Pathological Processes Research Articles

Molecular Mechanism of Tau Misfolding and Aggregation: Insights from Molecular Dynamics Simulation.

Tau dysfunction has a close association with many neurodegenerative diseases, which are collectively referred to as tauopathies. Neurofibrillary tangles (NFTs) formed by misfolding and aggregation of tau are the main pathological process of tauopathy. Therefore, uncovering the misfolding and aggregation mechanism of tau protein will help to reveal the pathogenic mechanism of tauopathies. Molecular dynamics (MD) simulation is well suited for studying the dynamic process of protein structure changes. It provides detailed information on protein structure changes over time at the atomic resolution. At the same time, MD simulation can also simulate various conditions conveniently. Based on these advantages, MD simulations are widely used to study conformational transition problems such as protein misfolding and aggregation. Here, we summarized the structural features of tau, the factors affecting its misfolding and aggregation, and the applications of MD simulations in the study of tau misfolding and aggregation.

Clinical Predictors of Postmortem Amyloid and Nonamyloid Cerebral Small Vessel Disease in Middle-Aged to Older Adults.

Sporadic cerebral small vessel disease (CSVD) is a class of important pathologic processes known to affect the aging brain and to contribute to cognitive impairment. We aimed to identify clinical risk factors associated with postmortem CSVD in middle-aged to older adults. We developed and tested risk models for their predictive accuracy of a pathologic diagnosis of nonamyloid CSVD and cerebral amyloid angiopathy (CAA) in a retrospective sample of 160 autopsied cases from the Edinburgh Brain Bank. Individuals aged 40 years and older covering the spectrum of healthy aging and common forms of dementia (i.e., highly-prevalent etiologies such as Alzheimer disease (AD), vascular cognitive impairment (VCI), and mixed dementia) were included. We performed binomial logistic regression models using sample splitting and cross-validation methods. Demographics, lifestyle habits, traditional vascular risk factors, chronic medical conditions, APOE4, and cognitive status were assessed as potential predictors. Forty percent of our sample had a clinical diagnosis of dementia (AD = 33, VCI = 26 and mixed = 5) while others were cognitively healthy (n = 96). The mean age at death was 73.8 (SD 14.1) years, and 40% were female. The presence of none-to-mild vs moderate-to-severe nonamyloid CSVD was predicted by our model with good accuracy (area under the curve [AUC] = 0.84, sensitivity [SEN] = 72%, specificity [SPE] = 95%), with the most significant clinical predictors being age, history of cerebrovascular events, and cognitive impairment. The presence of CAA pathology was also predicted with high accuracy (AUC = 0.86, SEN = 93%, SPE = 79%). Significant predictors included alcohol intake, history of cerebrovascular events, and cognitive impairment. In a subset of atypical dementias (n = 24), our models provided poor predictive performance for both nonamyloid CSVD (AUC = 0.50) and CAA (AUC = 0.43). CSVD pathology can be predicted with high accuracy based on clinical factors in patients within the spectrum of AD, VCI, and normal aging. Whether this prediction can be enhanced by the addition of fluid and neuroimaging biomarkers warrants additional study. Improving our understanding of clinical determinants of vascular brain health may lead to novel strategies in the prevention and treatment of vascular etiologies contributing to cognitive decline. This study provides Class II evidence that selected clinical factors accurately distinguish between middle-aged to older adults with and without cerebrovascular small vessel disease (amyloid and nonamyloid) pathology.

Alternative splicing-related long noncoding RNA ANRIL facilitates hepatocellular carcinoma by targeting the miR-199a-5p/SRSF1 axis and impacting Anillin.

Hepatocellular carcinoma (HCC) is characterized by aberrant alternative splicing (AS), which plays an important part in the pathological process of this disease. However, available reports about genes and mechanisms involved in AS process are limited. Our previous research has identified ANRIL as a long noncoding RNA related to the AS process of HCC. Here, we investigated the exact effect and the mechanism of ANRIL on HCC progress.The ANRIL expression profile was validated using the real-time quantitative polymerase chain reactionassay. The western blot analysis and IHC assay were conducted on candidate targets, including SRSF1 and Anillin. The clinicopathological features of 97 patients were collected and analyzed. Loss-of and gain-of-function experiments were conducted. The dual-luciferase reporter assay was applied to verify the interaction between ANRIL, miR-199a-5p, and SRSF1.Anomalous upregulation of ANRIL in HCC was observed, correlating with worse clinicopathological features of HCC. HCC cell proliferation, mobility, tumorigenesis, and metastasis were impaired by depleting ANRIL. We found that ANRIL acts as a sponger of miRNA-199a-5p, resulting in an elevated level of its target protein SRSF1. The phenotypes induced by ANRIL/miR-199a-5p/SRSF1 alteration are associated with Anillin, a validated HCC promoter.ANRIL is an AS-related lncRNA promoting HCC progress by modulating the miR-199a-5p/SRSF1 axis. The downstream effector of this axis in the development of HCC is Anillin.

Epigenetics and environmental health.

Epigenetic modifications including DNA methylation, histone modifications, chromatin remodeling, and RNA modifications complicate gene regulation and heredity and profoundly impact various physiological and pathological processes. In recent years, accumulating evidence indicates that epigenetics is vulnerable to environmental changes and regulates the growth, development, and diseases of individuals by affecting chromatin activity and regulating gene expression. Environmental exposure or induced epigenetic changes can regulate the state of development and lead to developmental disorders, aging, cardiovascular disease, Alzheimer's disease, cancers, and so on. However, epigenetic modifications are reversible. The use of specific epigenetic inhibitors targeting epigenetic changes in response to environmental exposure is useful in disease therapy. Here, we provide an overview of the role of epigenetics in various diseases. Furthermore, we summarize the mechanism of epigenetic alterations induced by different environmental exposures, the influence of different environmental exposures, and the crosstalk between environmental variation epigenetics, and genes that are implicated in the body's health. However, the interaction of multiple factors and epigenetics in regulating the initiation and progression of various diseases complicates clinical treatments. We discuss some commonly used epigenetic drugs targeting epigenetic modifications and methods to prevent or relieve various diseases regulated by environmental exposure and epigenetics through diet.

The mechanisms of white matter injury and immune system crosstalk in promoting the progression of Parkinson’s disease: a narrative review

Parkinson’s disease (PD) is neurodegenerative disease in middle-aged and elderly people with some pathological mechanisms including immune disorder, neuroinflammation, white matter injury and abnormal aggregation of alpha-synuclein, etc. New research suggests that white matter injury may be important in the development of PD, but how inflammation, the immune system, and white matter damage interact to harm dopamine neurons is not yet understood. Therefore, it is particularly important to delve into the crosstalk between immune cells in the central and peripheral nervous system based on the study of white matter damage in PD. This crosstalk could not only exacerbate the pathological process of PD but may also reveal new therapeutic targets. By understanding how immune cells penetrate through the blood–brain barrier and activate inflammatory responses within the central nervous system, we can better grasp the impact of structural destruction of white matter in PD and explore how this process can be modulated to mitigate or combat disease progression. Microglia, astrocytes, oligodendrocytes and peripheral immune cells (especially T cells) play a central role in its pathological process where these immune cells produce and respond to pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6), and white matter injury causes microglia to become pro-inflammatory and release inflammatory mediators, which attract more immune cells to the damaged area, increasing the inflammatory response. Moreover, white matter damage also causes dysfunction of blood–brain barrier, allows peripheral immune cells and inflammatory factors to invade the brain further, and enhances microglia activation forming a vicious circle that intensifies neuroinflammation. And these factors collectively promote the neuroinflammatory environment and neurodegeneration changes of PD. Overall, these findings not only deepen our understanding of the complexity of PD, but also provide new targets for the development of therapeutic strategies focused on inflammation and immune regulation mechanisms. In summary, this review provided the theoretical basis for clarifying the pathogenesis of PD, summarized the association between white matter damage and the immune cells in the central and peripheral nervous systems, and then emphasized their potential specific mechanisms of achieving crosstalk with further aggravating the pathological process of PD.

Differential expression of FSTL1 and its correlation with the pathological process of periodontitis.

This study aimed to elucidate the alterations in Follistatin-like protein 1 (FSTL1) and its association with the pathological process of periodontitis. This study included 48 patients with periodontitis and 42 healthy controls. The expression level of FSTL1 in the gingiva was determined by RT-qPCR, validated using the dataset GSE16134, and subsequently examined by western blotting. Bioinformatics analysis revealed a single-cell distribution of FSTL1, characteristic of angiogenesis and immune cell infiltration. The expression and distribution of FSTL1, vascular endothelial marker protein CD31 and myeloperoxidase (MPO), the indicator of neutrophil activity, were determined by immunohistochemistry (IHC). A series of correlation analyses was performed to determine the associations between FSTL1 and clinical parameters, including probing depth (PD) and clinical attachment loss (CAL), and their potential role in angiogenesis (CD31) and neutrophil infiltration (MPO). FSTL1 was significantly upregulated in the gingiva of patients with periodontitis compared to their healthy counterparts. In addition, FSTL1 was positively correlated with the clinical parameters PD (r = .5971, p = .0005) and CAL (r = .6078, p = .0004). Bioinformatic analysis and IHC indicated that high FSTL1 expression was significantly correlated with angiogenesis and neutrophil infiltration in periodontitis. Moreover, receiver operating characteristic (ROC) analysis demonstrated that FSTL1 could serve as an independent indicator for evaluating the severity of periodontitis (area under the curve [AUC] = 0.9011, p < .0001). This study demonstrated FSTL1 upregulation in periodontitis and its potential contribution to the disease via angiogenesis and neutrophil infiltration.

The Role of Curcumin in Oral Health and Diseases: A Systematic Review

Curcumin (Curcumin) belongs to the polyphenol family. It is extracted by drying the root of a plant of Asian origin, belonging to the Zingiberaceae family. The best-known species is Curcumincuma Longa. Curcumin has been recognized as having great therapeutic powers since ancient times. Studies on curcumin have since confirmed its powerful antioxidant properties, preventing both the formation of free radicals and their neutralization, having anti-inflammatory, antibacterial, immunological, and neuroprotective properties, as well as being a regulator of the intestinal microbiota with beneficial effects on the clinical manifestations of metabolic syndrome. Our study aimed to highlight how all these therapeutic aspects could benefit oral health, both preventing and improving the course of pathological processes. The effect of mouthwashes, and curcumin-based gels on the regulation of bacterial plaque and in the control of gingivitis, was largely comparable to that of using 0.20% chlorhexidine, with fewer side effects. Being a highly hydrophobic substance, it has a high permeability to cross the cell membrane. Bioavailability increases when combined with liposoluble substances (e.g., olive oil) and piperine, which improves absorption. Curcumin also has a negligible degree of toxicity, making it an excellent alternative to the use of gold standard products for oral disinfection.

Lactate metabolism and acute kidney injury.

Acute kidney injury (AKI) is a common clinically critical syndrome in hospitalized patients with high morbidity and mortality. At present, the mechanism of AKI has not been fully elucidated, and no therapeutic drugs exist. As known, glycolytic product lactate is a key metabolite in physiological and pathological processes. The kidney is an important gluconeogenic organ, where lactate is the primary substrate of renal gluconeogenesis in physiological conditions. During AKI, altered glycolysis and gluconeogenesis in kidneys significantly disturb the lactate metabolic balance, which exert impacts on the severity and prognosis of AKI. Additionally, lactate-derived posttranslational modification, namely lactylation, is novel to AKI as it could regulate gene transcription of metabolic enzymes involved in glycolysis or Warburg effect. Protein lactylation widely exists in human tissues and may severely affect non-histone functions. Moreover, the strategies of intervening lactate metabolic pathways are expected to bring a new dawn for the treatment of AKI. This review focused on renal lactate metabolism, especially in proximal renal tubules after AKI, and updated recent advances of lactylation modification, which may help to explore potential therapeutic targets against AKI.

Granzyme B Promotes Proliferation, Migration and EMT Process in Gastric Cancer.

Granzyme B (GZMB), a critical member of the Gr gene family, is known to play an essential role in diverse physiological and pathological processes such as inflammation, acute and chronic inflammatory diseases, and cancer progression. In this study, we delve deeper into the role of GZMB within the context of gastric cancer (GC) to examine its expression patterns and functional implications. To accomplish this, we applied a combination of quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry techniques. These methodologies allowed us to accurately gauge GZMB expression levels in GC tissues and investigate their correlation with various clinical-pathological variables. Our secondary focus was to discern the regulatory influence of GZMB on GC cell biology. We used an array of assays including cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine, and migration assays. The effect of GZMB on gastric cancer progression was further validated through a subcutaneous xenograft mouse model. Our findings underscored that GZMB mRNA and protein levels were upregulated in GC tissues, a feature that showed a significant correlation with GC staging. We also discovered that a decrease in GZMB expression via knockdown experiments suppressed the proliferation and migration capabilities of GC cells. This effect was manifested through diminished expression levels of epithelial-mesenchymal transition (EMT) markers. In stark contrast, the overexpression of GZMB through plasmid transfection appeared to enhance the proliferation and migration abilities of GC cells. This was coupled with an upregulation in EMT expression. Our study concludes by emphasizing that GZMB promotes the growth, migration, and EMT processes in gastric cancer. In vitro, cell-based experiments and in vivo xenograft mouse models confirm this. Our findings provide a more comprehensive understanding of GZMB's role in gastric cancer pathogenesis, potentially opening doors for novel therapeutic strategies targeting this molecular pathway.

Universal Covalent Grafting Strategy of an Aptamer on a Carbon Fiber Microelectrode for Selective Determination of Dopamine In Vivo.

The chemical information on brain science provided by electrochemical sensors is critical for understanding brain chemistry during physiological and pathological processes. A major challenge is the selectivity of electrochemical sensors in vivo. This work developed a universal covalent grafting strategy of an aptamer on a carbon fiber microelectrode (CFE) for selective determination of dopamine in vivo. The universal strategy was proposed by oxidizing poly(tannic acid) (pTA) to form an oxidized state (pTAox) and then coupling a nucleophilic sulfhydryl molecule of the dopamine-binding mercapto-aptamer with the o-quinone moiety of pTAox based on click chemistry for the interfacial functionalization of the CFE surface. It was found that the universal strategy proposed could efficiently graft the aptamer on a glassy carbon electrode, which was verified by using electroactive 6-(ferrocenyl) hexanethiol as a redox reporter. The amperometric method using a fabricated aptasensor for the determination of dopamine was developed. The linear range of the aptasensor for the determination of dopamine was 0.2-20 μM with a sensitivity of 0.09 nA/μM and a limit of detection of 88 nM (S/N = 3). The developed method has high selectivity originating from the specific recognition of the aptamer in concert with the cation-selective action of pTA and could be easily applicable to probe dopamine dynamics in the brain. Furthermore, complex vesicle fusion modes were first observed at the animal level. This work demonstrated that the covalently grafted immobilization strategy proposed is promising and could be extended to the in vivo analysis of other neurochemicals.

Aerobic exercise improves motor dysfunction in Parkinson's model mice via differential regulation of striatal medium spiny neuron.

The striatum plays a crucial role in providing input to the basal ganglia circuit and is implicated in the pathological process of Parkinson's disease (PD). Disruption of the dynamic equilibrium in the basal ganglia loop can be attributed to the abnormal functioning of the medium spiny neurons (MSNs) within the striatum, potentially acting as a trigger for PD. Exercise has been shown to mitigate striatal neuronal dysfunction through neuroprotective and neurorestorative effects and to improve behavioral deficits in PD model mice. In addition, this effect is offset by the activation of MSNs expressing dopamine D2 receptors (D2-MSNs). In the current study, we investigated the underlying neurobiological mechanisms of this effect. Our findings indicated that exercise reduces the power spectral density of the beta-band in the striatum and decreases the overall firing frequency of MSNs, particularly in the case of striatal D2-MSNs. These observations were consistent with the results of molecular biology experiments, which revealed that aerobic training specifically enhanced the expression of striatal dopamineD2 receptors (D2R). Taken together, our results suggest that aerobic training aimed at upregulating striatal D2R expression to inhibit the functional activity of D2-MSNs represents a potential therapeutic strategy for the amelioration of motor dysfunction in PD.

KLF4 Inhibits the Activation of Human Hepatic Stellate Cell In Vitro.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs. Kruppel-like factor 4 (KLF4) plays a pivotal role in a wide array of physiological and pathological processes. This study aimed to investigate the effect of KLF4 on the proliferation, apoptosis and phenotype of quiescent HSCs METHODS: We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector, to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection. Cell proliferation was assessed using the CCK-8 assay. Flow cytometry was used to detect the cell cycle distribution and apoptosis rate. Western blotting was used to determine the levels of some quiescence and activation markers of HSCs RESULTS: Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1, which are quiescent HSC markers, while significantly decreased the levels of N-cadherin and a-SMA, known activated HSC markers. In contrast, cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced CONCLUSION: KLF4 inhibits the proliferation and activation of human LX-2 HSCs. It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.

Extracellular vesicles: Function, resilience, biomarker, bioengineering, and clinical implications

ABSTRACT Extracellular vesicles (EVs) have emerged as key players in intercellular communication, disease pathology, and therapeutic innovation. Initially overlooked as cellular debris, EVs are now recognized as vital mediators of cell-to-cell communication, ferrying a cargo of proteins, nucleic acids, and lipids, providing cellular resilience in response to stresses. This review provides a comprehensive overview of EVs, focusing on their role as biomarkers in disease diagnosis, their functional significance in physiological and pathological processes, and the potential of bioengineering for therapeutic applications. EVs offer a promising avenue for noninvasive disease diagnosis and monitoring, reflecting the physiological state of originating cells. Their diagnostic potential spans a spectrum of diseases, including cancer, cardiovascular disorders, neurodegenerative diseases, and infectious diseases. Moreover, their presence in bodily fluids such as blood, urine, and cerebrospinal fluid enhances their diagnostic utility, presenting advantages over traditional methods. Beyond diagnostics, EVs mediate crucial roles in intercellular communication, facilitating the transfer of bioactive molecules between cells. This communication modulates various physiological processes such as tissue regeneration, immune modulation, and neuronal communication. Dysregulation of EV-mediated communication is implicated in diseases such as cancer, immune disorders, and neurodegenerative diseases, highlighting their therapeutic potential. Bioengineering techniques offer avenues for manipulating EVs for therapeutic applications, from isolation and purification to engineering cargo and targeted delivery systems. These approaches hold promise for developing novel therapeutics tailored to specific diseases, revolutionizing personalized medicine. However, challenges such as standardization, scalability, and regulatory approval need addressing for successful clinical translation. Overall, EVs represent a dynamic frontier in biomedical research with vast potential for diagnostics, therapeutics, and personalized medicine.

Vasculoendothelial dysfunction and bone health in obese children: A connection with cancers

Obesity in children, a growing global epidemic, is not merely characterized by excessive body fat but also by a cascade of metabolic and functional derangements with significant musculoskeletal consequences. One of the key underpinnings of these detrimental effects is vasculoendothelial dysfunction (VED), a multifaceted pathological process characterized by impaired endothelial cell function. The endothelium, lining the vasculature, plays a pivotal role in regulating vascular tone, inflammation, and coagulation, and its dysfunction in obese children leads to a plethora of downstream complications with profound implications for musculoskeletal and bone health. Diagnosing VED in obese children remains a challenge. Traditional cardiovascular risk factors such as hypertension and dyslipidemia are often detected long after the occurrence of endothelial dysfunction. In the context of bone sarcoma, the interaction between cancer cells and vascular endothelium in the bone microenvironment is critical. The bone is a highly vascularized tissue, and the vascular endothelium plays a role in regulating bone homeostasis. In the presence of cancer cells, altered bone microenvironment provides a milieu favorable to tumor growth and invasion. The relationship between the vascular endothelium and cancer is complex and can vary depending on the type of cancer and its microenvironment. Understanding the interactions between vascular endothelium and bone sarcoma is crucial for developing targeted therapies. New molecular and cellular mechanisms involved in these interactions are awaiting to be uncovered, shedding light on potential therapeutic strategies for cancer treatment. On a relevant note, emerging vasculoendothelial markers hold promise for early detection and intervention. Early identification of VED, through the detection of vasculoendothelial markers, opens doors for timely interventions aimed at preventing its detrimental effects on musculoskeletal and bone health in obese children

Type 2 diabetes mellitus related sarcopenia: a type of muscle loss distinct from sarcopenia and disuse muscle atrophy

Muscle loss is a significant health concern, particularly with the increasing trend of population aging, and sarcopenia has emerged as a common pathological process of muscle loss in the elderly. Currently, there has been significant progress in the research on sarcopenia, including in-depth analysis of the mechanisms underlying sarcopenia caused by aging and the development of corresponding diagnostic criteria, forming a relatively complete system. However, as research on sarcopenia progresses, the concept of secondary sarcopenia has also been proposed. Due to the incomplete understanding of muscle loss caused by chronic diseases, there are various limitations in epidemiological, basic, and clinical research. As a result, a comprehensive concept and diagnostic system have not yet been established, which greatly hinders the prevention and treatment of the disease. This review focuses on Type 2 Diabetes Mellitus (T2DM)-related sarcopenia, comparing its similarities and differences with sarcopenia and disuse muscle atrophy. The review show significant differences between the three muscle-related issues in terms of pathological changes, epidemiology and clinical manifestations, etiology, and preventive and therapeutic strategies. Unlike sarcopenia, T2DM-related sarcopenia is characterized by a reduction in type I fibers, and it differs from disuse muscle atrophy as well. The mechanism involving insulin resistance, inflammatory status, and oxidative stress remains unclear. Therefore, future research should further explore the etiology, disease progression, and prognosis of T2DM-related sarcopenia, and develop targeted diagnostic criteria and effective preventive and therapeutic strategies to better address the muscle-related issues faced by T2DM patients and improve their quality of life and overall health.

Job burnout, cognitive functioning, and Brain-derived neurotrophic factor expression among hospital Mexican nurses.

To analyze the relationship between burnout syndrome, cognitive functions, and sBDNF (Serum Brain-derived Neurotrophic Factor) in Mexican nurses. A descriptive cross-sectional design was used. This study target staff nurses working in hospitals in Guanajuato, México. Demographic and working condition data were collected via questionnaire. The Maslach Burnout Inventory (MBI) was used to evaluate burnout. A blood sample were collected and processed by ELISA technique to measure sBDNF. Finally, the General Cognitive Assessment (CAB) of the Cognifit© neuropsychological battery was used to evaluated cognitive functions. Findings showed that there are sociodemographic characteristics and working conditions associated with burnout syndrome among nurses. Furthermore, the data demonstrated a significant decrease in sBDNF levels in burnout nurses and a negative correlation between BDNF levels and burnout syndrome. Additionally, these burnout nurse also revealed significant cognitive impairment in reasoning, memory, and attention as well as total scores of CAB. Interestingly, we found a positive correlation between sBDNF levels and the cognitive deficits in burnout nurse. Reduced BDNF levels could be a biological indicator or part of the pathological process of burnout, which could affect cognitive abilities. Reduced cognitive function in nurses has relevant implications and emphasizes the need for specialized preventive strategies because nurses make clinical decisions concerning their patients, whose situations are constantly changing.

#2983 Correlation of microRNA expression with IgA nephropathy progression: preliminary results

Abstract Background and Aims IgA nephropathy (IgAN) is the most common cause of glomerulonephritis worldwide. IgAN is diagnosed based on the result of histopathological examination. The risk factors for disease progression primarily include the level of impairment of renal function at the time of diagnosis (GFR &amp;lt; 60 ml/min/1.73 m2), the presence of hypertension, daily administration of more than 1.0 g, and the occurrence of hematuria. MicroRNAs (miRNAs) are important regulators of gene expression. Their role has a significant impact on the proper functioning of the body, and disorders related to their expression can lead to the development of many pathological processes in the body. The study aimed to assess the dependence of microRNA expression on the stage of the disease in patients with IgA nephropathy. Method A study was conducted involving patients newly diagnosed with IgA nephropathy based on histopathological examination results. The expression of microRNA molecules (miR-146a, miR-155, miR-200b, miR-429) was determined in urine sediment and blood. The analysis focused on examining the relationship between microRNA expression and the stage of disease progression (proteinuria, renal parameters). Blood and urine tests were performed before the start of immunosuppressive treatment with glucocorticosteroids according to the Pozzi regimen. Results Twenty-seven patients were included in the study group- 14 women (51.9%) and 13 men (48.1%). Nephrotic-range proteinuria (&amp;gt;3000 mg/24 h) was observed in 5 patients (18.5%), while subnephrotic proteinuria was present in 22 patients (77.8%). Reserved kidney function (eGFR &amp;gt; 90) was found in 17 patients (63%). The exact distribution of results according to the stage of renal failure is given in the Table 1. Statistically significant positive correlation was found between serum creatinine concentration and miR-200b expression in urine (R = 0,398, p = 0,049). Initially, no statistically significant correlation was found between the expression of selected miRNA molecules and the degree of 24-hour proteinuria. Conclusion MiRNA molecules may be promising, non-invasive biomarkers widely used in clinical practice. Determining the viability of clinical use of microRNA molecules will require a panel of miRNAs or miRNAs used in combination with other biomarkers and clinical outcomes to improve diagnostic utility.

In-depth organic mass cytometry reveals differential contents of 3-hydroxybutanoic acid at the single-cell level

Comprehensive single-cell metabolic profiling is critical for revealing phenotypic heterogeneity and elucidating the molecular mechanisms underlying biological processes. However, single-cell metabolomics remains challenging because of the limited metabolite coverage and inability to discriminate isomers. Herein, we establish a single-cell metabolomics platform for in-depth organic mass cytometry. Extended single-cell analysis time guarantees sufficient MS/MS acquisition for metabolite identification and the isomers discrimination while online sampling ensures the high-throughput of the method. The largest number of identified metabolites (approximately 600) are achieved in single cells and fine subtyping of MCF-7 cells is first demonstrated by an investigation on the differential levels of 3-hydroxybutanoic acid among clusters. Single-cell transcriptome analysis reveals differences in the expression of 3-hydroxybutanoic acid downstream antioxidative stress genes, such as metallothionein 2 (MT2A), while a fluorescence-activated cell sorting assay confirms the positive relationship between 3-hydroxybutanoic acid and target proteins; these results suggest that the heterogeneity of 3-hydroxybutanoic acid provides cancer cells with different ability to resist surrounding oxidative stress. Our method paves the way for deep single-cell metabolome profiling and investigations on the physiological and pathological processes that occur during cancer.

#2069 Elucidating the function of novel Arg1+/Clec4d+ scar-associated monocyte-derived macrophage population in driving fibrosis in kidney disease models

Abstract Background and Aims Fibrosis is the final common pathway in all progressive kidney disease. Macrophages are a major myeloid cell component of the renal mononuclear phagocyte system, with roles in defence against infection, renal injury, and repair. Using single-cell RNA sequencing, we identified a novel myeloid cell subset exclusively present in acute injury of the unilateral ureteric obstruction (UUO) model of kidney fibrosis. This population transcriptomically aligns to monocytes but is enriched for both Arginase-1 (Arg1) and the C-type lectin 4d (Clec4d) and a large number of pro-inflammatory and pro-fibrotic genes. We hypothesise that this novel Arg1+/Clec4d+ population contributes to fibrosis deposition in progressive kidney disease. Methods Monocyte-macrophage populations were characterised in different pre-clinical models; UUO, unilateral ischaemic reperfusion injury (uIRI) and subtotal nephrectomy with flow cytometry. Arg1+/Clec4d+ cells were identified by RT-qPCR, flow cytometry, and immunofluorescence. The origin and consequence of Arg1 deletion in monocytes on renal fibrosis was determined by utilising Ccr2-ERT2-TdTomato and Ccr2-ERT2-TdTomato; Arg1 fl/fl mice, respectively. Results The presence of the Arg1+/Clec4d+ cells was validated in the UUO, uIRI and subtotal nephrectomy models of kidney injury. Analysis of intra-renal inflammation revealed CD45+CD11b+Arg1+ cells persisted and increased in number across 7 days. Post-injury, Arg1+ cells were confirmed to be Clec4dhi. To confirm that Arg1+/Clec4d+ cells were derived from monocytes (Ccr2+), we administered tamoxifen to Ccr2CreERT2-TdTomato mice under a single or multiple-dose regimen following UUO surgery. At day 7 post-injury, ∼50% and ∼90% of the Arg1+ cells were TdTomato+ following a single or multiple doses of tamoxifen, respectively, suggesting that these cells are primarily monocyte derived. Moreover, the Arg1+/Clec4d+ cells were found to localise to areas of scarring (Fig. 1). To investigate the therapeutic potential of targeting Clec4d+ expressed on the Arg1+/Clec4d+ macrophages, mice underwent UUO surgery and given multiple doses of a Clec4d-neutralising antibody. A decrease in Arg1 and fibrosis-related gene expression was evident by day 7 post-injury. To understand the role of increased Arg1 expression in the cells we generated Ccr2-ERT2-TdTomato; Arg1 fl/fl mice. These mice had as expected a reduced Arg1 expression in the Ccr2+ population and metabolic profiling by SCENITH of the tissue monocytes revealed that the loss of Arg1 in the cells reduced their glucose dependence. The loss of Arg1 in these cells reduced Havcr1 gene expression in the kidney. Conclusion As fibrosis is a pathological process that can affect any organ, the work included here, in addition to the proposed future studies, has the potential to develop novel therapeutics that limit fibrosis and enhance repair to halt the progression of disease in CKD.

#2009 FGF23 in patients with diabetic kidney disease and atherosclerosis

Abstract Background and Aims Diabetes mellitus (DM) and chronic kidney disease (CKD) are risk factors for vascular atherosclerosis, a pathological process with an important inflammatory component. The coexistence of these conditions in patients with diabetic kidney disease (DKD) has additive effects with worst cardiovascular mortality and morbidity rates when compared to non-diabetic CKD patient on cardiovascular outcomes. Accumulating research has shown that increased levels of bone-derived fibroblast growth factor 23 (FGF23), the major phosphate-regulating hormone, constitute a novel predictor of cardiovascular and all-cause mortality in CKD patients, with potential effects on disorders of glucose metabolism as well. The objective of this comparative study is to determine the differences in FGF23 levels in two groups of patients with CKD, with and without DM, matched by age, sex and renal function, all of them with clinical atherosclerotic disease. We also evaluated the relationships of FGF23 with several inflammatory markers and parameters of glucose metabolism. Method Sixty-six patients with CKD stages 3-4 (33 of them with DM) and diagnosed with peripheral vascular disease were included in the study. Whole blood and serum samples were obtained from all participants. CRP values, neutrophil/lymphocyte (N/L) ratio, HbA1c (%), fasting glucose, and Triglycerides and glucose (TyG) index, among other parameters, were obtained using standardized routine methods. Circulating levels of the intact form of FGF23, tumor necrosis factor (TNF) α, interleukin (IL) 6, and IL10 were determined by ELISA immunoassays in both groups. We also performed gene expression analysis for these three cytokines in peripheral blood cells (PBCs) using qPCR. Results The mean values of eGFR, ACR and phosphate were 37.6 ± 11.2 ml/min/1.73 m2; 294.1 ± 271.2 mg/g; 3.8 ± 0.82 mg/dL; respectively without differences between patients with and without DM The median circulating FGF23 values were higher in the DM group (51.59 [37.73-89.68] vs. 38.1 [33-54.14] pg/mL; p&amp;lt;0.001). Likewise, patients with DM presented higher levels of TNFα both in serum concentration (1.78 [0.72-2.47] vs. 1.28 [1.01-1.88] pg/mL; p&amp;lt;0.01) as in the expression of mRNA in PBCs (2.1 ± 1.26 vs. 1.87 ± 1.16 a.u.; p&amp;lt;0.05). No differences were observed for IL6 and IL10 levels, nor for CRP concentration or N/L ratio. As expected, fasting glucose, HbA1c and TyG index were higher in the group of subjects with DM. FGF23 was inversely correlated with eGFR (r=−0.435, p&amp;lt;0.0001), while it showed a positive association with ACR (r=0.416, p&amp;lt;0.001) and, interestingly, with proinflammatory cytokines including circulating TNFα (r=0.44, p&amp;lt;0.001), circulating IL6 (r=0.27, p=0.04), and TNF mRNA expression in PBCs (r=0.245, p=0.047). Furthermore, circulating FGF23 was also direct and significantly correlated with HbA1c (r=0.34, p&amp;lt;0.01). Conclusion These results suggest that FGF23 levels are higher in DKD than in non-DM CKD patients with similar renal function. DKD patients also had higher levels of the proinflammatory cytokine TNFα. Our results also show positive correlations of FGF23 with the inflammatory cytokines TNFα and IL6, as well as with HbA1c. Previous studies, shown that elevated TNFα and glucose promote FGF23 production, which may help to explain the higher levels of FGF23 in patients with DM Future studies should clarify whether FGF23 is simply a marker of atherosclerosis disease severity or a contributing factor to this complication in CKD.