Proximal Fluid Research Articles

Potential protein biomarkers in saliva for detection of frailty syndrome by targeted proteomics

Frailty is a physiological geriatric syndrome, caused by immunosenescence, inflammation and alterations at the protein level leading to metabolic and microbiota changes. Currently, this syndrome is evaluated clinically with the Frailty-VIG index. The aim of the study was therefore to investigate the potential suitability of saliva as a non-invasive proximal biological fluid for the characterisation and identification of possible protein-level biomarkers in frailty syndrome. This cross-sectional study was conducted in a rural population of older Spanish adults using the SMR proteomics technique. A differential protein profile of eight potential and surrogate proteins (CYTC, CYTD, CYTS, CYTB, MIF, ALBU, CD44 and B2MG) was detected in saliva, all of which correlated with factors characterising frailty syndrome, such as vascular ageing (arterial stiffness and cardiovascular disease), obesity, mood problems, global cognitive impairment, changes in gait and hand pressure strength. The proteins CYTD (r = 0.415, p = 0.013) and CYTC (r = 0.280, p = 0.026), which were detected differentially in the protein profile, were associated with the Frailty-VIG index. All analysed proteins are associated not only with the clinical symptoms of frailty syndrome, but also with an acute inflammatory response, endothelial cell proliferation and the complement system, among others.

Estimated Proximal Tubule Fluid Phosphate Concentration and Renal Tubular Damage Biomarkers in Early Stages of Chronic Kidney Disease

IntroductionAn increase in proximal tubule fluid phosphate concentration is caused by increased serum fibroblast growth factor 23 (FGF23) levels, which resulted in renal tubular damage in a mouse model of chronic kidney disease (CKD). However, few human studies have supported this concept. This study aimed to explore the association among estimated proximal tubule fluid phosphate concentration (ePTFp), serum FGF23 levels, and renal tubular damage biomarkers in middle-aged and older populations with mild decline in renal function. MethodsThis cross-sectional study included 218 participants aged ≥45 with CKD stages G2–G4. Anthropometric measurements, blood tests, spot urine biomarkers, renal ultrasonography, cardiovascular assessment, smoking status, and medication usage were obtained in the morning in fasted states. The ePTFp was calculated using serum creatinine, urine phosphate, and creatinine concentrations. Urinary β2-microglobulin and liver-type fatty acid-binding protein (L-FABP) levels were evaluated to assess renal tubular damage.Results: ePTFp, serum FGF23, urinary β2-microglobulin, and urinary L-FABP levels increased with CKD stage progression (stages G2, G3, and G4). However, serum and urine phosphate concentrations were comparable across the CKD stages. Univariate analysis revealed a stronger correlation of ePTFp with serum FGF23, urinary β2-microglobulin, and urinary L-FABP levels than with the corresponding serum and urine phosphate concentrations. Multivariate analyses demonstrated that increased ePTFp was independently associated with elevated serum FGF23 and urinary β2-microglobulin levels, even after adjusting for potential covariates, including the estimated glomerular filtration rate and urinary albumin-to-creatinine ratio. ConclusionsOur results are consistent with the concept in mouse model and suggest that increased ePTFp are associated with increased serum FGF23 levels and renal tubular damage during the early stages of CKD.

SGLT2-independent effects of canagliflozin on NHE3 and mitochondrial complex I activity inhibit proximal tubule fluid transport and albumin uptake.

Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo.NEW & NOTEWORTHY Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead–based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (μg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 μg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 μg of beads for sample preparation and long-term digestion (16h) with 0.15 μg Lys-C and 2.5 μg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Exploration of the method and efficacy of treatments for intractable pelvic pain caused by rectal or bladder fistula

Objective: To explore the causes and therapeutic effects of pelvic pain caused by rectal fistula or bladder fistula after comprehensive treatment of cervical cancer and rectal cancer (radiotherapy, surgery, chemotherapy, and other treatments). Methods: A retrospective analysis was conducted on the clinical and pathological data of patients with pelvic tumors admitted to the First People's Hospital of Yinchuan City, Ningxia and the Affiliated Cancer Hospital of Zhengzhou University from June 2016 to June 2022. The causes of persistent pelvic pain in patients after comprehensive treatment was investigated, and the corresponding therapeutic effects after clinical treatment was observed. Results: Thirty-two tumor patients experienced persistent pain after comprehensive treatment, including 22 cases of cervical cancer and 10 cases of rectal cancer. The preoperative pain of the entire group of patients was evaluated using the digital grading method, with a pain score of (7.88±1.31) points. Among the 32 patients, there were 16 cases of rectovaginal fistula or ileovaginal fistula, 9 cases of vesicovaginal fistula, 5 cases of rectoperineal fistula, and 2 cases of vesicovaginorectal fistula. Thirty-two patients were initially treated with medication to relieve pain, and according to the ruptured organs, a fistula was made to the corresponding proximal intestinal canal and renal pelvis to intercept the intestinal contents and urine. However, the pain did not significantly be improved. The pain score of treatment with the above methods for one week was (8.13±1.13) points, and there was no statistically significant difference compared to preoperative treatment (P=0.417). In the later stage, based on a comprehensive evaluation of whether the tumor had recurred, the value of organ preservation, the benefits of surgery, the balance between survival time and improving quality of life, pathological organ resection or repair was performed. The surgical methods included repair of leaks, local debridement combined with irrigation of proximal intestinal fluid, distal closure of the sigmoid colon combined with proximal ostomy, posterior pelvic organ resection, anterior pelvic organ resection, and total pelvic organ resection. One week after surgery, the patients' pain completely relieved or disappeared, with the pain score of (1.72±1.37) points, which was significantly divergent from the preoperative and initial surgical treatments (P＜0.001). Conclusions: Palliative pyelostomy and proximal enterostomy cannot effectively alleviate persistent pelvic floor pain. The fundamental way to alleviate pain is complete blocking of the inflammatory erosion of the intestinal fluid and urine.

Abstract 7565: Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing

Abstract Background: Cell-free DNA (cfDNA) detected in proximal body fluids has demonstrated potential for cancer detection using minimally invasive methodology. Our past work showed that tumor cfDNA is present in the cerebrospinal fluid (CSF) and other body fluids of patients with inconclusive standard of care testing. However, past work measuring copy number aberrations or somatic mutations was limited in cancer classification. To facilitate reliable classification, even at low tumor fractions and with fragmented DNA, we developed XR-methylSeq, a methylation sequencing platform to enrich for cell type-specific markers. Methods: We benchmarked XR-methylSeq with the K562 cell line and correlated the methylation values with gold standard measurement − whole genome bisulfite sequencing (WGBS). Methylation classifiers were applied for at least 22 cytology-positive body fluids, incorporating methylation array data from public references. T-distributed stochastic neighbor embedding (t-SNE) analysis was used for visualization in R. Deconvolution of cell type fractions for at least 29 (seven cytology-negative) body fluids and plasma samples was conducted using wgbstools. Cell type-specific markers were identified from a human DNA methylation atlas. Results: Benchmarks: XR-methylSeq has a 5-fold enrichment of the cell type-specific markers compared with WGBS. XR-methylSeq at 20 ng input highly correlates with WGBS at 2 μg (Pearson’s r = 0.97). Body Fluids: Thirteen cytology-positive CSF samples had copy number aberrations, 77% of them had concordant tumor classification, while the remaining 23% clustered with low tumor fraction samples. All nine lung primaries, including a low tumor fraction case that did not originally classify, showed a consistent cell-of-origin through deconvolution, as indicated by increased contributions from lung alveolar epithelial cells. Among six other body fluids, three exhibited the highest fractions aligning with the clinically identified cancer cell-of-origin. Additionally, the plasma cfDNA of a patient with acute liver injury had a higher fraction of hepatocyte signatures (22%) than the healthy control (8%). Conclusions: This research highlights the potential of XR-methylSeq as an enriched methylation profiling method useful for liquid biopsy applications. Citation Format: Jingru Yu, Lauren S. Ahmann, Yvette Y. Yao, Angus Toland, Alicia Snowden, Chandler Ho, Benjamin Pinsky, Hannes Vogel, Ruben Y. Luo, Linlin Wang, Brooke Howitt, Brittany Holmes, Alarice C. Lowe, Wei Gu. Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7565.

Sulfur and Carbon–Oxygen Isotopic Geochemistry and Fluid Inclusion Characteristics of the Yolindi Cu-Fe Skarn Mineralization, Biga Peninsula, NW Turkey: Implications for the Source and Evolution of Hydrothermal Fluids

The current study sought to investigate the physiochemical conditions and fluid evolution within the Yolindi Cu-Fe skarn mineralization located in the Biga Peninsula, NW Turkey. This was accomplished through a comprehensive investigation of geological and mineralogical data, along with isotopic analyses of sulfur (δ34S), carbon (δ13C), and oxygen (δ18O) of sulfide and calcite minerals, respectively, as well as fluid inclusion data pertaining to various minerals (e.g., andradite, quartz, and calcite). The Yolindi area features a complex geological framework, including the Paleozoic Kalabak Group (which includes the Torasan, Yolindi, and Sazak formations) and the Triassic Karakaya Complex. These formations were subsequently intruded via Early Miocene Şaroluk granitoids and Hallaçlar volcanics. Skarn formation is zoned into endoskarn and exoskarn types (being categorized into proximal, intermediate, and distal zones), with distinct mineral assemblages indicating concentric and contact metamorphic alteration patterns around the western part of Şaroluk granitoid intrusion in contact with the Torasan formation. The ore mineralogy and paragenesis suggest three distinct stages of evolution: an initial phase of prograde metasomatism characterized by the formation of magnetite and pyrite alongside anhydrous calc-silicate minerals; a subsequent phase of retrograde alteration marked by the formation of epidote, actinolite, and scapolite, accompanied by the occurrence of chalcopyrite and specular hematite; and finally, a post-metasomatic stage involving oxidation processes that led to the development of secondary mineral assemblages containing cerussite, covellite, and malachite. Sulfur isotopes (δ34S) of sulfides from endoskarn (from +0.27 to +0.57‰VCDT) to intermediate exoskarn (from −9.44 to −5.46‰VCDT) zones indicate a diverse sulfur source, including magmatic, sedimentary, and possibly organic matter. δ34S values in hydrothermal fluids suggest a magmatic–hydrothermal origin, with endoskarn and proximal zone fluids showing a slight negative signature and intermediate zone fluids indicating a strong influence from organic-rich or metamorphic sulfur reservoirs. Carbon and oxygen isotopic compositions (δ13C and δ18O) of calcite revealed a progression from marine carbonate signatures in marble samples (from +1.89 to +2.23‰VPDB; from +21.61 to +21.73‰VSMOW) to depleted values in prograde (from −6.0 to +0.09‰VPDB; from +6.22 to +18.14‰VSMOW) and retrograde skarns (from −3.8 to −2.25‰VPDB; from +0.94 to +3.62‰VSMOW), reflecting interactions with high-temperature magmatic fluids and meteoric water mixing. The fluid inclusions in prograde minerals generated under the conditions of fluid boiling exhibited high temperatures, reaching up to 412 °C, and salinities up to 26 wt.% NaCl equivalent. Conversely, the fluid inclusions in retrograde minerals, which were generated due to fluid mixing, exhibited lower temperatures (with an average of 318 °C) and salinities with an average of 4.9 wt.% NaCl equivalent. This indicated that the cooler and more diluted fluids mix with meteoric waters and interact with organic materials in the host rocks. This suggests a multifaceted origin involving various sources and processes. Therefore, this study concluded that the skarn mineralization in the Yolindi area resulted from complex interactions between magmatic, metamorphic, and meteoric fluids, reflecting a dynamic ore-forming environment with implications for the regional metallogeny of Cu-Fe skarn deposits.

The Sasaki-W anastomosis for recurrent Crohn’s disease stenosis after the Kono-S anastomosis

BackgroundPostoperative recurrence is frequently encountered in the management of patients with Crohn’s disease and is most often found at the anastomotic site. A novel technique, the Sasaki-W anastomosis, is an antimesenteric cutback end-to-end isoperistaltic anastomosis. We report a patient with Crohn’s disease who underwent partial intestinal resection for postoperative anastomotic stenosis, reconstructed with the Sasaki-W anastomosis, after initial intestinal resection reconstructed with a Kono-S anastomosis.Case presentationA 30-year-old male was diagnosed with Crohn’s disease and treated with mesalamine and adalimumab, and he underwent ileocecal resection using the Kono-S anastomosis at the time of diagnosis. He was treated with infliximab without any symptoms or recurrence for 7 years. He was admitted presenting with upper abdominal pain. Physical examination showed mild tenderness and distension in the upper abdomen. Laboratory data showed no remarkable findings. Computed tomography scan showed wall thickening in the ileum with proximal dilation and fluid retention. Non-operative management with antibiotics and fasting did not improve the symptoms within 7 days. Ten days after admission, ileocecal resection reconstructed with the Sasaki-W anastomosis was performed. At operation, there was a 15-cm intestinal stenosis at the site of the previous Kono-S anastomosis. The transverse colon and ileum were reconstructed with the Sasaki-W anastomosis. The postoperative course was uneventful, and the patient was discharged 17 days postoperatively. The patient had no obstructive symptoms and no findings consistent with bowel obstruction were observed on computed tomography scan one year postoperatively.ConclusionsThe Sasaki-W anastomosis is a viable option for intestinal reconstruction in patients with postoperative recurrence after a Kono-S anastomosis.

Myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia are indistinguishable by their cerebrospinal fluid proteomes

Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and fibromyalgia have overlapping neurologic symptoms particularly disabling fatigue. This has given rise to the question whether they are distinct central nervous system (CNS) entities or is one an extension of the other. Material and methods To investigate this, we used unbiased quantitative mass spectrometry-based proteomics to examine the most proximal fluid to the brain, cerebrospinal fluid (CSF). This was to ascertain if the proteome profile of one was the same or different from the other. We examined two separate groups of ME/CFS, one with (n = 15) and one without (n = 15) fibromyalgia. Results We quantified a total of 2083 proteins using immunoaffinity depletion, tandem mass tag isobaric labelling and offline two-dimensional liquid chromatography coupled to tandem mass spectrometry, including 1789 that were quantified in all the CSF samples. ANOVA analysis did not yield any proteins with an adjusted p value <.05. Conclusion This supports the notion that ME/CFS and fibromyalgia as currently defined are not distinct entities. Key message ME/CFS and fibromyalgia as currently defined are not distinct entities. Unbiased quantitative mass spectrometry-based proteomics can be used to discover cerebrospinal fluid proteins that are biomarkers for a condition such as we are studying.

Association between Bioimpedance Spectroscopy and Magnetic Resonance Lymphangiography in the Diagnosis and Assessment of Lymphedema.

This study assesses associations between bioimpedance spectroscopy (BIS) and magnetic resonance lymphangiography (MRL) in the staging and assessment of lymphedema. Adults who received MRL and BIS between 2020 and 2022 were included. We collected fluid, fat, and lymphedema severity ratings, and measured fluid stripe thickness, subcutaneous fat width, and lymphatic diameter on MRL. BIS lymphedema index (L-Dex) scores were collected from patient charts. We assessed sensitivity and specificity of L-Dex scores to detect MRL-identified lymphedema, and examined associations between L-Dex scores and MRL imaging measures. Forty-eight limbs across 40 patients were included. L-Dex scores had 72.5% sensitivity and 87.5% specificity for detecting MRL-defined lymphedema, with a 96.7% estimated positive predictive value and 38.9% negative predictive value. L-Dex scores were associated with MRL fluid and fat content scores (p ≤ 0.05), and lymphedema severity (p = 0.01), with better discrimination between fluid than fat content levels on pairwise analysis, and poor discrimination between adjacent severity levels. L-Dex scores were correlated with distal and proximal limb fluid stripe thickness (distal: rho = 0.57, p < 0.01; proximal: rho = 0.58, p < 0.01), partially correlated with distal subcutaneous fat thickness when accounting for body mass index (rho = 0.34, p = 0.02), and were not correlated with lymphatic diameter (p = 0.25). L-Dex scores have high sensitivity, specificity, and positive predictive value for the identification of MRL-detected lymphedema. L-Dex has difficulty distinguishing between adjacent severity levels of lymphedema and a high false negative rate, explained in part by reduced discrimination between levels of fat accumulation.

Loss of CSF-contacting neuron sensory function is associated with a hyper-kyphosis of the spine reminiscent of Scheuermann’s disease

Scheuermann’s disease, also referred to as Scheuermann’s kyphosis, is the second most frequent spine deformity occurring in humans after adolescent idiopathic scoliosis (AIS), both with an unclear etiology. Recent genetic studies in zebrafish unraveled new mechanisms linked to AIS, highlighting the role of the Reissner fiber, an acellular polymer bathing in the cerebrospinal fluid (CSF) in close proximity with ciliated cells and mechanosensory neurons lining the central canal of the spinal cord (CSF-cNs). However, while the Reissner fiber and ciliary beating have been linked to AIS-like phenotypes in zebrafish, the relevance of the sensory functions of CSF-cNs for human spine disorders remains unknown. Here, we show that the thoracic hyper-kyphosis of the spine previously reported in adult pkd2l1 mutant zebrafish, in which the mechanosensory function of CSF-cNs is likely defective, is restricted to the sagittal plane and is not associated with vertebral malformations. By applying orthopedic criteria to analyze the amplitude of the curvature at the apex of the kyphosis, the curve pattern, the sagittal balance and sex bias, we demonstrate that pkd2l1 knock-outs develop a phenotype reminiscent of Scheuermann’s disease. Altogether our work consolidates the benefit of combining genetics and analysis of spine deformities in zebrafish to model idiopathic spine disorders in humans.

Sedimentation and migration dynamics of a spherical particle in an elastoviscoplastic fluid near a wall

The sedimentation of a spherical particle in an elastoviscoplastic fluid in proximity of a flat wall is investigated by direct numerical simulations. The governing equations under inertialess conditions are solved by the finite element method with an Arbitrary Lagrangian-Eulerian formulation to manage the particle motion. The fluid is modeled with the Giesekus constitutive equation modified as proposed by Saramito (2007).

Polarity in respiratory development, homeostasis and disease.

The respiratory system is composed of a multitude of cells that organize to form complex branched airways that end in alveoli, which respectively function to guide air flow and mediate gas exchange with the bloodstream. The organization of the respiratory sytem relies on distinct forms of cell polarity, which guide lung morphogenesis and patterning in development and provide homeostatic barrier protection from microbes and toxins. The stability of lung alveoli, the luminal secretion of surfactants and mucus in the airways, and the coordinated motion of multiciliated cells that generate proximal fluid flow, are all critical functions regulated by cell polarity, with defects in polarity contributing to respiratory disease etiology. Here, we summarize the current knowledge of cell polarity in lung development and homeostasis, highlighting key roles for polarity in alveolar and airway epithelial function and outlining relationships with microbial infections and diseases, such as cancer.

BIOM-59. NOVEL BIOMARKER DISCOVERY AND DIAGNOSTICS FOR INTRA-AXIAL BRAIN TUMORS, USING CSF PROTEOMICS

Abstract BACKGROUND Invasive brain sampling is typically necessary for reliable diagnosis and prognostication of intra-axial brain tumors but carries risk of morbidity. Liquid biopsy of proximal fluids may mitigate this risk. Through direct contact with the tumor microenvironment and, as an ultra-filtrate of plasma, the cerebrospinal fluid may be the ideal matrix. Reflecting the tumor phenotype, proteomic analyses are critical. Here we identified diagnostic CSF proteomic signatures and putatively novel biomarkers for glioblastoma (GBM), brain metastases (BM), and central nervous system lymphoma (CNSL). METHODS CSF samples were retrospectively retrieved from the Penn State Neuroscience Biorepository and profiled using shotgun proteomics and the MStern approach. Proteomic signatures were identified using machine learning classifiers and survival analyses. RESULTS With as little as 30 µL of CSF, 755 unique proteins were recovered across 73 samples (22 GBM, 17 BM, 14 CNSL, 20 NPH). Proteomic-based classifiers identified malignancy with area under the receiver operating characteristic (AUROC) of 0.94 and distinguished between tumor entities with AUROC ≥0.95. More clinically relevant triplex classifiers, comprised of just 3 proteins, distinguished between tumor entities with AUROC of 0.75-0.89. Novel biomarkers were identified, including GAP43, TFF3 and CACNA2D2, and characterized using single-cell RNA sequencing. DISCUSSION Reliable classification of intra-axial malignancies using low CSF volumes is feasible, allowing for longitudinal tumor surveillance. Based on emerging evidence, upfront implantation of CSF reservoirs in brain tumor patients warrants consideration.

BSCI-02 CSF PROTEOMICS AS A MINIMALLY-INVASIVE STRATEGY FOR DISTINGUISHING BRAIN METASTASES FROM OTHER PRIMARY BRAIN MALIGNANCIES

BACKGROUNDAccurate diagnosis and prognostication of intra-axial brain tumors hinges on invasive brain sampling, which carries risk of morbidity. Minimally invasive sampling of proximal fluids, also known as liquid biopsy, can mitigate this risk. Although the cerebrospinal fluid (CSF) is the ideal liquid biopsy source, the traditionally high volumes required for impactful analyses have deterred progress. The objective of this study was to identify diagnostic and prognostic CSF proteomic signatures in glioblastoma (GBM), brain metastases (BM), and central nervous system lymphoma (CNSL).METHODSCSF samples were retrospectively retrieved from the Penn State Neuroscience Biorepository and profiled using shotgun proteomics with low sample volumes. Proteomic signatures were identified using machine learning classifiers and survival analyses.RESULTSUsing 30µL CSF volumes, we recovered 800 unique peptides across 73 samples [20 normal pressure hydrocephalus (NPH, non-tumor control), 22 GBM, 17 BM, and 14 CNSL]. Externally-validated proteomic-based classifiers identified malignancy with AUROC of 0.94 and distinguished individual tumor entities from others with AUROC ≥0.96. More clinically relevant triplex classifiers, comprised of just 3 peptides, distinguished individual tumor entities with AUROC ≥0.90. Novel biomarkers were identified among the top classifiers, including TFF3 and CACNA2D2, and characterized using single-cell RNA sequencing data. Survival analyses validated previously implicated prognostic signatures, including blood brain barrier disruption.DISCUSSIONReliable classification of intra-axial malignancies using low CSF volumes is feasible, which has ramifications for longitudinal tumor surveillance. Novel biomarkers identified here necessitate future validation. Based on emerging evidence, upfront implantation of CSF reservoirs in brain tumor patients warrants consideration.

Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model

BackgroundNowadays, nanoparticles (NPs) have evolved as multifunctional systems combining different custom anchorages which opens a wide range of applications in biomedical research. Thus, their pharmacological involvements require more comprehensive analysis and novel nanodrugs should be characterized by both chemically and biological point of view. Within the wide variety of biocompatible nanosystems, iron oxide nanoparticles (IONPs) present mostly of the required features which make them suitable for multifunctional NPs with many biopharmaceutical applications.ResultsCisplatin-IONPs and different functionalization stages have been broadly evaluated. The potential application of these nanodrugs in onco-therapies has been assessed by studying in vitro biocompatibility (interactions with environment) by proteomics characterization the determination of protein corona in different proximal fluids (human plasma, rabbit plasma and fetal bovine serum),. Moreover, protein labeling and LC–MS/MS analysis provided more than 4000 proteins de novo synthetized as consequence of the nanodrugs presence defending cell signaling in different tumor cell types (data available via ProteomeXchanges with identified PXD026615). Further in vivo studies have provided a more integrative view of the biopharmaceutical perspectives of IONPs.ConclusionsPharmacological proteomic profile different behavior between species and different affinity of protein coating layers (soft and hard corona). Also, intracellular signaling exposed differences between tumor cell lines studied. First approaches in animal model reveal the potential of theses NPs as drug delivery vehicles and confirm cisplatin compounds as strengthened antitumoral agents.

Renal and Vascular Effects of Combined SGLT2 and Angiotensin-Converting Enzyme Inhibition.

Background:The cardiorenal effects of sodium-glucose cotransporter 2 inhibition (empagliflozin 25 mg QD) combined with angiotensin-converting enzyme inhibition (ramipril 10 mg QD) were assessed in this mechanistic study in patients with type 1 diabetes with potential renal hyperfiltration.Methods:Thirty patients (out of 31 randomized) completed this double-blind, placebo-controlled, crossover trial. Recruitment was stopped early because of an unexpectedly low proportion of patients with hyperfiltration. Measurements were obtained after each of the 6 treatment phases over 19 weeks: (1) baseline without treatment, (2) 4-week run-in with ramipril treatment alone, (3) 4-week combined empagliflozin-ramipril treatment, (4) a 4-week washout, (5) 4-week combined placebo-ramipril treatment, and (6) 1-week follow-up. The primary end point was glomerular filtration rate (GFR) after combination treatment with empagliflozin-ramipril compared with placebo-ramipril. GFR was corrected for ramipril treatment alone before randomization. At the end of study phase, the following outcomes were measured under clamped euglycemia (4 to 6 mmol/L): inulin (GFR) and para-aminohippurate (effective renal plasma flow) clearances, tubular sodium handling, ambulatory blood pressure, arterial stiffness, heart rate variability, noninvasive cardiac output monitoring, plasma and urine biochemistry, markers of the renin-angiotensin-aldosterone system, and oxidative stress.Results:Combination treatment with empagliflozin-ramipril resulted in an 8 mL/min/1.73 m2 lower GFR compared with placebo-ramipril treatment (P=0.0061) without significant changes to effective renal plasma flow. GFR decrease was accompanied by a 21.3 mL/min lower absolute proximal fluid reabsorption rate (P=0.0092), a 3.1 mmol/min lower absolute proximal sodium reabsorption rate (P=0.0056), and a 194 ng/mmol creatinine lower urinary 8-isoprostane level (P=0.0084) relative to placebo-ramipril combination treatment. Sodium-glucose cotransporter 2 inhibitor/angiotensin-converting enzyme inhibitor combination treatment resulted in additive blood pressure–lowering effects (clinic systolic blood pressure lower by 4 mm Hg [P=0.0112]; diastolic blood pressure lower by 3 mm Hg [P=0.0032]) in conjunction with a 94.5 dynes × sex/cm5 lower total peripheral resistance (P=0.0368). There were no significant changes observed to ambulatory blood pressure, arterial stiffness, heart rate variability, or cardiac output with the addition of empagliflozin.Conclusions:Adding sodium-glucose cotransporter 2 inhibitor treatment to angiotensin-converting enzyme inhibitor resulted in an expected GFR dip, suppression of oxidative stress markers, additive declines in blood pressure and total peripheral resistance. These changes are consistent with a protective physiologic profile characterized by the lowering of intraglomerular pressure and related cardiorenal risk when adding a sodium-glucose cotransporter 2 inhibitor to conservative therapy.Registration:URL: https://www.clinicaltrials.gov; Unique identifier: NCT02632747.

CSF proteomics for differentiation of brain malignancies.

2021 Background: Accurate diagnosis and prognostication of intra-axial brain tumors hinges on invasive brain sampling, which carries risk of morbidity. Minimally invasive sampling of proximal fluids, also known as liquid biopsy, can mitigate this risk. Although the cerebrospinal fluid (CSF) is the ideal liquid biopsy source, the traditionally high volumes required for impactful analyses have deterred progress. The objective of this study was to identify diagnostic and prognostic CSF proteomic signatures in glioblastoma (GBM), brain metastases (BM), and central nervous system lymphoma (CNSL). Methods: CSF samples were retrospectively retrieved from the Penn State Neuroscience Biorepository and profiled using shotgun proteomics with ultra-low sample volumes. Proteomic signatures were identified using machine learning classifiers and survival analyses. Results: Using 30µL CSF volumes, we recovered 800 unique peptides across 73 samples [20 normal pressure hydrocephalus (NPH, non-tumor control), 22 GBM, 17 BM, and 14 CNSL]. Externally-validated proteomic-based classifiers identified malignancy with AUROC of 0.94 and distinguished individual tumor entities from others with AUROC ≥0.96. More clinically relevant triplex classifiers, comprised of just 3 peptides, distinguished individual tumor entities with AUROC ≥0.90. Novel biomarkers were identified among the top classifiers, including TFF3 and CACNA2D2, and characterized using single-cell RNA sequencing data. Survival analyses validated previously implicated prognostic signatures, including blood brain barrier disruption. Conclusions: Reliable classification of intra-axial malignancies using ultra-low CSF volumes is feasible, which has ramifications for longitudinal tumor surveillance. Novel biomarkers identified here necessitate future validation. Based on emerging evidence, upfront implantation of CSF reservoirs in brain tumor patients warrants consideration.

20-OR: The Renal and Vascular Effects of Combined SGLT2 and Angiotensin Converting Enzyme Inhibition

The goal of this mechanistic trial was to determine the kidney and cardiovascular effects of combined treatment with an SGLT2i (empagliflozin 25mg QD) and an ACEi (ramipril 10mg QD) for 4 weeks in patients at risk of renal hyperfiltration. In this randomized, double-blind, placebo-controlled, cross-over trial, measurements were obtained following each of the 4 treatment phases: 1) no treatment, 2) 4-week ramipril treatment alone, 3) 4-week empagliflozin-ramipril combination treatment, and 4) 4-week placebo-ramipril combination treatment. The primary endpoint was GFR after empagliflozin-ramipril treatment compared to placebo-ramipril. At the end of each study phase the following measurements were performed under clamped euglycemia (4-6 mmol/L) : inulin (GFR) and paraaminohippurate (effective renal plasma flow, ERPF) clearances, tubular sodium handling, ambulatory blood pressure, arterial stiffness, heart rate variability, non-invasive cardiac output monitoring, plasma and urine biochemistry. Empagliflozin-ramipril treatment resulted in an 8ml/min/1.73m2 lower GFR (p=0.0061) , lower absolute proximal fluid reabsorption rate (p=0.0092) , lower absolute proximal sodium reabsorption rate (p=0.0056) , and lower urinary 8-isoprostane (p=0.0084) relative to placebo-ramipril treatment. Empagliflozin-ramipril treatment resulted in additive blood pressure lowering effects (SBP p=0.0112; DBP p=0.0032) and a lower total peripheral resistance (p=0.0368) . There were no other significant changes observed with the addition of empagliflozin. SGLT2i-ACEi combination treatment resulted in an expected GFR “dip”, suppression of oxidative stress markers, and additive declines in blood pressure and total peripheral resistance in this mechanistic study. These changes are consistent with a protective physiological profile characterized by the lowering of intraglomerular pressure and related cardiorenal risk when adding an SGLT2i to conservative therapy. Disclosure Y. Lytvyn: None. K. Kimura: Employee; 3Boehringer Ingelheim Canada Ltd./Ltée. N. Peter: Employee; Boehringer Ingelheim Pharma GmbH &amp; Co.KG. V.S. Lai: Advisory Panel; Baxter. J. Tse: None. L. Cham: None. B.A. Perkins: Advisory Panel; Abbott Diabetes, Insulet Corporation, Sanofi. Board Member; Novo Nordisk. Research Support; BMO Bank of Montreal, Novo Nordisk. Other Relationship; Abbott Diabetes, Insulet Corporation, Medtronic, Novo Nordisk. N. Soleymanlou: Employee; Boehringer Ingelheim Pharmaceuticals Inc., Boehringer Ingelheim Pharmaceuticals Inc. D. Cherney: Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca and Novo-Nordisk. Other Relationship; AbbVie Inc., AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Janssen Research &amp; Development, LLC, Lilly, Maze, BMS, CSL-Behring, Merck, Otsuka, Novartis and Novo-Nordisk, Mitsubishi Tanabe Pharma Corporation, Sanofi. Funding Boehringer Ingelheim and CIHR, Diabetes Canada and the Heart and Stroke Richard Lewar Centre of Excellence and the Heart and Stroke Foundation of Canada

MO545: Estimated Proximal Tubule Fluid Phosphate Concentration: An Early Marker of CKD-MBD?

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) typically associates with mineral bone disorders (CKD-MBD), kidney damage progression and increased morbidity and mortality. CKD-MBD develops early in CKD and, if promptly detected, could receive therapeutic attention. However, early markers of CKD-MBD like fibroblast growth factor 23 (FGF23) and α-Klotho are not widely available in clinical practice. Recently, estimated proximal tubule fluid phosphate concentration (ePTFp) has been proposed as a novel early marker of MBD. In fact, in CKD patients, along with the progressive reduction of nephron number, FGF23 increments are necessary to reduce renal tubular phosphate reabsorption and to maintain phosphate balance. As a consequence, PTFp concentration per single nephron progressively increases, leading to renal tubular cells overload, interstitial inflammatory response and progression of kidney damage. For this reason, PTFp concentration could represent an early marker of CKD-MBD. OBJECTIVE To evaluate the relationship between ePTFp and early markers of MBD in patients with CKD stage G2–4. METHOD In CKD patients stage G2–4, we evaluated serum FGF23, α-Klotho, parathyroid hormone (PTH) and ePTFp [ePTFp = (phosphaturia/creatininuria) x creatininemia x 3,33]; 30 healthy subjects (age 40 ± 5 years) were the control group for normal value of ePTFp. RESULTS In 68 CKD patients (age 58.9 ± 15.6 years eGFR 45 ± 21 mL/min), ePTFp averaged 2.4 ± 1.3 mg/mg which was significantly higher than in the control group (ePTFp = 1.2 ± 0.5; P &amp;lt; 0.01). ePTFp increased progressively along with the increasing CKD stages (stage 2: 1.6 ± 0.6; stage 3: 2.3 ± 0.9; stage 4: ± 1.5; P &amp;lt; 0.001) with values significantly higher than the control group since CKD stage 2 (ePTFp: Control group = 1.2 ± 0.5 versus stage 2 = 1.6 ± 0.6; P &amp;lt; 0.05). ePTFp showed a positive correlation with FGF23 (r: 0.69; P &amp;lt; 0.001) and PTH (r:0.310; P &amp;lt; 0.05) and a negative correlation with α-Klotho (r: −237; P &amp;lt; 0.05) and eGFR (r: −647; P &amp;lt; 0.001). CONCLUSION Our data showed early and progressive increments of ePTFp in CKD patients since CKD stage 2. These increments are paralleled by increased FGF23 and PTH and decreased α-Klotho, suggesting reciprocal pathophysiologic dependence. Accordingly, ePTFp could represent a surrogate of renal tubular cells phosphate overload, indicative of CKD-MBD development. ePTFp can be easily obtained in everyday clinical routine and could represent a reference to make therapeutic choices (e.g. dietary phosphate control). In conclusion, ePTFp could identify patients at risk of developing the CKD-MBD maladaptive response with increased risk of kidney damage progression.