum. In this study, we found that the distribution of fibronectin (FN1)-positive non-GABAergic, presumptive pyramidal cells exactly coincided with the extent of the Sub1 region of male mice. Using FN1 immunohistochemistry, the Sub1 was found to keep relatively constant size throughout the septotemporal axis of the subiculum. In contrast, the size of the Sub2 became smaller as it approached the septal side, and the Sub2 finally disappeared at the most septal level of the subiculum. Retrograde tracer experiments confirmed that FN1-positive Sub1 neurons projected to the retrosplenial cortex, which is thought to be associated with spatial memory, whereas FN1-negative Sub2 neurons projected to the nucleus accumbens associated with emotional memory. Considering both the functional segregation of these two subicular targets and the relative abundance of the Sub2 on the temporal side, the subiculum can be one of the neural substrates for functional differences between the septal and temporal hippocampal formation associated with the spatial and emotional memory, respectively.

Enhancing osteoblast differentiation and bone repair: The priming effect of photobiomodulation on adipose stromal cells

Cellular therapy using adipose tissue–derived mesenchymal stromal cells (at-MSCs) has garnered attention for the treatment of bone defects. Therefore, preconditioning strategies to enhance the osteogenic potential of at-MSCs could optimize cell therapy outcomes, and photobiomodulation (PBM) therapy has emerged as an effective, noninvasive, and low-cost alternative. This study explored the impacts of PBM on at-MSCs differentiation and the subsequent repair of bone defects treated with cell injection. Rat at-MSCs were cultured and irradiated (at-MSCsPBM) following the PBM protocol (660 nm; 20 mW; 0.714 W/cm2; 0.14 J; 5 J/cm2). Cellular differentiation was assessed based on the expression of gene and protein markers. Reactive oxygen species (ROS) were detected using fluorescence. At-MSCsPBM were injected into 5-mm calvarial lesions, and bone formation was analyzed using micro-CT and histological evaluations. At-MSCs were used as control. Data were analyzed using the ANOVA or t-test. At-MSCsPBM exhibited high levels of gene and protein runt-related transcription factor-2 (Runx2) and alkaline phosphatase (Alp) expression. PBM increased ALP activity and significantly reduced ROS levels. In addition, PBM increased the expression of Wnt pathway–associated genes. In vivo, there was an increase in the morphometric parameters, including bone volume, percentage of bone volume, bone surface area, and trabecular number, in at-MSCsPBM-treated defects compared with those in the control. These findings suggest that PBM enhances the osteogenic potential of at-MSCs, thereby supporting the advancement of improved cellular therapies for bone regeneration.

Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

Osteoarthritis (OA) is a prevalent chronic degenerative disease, marked by a complex interplay of mechanical stress, inflammation, and metabolic imbalances. Recent studies have highlighted the potential of spermidine (SPD), a naturally occurring polyamine known for its anti-inflammatory and antioxidant properties, as a promising therapeutic agent for OA. This study delves into the therapeutic efficacy and mechanistic pathways of SPD in mitigating OA symptoms. Forty Sprague-Dawley rats were randomly assigned to four groups, including the CG (sham operation), model (anterior cruciate ligament transection [ACLT], and treatment (ACLT + two different doses of SPD) groups. In vivo, correlations between OA severity and different interventions were assessed by ELISA, X-rays, CT imaging, histological staining, and immunohistochemistry. In vitro, IL-1β was used to trigger chondrocyte inflammation, and SPD's cytotoxicity was assessed in primary rat chondrocytes. Next, inflammatory markers, extracellular matrix (ECM) proteins, and pathway marker proteins were detected in chondrocytes administered IL-1β alone, SPD, or aryl hydrocarbon receptor (AhR) silencing, by qRT-PCR, Griess reaction, ELISA, Western blot, and immunofluorescence. Morphological alterations and pyroptosis in chondrocytes were examined by transmission electron microscopy (TEM) and flow cytometry. Our research reveals that SPD exerts significant anti-inflammatory and antipyroptotic effects on IL-1β-treated chondrocytes and in anterior cruciate ligament transection (ACLT) rat models of OA, primarily through interaction with the Aryl hydrocarbon receptor (AhR). Specifically, SPD's binding to AhR plays a crucial role in modulating the inflammatory response and cellular pyroptosis by inhibiting both the AhR/NF-κB and NLRP3/caspase-1/GSDMD signaling pathways. Furthermore, the knockdown of AhR was found to negate the beneficial effects of SPD, underscoring the centrality of the AhR pathway in SPD's action mechanism. Additionally, SPD was observed to promote the preservation of cartilage integrity and suppress ECM degradation, further supporting its potential as an effective intervention for OA. Collectively, our findings propose SPD as a novel therapeutic approach for OA treatment, targeting the AhR pathway to counteract the disease's progression and highlighting the need for further clinical evaluation to fully establish its therapeutic utility.

B-249 Assessing Targeted Mass Spectrometry Assay Performance for Putative Biomarker Proteins on a New Hybrid Nominal Mass Instrument

Abstract Background The development of targeted mass spectrometry-based proteomics assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to highly multi-analyte clinical studies. Targeted assays are currently unable to scale to thousands of peptide targets from hundreds of proteins, requiring extensive refinement down assays for the minimum useful analytes. Large parallel reaction monitoring (PRM) assays are now possible with a new hybrid nominal-mass instrument. The scale of assay is achievable with this instrument by using real-time alignment, while being sensitive and fast enough to handle many concurrent targets. To assess the performance of this new instrument we constructed a large scale PRM assay for proposed neurodegenerative disease marker proteins, and an assay for proteins associated with amyloid tissue typing. Methods To evaluate the performance of this instrument on proteins of interest in cerebrospinal fluid, we performed an analysis on a pool from individuals diagnosed as Alzheimer’s, Parkinson’s, or cognitively normal. A calibration curve of cerebrospinal fluid diluted into chicken serum at 14 dilutions and individual human cerebrospinal fluid were digested with a protein aggregation and capture based digestion protocol. CSF peptides were separated by 60 minute gradient on C18 IonOpticks column, and amyloid tissue peptides separated by 30 minute gradient on C18 PepMap column with a Vanquish Neo UHPLC and analyzed by data-independent acquisition (DIA) and PRM with a new hybrid nominal mass instrument designed for targeted tandem mass spectrometry (tMS2). Scheduled methods were generated using a Skyline external tool, transitions were refined to minimize interference and maximize the LLOQ. DIA data was searched, and data extracted and analyzed in Skyline. PRM data was extracted and quantified with Skyline; CSF calibration curve LLOQ is calculated by bilinear fit. Results For the large scale cerebrospinal fluid (CSF) assay the selection of protein targets was based on literature from neurodegenerative disease cohort studies. We target 1065 peptides from 104 proteins, including proteins currently measured by ELISA in clinical research, e.g.: APP, TAU, and APOE. Sensitivity and reproducibility of the assay is assessed using figures of merit from replicate matched-matrix calibration curves. Over 80% of proteins have at least one peptide with a LLOQ below 30% dilution in the matched matrix, and of all targeted peptides over 55% had a LLOQ below 30% dilution. To demonstrate the application of the real-time alignment on the new instrument we constructed an assay for samples where not every analyte is shared. An assay for amyloidosis associated proteins was constructed for a total of 180 peptides mapping to 52 proteins. This includes 11 proteins most commonly associated with amyloid deposition; APCS, APOA4, APOE, CLU, LECT2, IGKC, IGLC2/4/7, SAA1, TTR, and VTN. From the preliminary set of 12 previously typed samples we observe peptide abundances consistent with the assigned type. Conclusions This novel instrument provides a new approach for building large targeted mass spectrometry assays. We demonstrate the ability of the real-time alignment to adjust scheduled retention time windows, ensuring that chromatographic shifts do not lead to a loss of peptide detection and quantification.

B-069 Characterization of a panel of antibodies as diagnostics for NASH and NAFL

Abstract Background Nonalcoholic fatty liver disease (NAFLD) is on the rise in Western industrialized countries and associated comorbidities like e.g. cirrhosis are a major cause of liver transplantations in the USA. In NAFLD, excess fat is accumulated in the liver whereas this build-up is not caused by alcohol. NASH (nonalcoholic steatohepatitis) and NAFL (nonalcoholic fatty liver) are two forms of NAFLD. NAFL represents a stage of the disease in which the liver does not exhibit major signs of inflammation or damage, while NASH does. NAFL does not necessarily progress to cause liver damage. NASH on the other hand may cause fibrosis and lead to cirrhosis with a risk of progression into liver cancer. Since NAFLD shows a prevalence of over 24-25% in both the USA and Europe, diagnosis moves more and more into focus. The current gold standard for NASH diagnosis is liver biopsy, and NAFLD as such can also be diagnosed by ultrasound and MRI, among others. A quest for biomarkers is ongoing to develop blood tests that allow NAFLD diagnosis by immunoassays for example. We have developed high affinity and specificity antibodies against four highly discussed marker proteins for NAFLD: RBP4 (Retinol Binding Protein 4), FGF21 (Fibroblast Growth factor 21), CHI3L1 (Chitinase-3-like protein 1) and PAI-1 (Plasminogen activator inhibitor-1). All four markers are discussed to be elevated in NAFLD patient serum. Methods We characterized the developed anti-RBP4 and anti-FGF21 antibodies via an in-house developed Chemiluminescence Immunoassay (CLIA). Results We have determined the limit of quantification (LoQ), limit of detection (LoD) and linear range of measurable concentrations for our antibody pairs. We further conducted spike and recovery experiments showing the precision of the developed antibodies against RBP4 and FGF21. Conclusions Together, we present antibody pairs for RBP4 and FGF21 suitable as a part of a diagnostic panel that has high potential to be applied in the diagnosis of NAFLD.

Effects of anti-inflammatory drugs on distinct endotypes of chronic rhinosinusitis without nasal polyps: comparison using an ex-vivo model.

The objectives of this study were to characterize the endotypes of different chronic rhinosinusitis without nasal polyps (CRSsNP) samples, to investigate the effects of certain anti-inflammatory drugs on these endotypes, and to investigate the effect of the same drugs on recently identified CRSsNP marker proteins. Initially, ethmoid tissues (ETs) from CRSsNP patients (n=12) were dissected into sections and incubated with the addition of mometasone, verapamil, cenicriviroc, and dupilumab. Cell culture media were collected after 24 hours, and the contents of the secreted proteins interferon-gamma (IFN-γ), interleukin (IL)-5, IL-17A, macrophage inflammatory protein-1 beta (MIP-1β), resistin and platelet (P)-selectin were measured using enzyme-linked immunosorbent assay (ELISA). The endotypes were characterized using the unstimulated samples. The fold changes of protein secretion caused by the analyzed substances were calculated. For each protein, the samples of the distinct endotypes were compared with the remaining samples. Both single and mixed endotypes were identified within the CRSsNP samples, whereas none of the typical endotype-defining cytokines were elevated in a significant portion of the samples. All of the incubated medicaments greatly reduced the tissue secretions of IFN-γ and IL-5 in type 1 CRS while causing a lower secretion of IL-17A in all endotypes compared to the remaining samples. Among the analyzed CRSsNP marker proteins, the distinct endotypes revealed different reactions to the drugs. Dupilumab induced more effects among the examined cytokines than the marker proteins but did not stand out from the other substances overall. Medications used to treat CRS may have different effects on distinct CRS endotypes.

Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles

Polyphosphate (polyP) is a ubiquitous polyanion present throughout the tree of life. While polyP's widely varied functions have been interrogated in single-celled organisms, little is known about the cellular distribution and function of polyP in multicellular organisms. To study polyP in metazoans, we developed the nematode Caenorhabditis elegans as a model system. We designed a high-throughput, longitudinal-orientation cryosectioning method that allowed us to scrutinize the intracellular localization of polyP in fixed C.elegans using fluorescent polyP probes and co-immunostaining targeting appropriate marker proteins. We discovered that the vast majority of polyP is localized within the endo-lysosomal compartments of the intestinal cells and is highly sensitive toward the disruption of endo-lysosomal compartment generation and food availability. This study lays the groundwork for further mechanistic research of polyPs in multicellular organisms and provides a reliable method for immunostaining hundreds of fixed worms in a single experiment.

Nanoparticle Tracking Analysis: An Effective Tool to Characterize Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-enclosed particles that have attracted much attention for their potential in disease diagnosis and therapy. However, the clinical translation is limited by the dosing consistency due to their heterogeneity. Among various characterization techniques, nanoparticle tracking analysis (NTA) offers distinct benefits for EV characterization. In this review, we will discuss the NTA technique with a focus on factors affecting the results; then, we will review the two modes of the NTA techniques along with suitable applications in specific areas of EV studies. EVs are typically characterized by their size, size distribution, concentration, protein markers, and RNA cargos. The light-scattering mode of NTA offers accurate size, size distribution, and concentration information in solution, which is useful for comparing EV isolation methods, storage conditions, and EV secretion conditions. In contrast, fluorescent mode of NTA allows differentiating EV subgroups based on specific markers. The success of fluorescence NTA heavily relies on fluorescent tags (e.g., types of dyes and labeling methods). When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids, such as saliva, blood, and serum. Finally, we will discuss the limitations and future directions of the NTA technique in EV characterization.

Effect of MSC-derived EVs induced by AGEs on energy metabolism in vascular endothelial cells

IntroductionAdvanced glycation end products (AGEs) play a critical role in the development of vascular diseases in diabetes. While stem cell therapies often involve exposure to AGEs, the impact of this environment on extracellular vesicles (EVs) and endothelial cell metabolism remains unclear. MethodsHuman umbilical cord mesenchymal stem cells (MSC) were treated with either 0 ng/mL or 100 ng/mL AGEs in a serum-free medium for 48 hours, after which MSC-EVs were isolated. The EVs were characterized for morphology, particle size, and protein markers of MSC-EVs, and performed miRNA sequencing to identify differentially expressed miRNAs. MSC-EVs were co-cultured with HUVEC cells to assess effects on cell viability, metabolic activity, oxidative stress, and antioxidant capacity. Tube formation and glucose transporter protein analyses were conducted to evaluate the angiogenic ability and glucose metabolism capacity. ResultsMSC-EVs ranged from 30 to 150 nm, consistenting with exosomal properties. AGEs treatment reduced MSC viability but had minimal effect on EV morphology and protein markers. miRNAs sequencing showed downregulation of hsa-miR-223-3p, hsa-miR-126-3p_R-1, with upregulation of hsa-miR-574-5p, implicating changes in glycolytic and oxidative phosphorylation pathways. MSC-EVs treated with AGEs decreased HUVEC viability (P<0.05), pH (P<0.05), ATP metabolism (P<0.05), glucose metabolism (P<0.05), while enhancing glycolysis processes, including glycolytic activity, capacity, and reserve (P<0.05). This likely resulted from impaired mitochondrial function, including reduced ATP production, maximal respiration, basal respiration, and spare respiratory capacity (P<0.05), or increased ROS (P<0.05) and G6PD activity (P<0.05). Additionally, AGEs reduced GLUT1, GLUT3, GLUT4, and SCO2 expression (P<0.05), along with angiogenic capacity (P<0.05) in HUVEC cells. ConclusionExposure to AGEs diminishes the therapeutic potential of MSC-derived EVs by disrupting energy metabolism and promoting metabolic reprogramming in endothelial cells. These findings suggest that adjusting the dosage or frequency of MSC-EVs may enhance their efficacy for treating diabetes-related vascular conditions. Further research is warranted to evaluate AGEs' broader impact on various cell types and metabolic pathways for improved exosome-based therapies.

Advancing diagnostics: integrating microRNA profiling and protein markers in ectopic pregnancy detection

IntroductionEctopic pregnancy (EP) poses significant health risks, particularly in developing nations, necessitating improved diagnostic methods. This study aimed to explore potential biomarkers for EP diagnosis.MethodsA case–control study was conducted at...

Osteoarthritis Year in Review 2024: Molecular biomarkers of osteoarthritis

ObjectiveTo provide a comprehensive and insightful summary of studies on molecular biomarkers at the gene, protein, and metabolite levels across different sample types and joints affected by osteoarthritis (OA). MethodsA literature search using the PubMed database for publications on OA biomarkers published between April 1, 2023 and April 30, 2024 was performed. Publications were then screened, examined at length, and summarized in a narrative review. ResultsOut of the 364 papers initially identified, 44 publications met inclusion criteria, were relevant to OA, and were further examined for data extraction and discussion. These studies included 1 genomic analysis, 22 on protein markers, 6 on metabolite markers, 9 on inflammatory mediators, and 6 integrating multiple molecular levels. ConclusionsSignificant advancements have been made in identifying molecular biomarkers for OA, encompassing various joints, sample types, and molecular levels. Despite this progress, gaps remain, particularly in the need for validation, larger sample sizes, the integration of more clinical data, and consideration of covariates. For early detection and improved treatment of OA, continued efforts in biomarker identification are needed. This effort should seek to identify effective biomarkers that advance early detection, support prevention, evaluate interventions, and improve patient outcomes.