Semiquantitative Immunoblotting Research Articles

TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.

Sex Differences in Ammoniagenesis in Obese, Diabetic, ZSF1 Rats

Diabetic ketoacidosis develops when insulin is insuffcient to clear blood sugar into cells for energy, then the liver breaks down fat for fuel with production of ketoacids. Lower pH activates proximal tubule PEPCK expression to replenish bicarbonate and excrete acid via NHE3 as ammonia, known as, “ammoniagenesis.” Baseline female (F) rodents exhibit greater ammoniagenesis and less NHE3 activity than males (M). We examined ammoniagenesis in male (M) and female (F) ZSF1 rats, a model of obesity and diabetic kidney disease. ZSF1 rats are born lean (L) or obese (O). OM display hyperglycemia and glucosuria at 10 wk, not yet evident in OF at 18 wk. We tested three hypotheses in ZSF1 rats (n=4-6/group): 1) ammoniagenesis rate is greater in OM and OF than LM and LF, 2) LF exhibit a higher rate of ammoniagenesis than OF, and 3) the blood glucose lowering SGLT2 inhibitor empagliflozin (SGLT2i) reduces ammoniagenesis in OM. Ammoniagenesis was measured as ammonia/24 hr/tibia length because OM and OF weigh 50% more than LM and LF. Abundance of PEPCK and NHE3 was assessed by semiquantitative immunoblots. Titratable acidity (TA) was measured as classically defined. In OM, ammoniagenesis was 6X greater (P< 0.0001), and TA elevated (P=0.0003) versus LM. Ammoniagenesis was not different in OF vs LF (P=0.99). Ammoniagenesis was only 2X greater in OM vs OF (P=0.001) indicative of higher baseline ammoniagenesis in F vs M. PEPCK abundance was 2.8X greater in both OM and OF vs LM and LF (P< 0.001), and NHE3 abundance was lower by 35 and 20%, respectively, in OM and OF vs LM and LF (P< 0.003). Oral SGLT2i treatment (10 mg/d, n=5-6/group, 6 wks starting at 20 wk old) vs vehicle lowered plasma glucose 35%, doubled urine glucose excretion, but did not significantly alter ammoniagenesis, PEPCK or NHE3 abundance (but tended to reduce urine TA). These findings demonstrate that at 16 weeks, OM exhibit highly elevated ammoniagenesis and TA along with hyperglycemia. In contrast, OF do not exhibit hyperglycemia nor elevated ammoniagenesis, illustrating a resistance to diabetic ketoacidosis at 16 wk, despite obesity, higher TA (P= 0.003), higher PEPCK and lower NHE3. In 20 wk old OM ZSF1, SGLT2i treatment does not reverse ammoniagenesis despite lowering plasma glucose for 6 weeks. R01 DK083785, R25DK113659, NIH Step Up U24 DK115255,. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

#4082 TAZ-KNOCKDOWN AFFECTS THE VASOPRESSIN-INDUCED AQUAPORIN-2 (AQP2) TRAFFICKING AND PROTEIN ABUNDANCE IN KIDNEY COLLECTING DUCT CELLS

Abstract Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Abnormal Expression of Synaptic and Extrasynaptic GABAA Receptor Subunits in the Dystrophin-Deficient mdx Mouse.

Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.

Anti-Inflammatory and Chondro-Protective Effects of Acidic Polysaccharide from Enteromorpha Prolifera in Experimental Models of Osteoarthritis In-Vitro and In-Vivo.

Osteoarthritis (OA) progression has been shown to increase the expression of inflammatory cytokines in joints, leading to the destruction of cartilage matrix. Interleukin (IL)-1β is a potent inflammatory cytokine associated with osteoarthritic synovial fluid. The protective effects of polysaccharides from Enteromorpha prolifera against acute hepatic injury was reported. In this study, we examined the effects of Enteromorpha polysaccharide extracts (EPEs) in the treatment of OA. The effects of the EPEs were assessed using an IL-1β-stimulated SW1353 and SW982 cells. The expression levels of specific mRNA and proteins were evaluated using semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and western immunoblotting. An OA animal study involving C57BL/6J mice was also conducted to assess the effects on tactile sensitivity and anterior cruciate ligament transection (ACLT). Acidic polysaccharide extract (APE) was shown to significantly reduce cytokine and chemokine mRNA levels in IL-1β-stimulated SW1353 and SW982 cells and attenuate the expression of proinflammatory cytokines and p38/AP-1 in SW1353 cells. APE was also shown to minimize the effect of osteolytic lesions in the knee joints of ACLT-induced osteoarthritic mice. APE is a potent inhibitor of joint degeneration associated with OA.

Psychological suppressive profile and autoantibodies variability in women living with breast cancer: A prospective cross-sectional study

Breast cancer (BC) is a leading cause of women's morbimortality worldwide. Unfortunately, attempts to predict women's susceptibility to developing BC well before it becomes symptomatic, based on their genetic, family, and reproductive background have proved unsatisfactory. Here we analyze the matching of personality traits and protein serum profiles to predict women's susceptibility to developing cancer. We conducted a prospective study among 150 women (aged 18–70 years), who were distributed into three groups (n = 50): women without breast pathology and women diagnosed with BC or benign breast pathology. Psychological data were obtained through standardized psychological tests and serum protein samples were analyzed through semiquantitative protein immunoblotting. The matching for psychological and immunological profiles was constructed from these data using a mathematical generalized linear model.The model predicted that women who have stronger associations between high-intensity stress responses, emotional containment, and an increased number and reduced variability of serum proteins (detected by IgG autoantibodies) have the greatest susceptibility to develop BC before the disease has manifested clinically. Hence, the present study endorses the possibility of using psychological and biochemical tests in combination to increase the possibility of identifying women at risk of developing BC before the disease shows clinical manifestations. A longitudinal study must be instrumented to test the prediction ability of the instrument in real scenarios. Trial registrationCommittee of Ethical Research of the Hospital General de México “Dr. Eduardo Liceaga,” Ministry of Health (DI/12/111/03/064).

Housekeeping Proteins Exhibit a High Level of Expression Variability Within Control Group and Between Ischemic Human Heart Biopsies.

Background Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. Methods and Results We evaluated the stability of HKP expression (actin, β-tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, β-tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age-matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca2+ ATPase)and cMyBPC (cardiac myosin binding protein-C)expression level after myocardial infarction. Conclusions These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.

Erythropoietin prevented the decreased expression of aquaporin1-3 in ureteral obstructive kidneys in juvenile rats.

Urinary tract obstruction is associated with impaired renal urinary concentration; even after the release of the obstruction, patients still suffer from polyuria. It has been reported that the decreased expression of aquaporins (AQPs) is associated with postobstructive polyuria, and erythropoietin (EPO) can promote the recovery of decreased AQP2 expression induced by bilateral ureteral obstruction. However, whether EPO can promote the recovery of the expression of AQP1-3 after the release of unilateral ureteral obstruction (UUO) has not yet been reported. To investigate the effects of EPO treatment on the expression of renal AQP1-3 after the release of UUO. UUO was established in rats by 24-h temporary unilateral obstruction of renal ureters. Three days following EPO treatment, the kidneys were removed to determine the expression levels of AQP1-3, NLRP3, caspase-1, and IL-1β via semiquantitative immunoblotting and immunohistochemistry. EPO inhibited the expression of NLRP3, caspase-1, and IL-1β; reduced plasma creatinine and urea; and promoted the recovery of AQP1-3 expression in UUO rats. EPO treatment prevented the decreased expression of renal AQPs and the development of impaired urinary concentration capacity after the release of UUO, which may partially occur by way of anti-inflammasome effects. EPO treatment could prevent the decreased expression of renal water transporter proteins AQP1-3 and the development of impaired renal functions, which may be associated with its anti-inflammasome effects. EPO regulated the expression of renal water transporter proteins AQP1-3, which could provide the potential for the treatment of postobstructive polyuresis. EPO treatment could be one of the effective methods by participating in multiple dimensions for patients with obstructive nephropathy.

Exploring Sex Differences in Renal Sodium Transporters with Four Core Genotype (FCG) Model

In the kidney, sex‐specific differences in Na+ transporter profiles along the nephron are now evident (“transporters” = co‐transporters, channels, claudins and pumps, their phosphorylation (p)): females (F) exhibit variable differences vs males (M) along the proximal tubule (PT) and higher distal (DT) and collecting duct (CD) Na+ transporter abundance and activity vs. M. Circulating levels of gonadal hormones determine sex differences in many physiologic traits, however, recent studies indicate that sex differences may also be due to the sex chromosome complement (SCC; XX vs.XY). The novel FCG C57BL/6 mouse model dissociates gonadal sex (ovaries (F) or testes (M)) from sex chromosomal complement (SCC; XX, XY). That is, traits influenced by gonadal hormones are similar between MXX and MXY (testes) vs FXX and FXY (ovaries); whereas traits influenced by SCC are similar between MXX and FXX vs. MXY and FXY (independent of gonads).MethodWe implemented the 4CG model with transporter profiling (semi‐quantitative immunoblotting; n = 4‐5 mice/genotype) to reveal the contributions of gonadal hormones vs. SCC on the sexual dimorphisms in transporter protein abundance along the nephron.Results(all P<0.05): Along the PT: OAT1 was lower and AQP1 and SGLT2 higher in abundance in gonadal F (FXX) vs M (MXY); SCC also contributed to differences: NHE3 was lower by 15‐30% in MXX vs. MXY and SGLT2 was 15% lower in FXY vs FXX. Inthe medullary thick ascending limb (TAL): alpha and beta NKATPase are 60% higher in FXX vs MXY and 25‐30% lower in FXY vs FXX; alpha was 15% lower in MXX vs. MXY. Along the cortical TAL and DT: NKCC2, NKCC2p, NCC, NCCp, alpha and beta NKATPase, claudin 7, SPAK and SPAKp kinase abundance were 1.4 ‐ 2.6 ‐ fold higher in FXX vs MXY; SCC differences were limited to 15%, 30%, and 20% lower NCC, and NCCp, and claudin 7 in MXX vs MXY, respectively. Along the CD: ENaC subunits were 30‐50% higher, claudin‐8 2‐fold higher, pendrin and UMOD 20% higher in FXX vs. MXY; SCC differences included 15‐20% lower ROMK and Kir 4.1 and ENaC beta and gamma subunits in MXX vs MXY. Results provide evidence for impact of both gonadal hormones and SCC on kidney transporter abundance, in some cases, offsetting the effects of the other. Further progress will come from defining whether X vs Y genes contribute to the differences. These findings have potential impact on electrolyte and blood pressure (patho)physiology in F vs. M.

Brevican and Neurocan Cleavage Products in the Cerebrospinal Fluid - Differential Occurrence in ALS, Epilepsy and Small Vessel Disease

The neural extracellular matrix (ECM) composition shapes the neuronal microenvironment and undergoes substantial changes upon development and aging, but also due to cerebral pathologies. In search for potential biomarkers, cerebrospinal fluid (CSF) and serum concentrations of brain ECM molecules have been determined recently to assess ECM changes during neurological conditions including Alzheimer’s disease or vascular dementia. Here, we measured the levels of two signature proteoglycans of brain ECM, neurocan and brevican, in the CSF and serum of 96 neurological patients currently understudied regarding ECM alterations: 16 cases with amyotrophic lateral sclerosis (ALS), 26 epilepsy cases, 23 cerebral small vessel disease (CSVD) patients and 31 controls. Analysis of total brevican and neurocan was performed via sandwich Enzyme-linked immunosorbent assays (ELISAs). Major brevican and neurocan cleavage products were measured in the CSF using semiquantitative immunoblotting. Total brevican and neurocan concentrations in serum and CSF did not differ between groups. The 60 kDa brevican fragment resulting from cleavage by the protease ADAMTS-4 was also found unchanged among groups. The presumably intracellularly generated 150 kDa C-terminal neurocan fragment, however, was significantly increased in ALS as compared to all other groups. This group also shows the highest correlation between cleaved and total neurocan in the CSF. Brevican and neurocan levels strongly correlated with each other across all groups, arguing for a joint but yet unknown transport mechanism from the brain parenchyma into CSF. Conclusively our findings suggest an ALS-specific pattern of brain ECM remodeling and may thus contribute to new diagnostic approaches for this disorder.

Impairment of Annexin A2-Mediated Cell Surface Fibrinolysis in Nonalcoholic Steatohepatitis Cirrhosis with Portal Vein Thrombosis

The annexin A2 (A2) complex assembles tissue plasminogen activator and plasminogen on cell surfaces, resulting in efficient plasmin generation, and is markedly reduced in some patients with thrombophilia. Nonalcoholic steatosis (NASH) cirrhosis is the leading cause of cirrhosis in developed countries, and patients with NASH cirrhosis are at increased risk of portal vein thrombosis (PVT). However, the exact mechanism for PVT in NASH cirrhosis is poorly understood. Our objective was to determine the potential contribution of A2 to NASH thrombogenesis.Using semiquantitative immunoblot analysis, we examined A2 expression in peripheral blood mononuclear cell (PBMC) lysates from obese controls and NASH subjects with or without cirrhosis, and in human umbilical vein endothelial cells (HUVECs) treated with platelet-poor plasma (PPP) from patients with varying degrees of NASH cirrhosis. We also analyzed formalin-fixed liver tissue from patients with or without steatosis or NASH cirrhosis for A2 expression by immunofluorescence. Finally, we evaluated A2-dependent PBMC surface plasmin generation using a fluorogenic assay, and systemic fluid-phase fibrinolysis via plasmin-anti-antiplasmin (PAP) complex and D-dimer ELISAs.Total A2 expression in PBMC lysates did not differ among subjects with a range of severity of NASH cirrhosis versus obese controls. Src kinase-dependent tyrosine phosphorylation of A2, which is critical for its translocation to the cell surface, decreased in HUVECs after co-culture with NASH Child-Turcotte-Pugh C PPP despite no change in total A2. Immunofluorescence of liver tissue from patients with NASH cirrhosis who developed PVT revealed an 85% decrease in microvascular A2 expression compared to NASH cirrhosis patients without PVT. Similarly, PBMC surface plasmin generation decreased as NASH severity worsened, even though d-dimer and PAP complex level increased with worsening disease severity, indicating more activated systemic fibrinolysis in decompensated NASH cirrhosis.Despite preservation of total A2 and acceleration of systemic fibrinolysis in NASH, A2-mediated cell surface fibrinolysis decreased as NASH cirrhosis worsened. We show for the first time that impaired A2-related fibrinolysis may reflect inhibition of A2 phosphorylation by NASH plasma, and that reduction of A2 expression in the hepatic microvasculature may contribute to PVT in these subjects. DisclosuresDesancho: Sanofi-Genzyme: Membership on an entity's Board of Directors or advisory committees.

ProNGF Measurement in Cerebrospinal Fluid Samples of a Large Cohort of Living Patients With Alzheimer's Disease by a New Automated Immunoassay.

The discovery of new biomarkers for Alzheimer's disease (AD) is essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. proNGF levels in the cerebrospinal fluid (CSF) represent a promising diagnostic biomarker for AD, but its validation was hampered by the absence of a reliable immunoassay. In the literature, proNGF is currently measured in postmortem brain tissue by semiquantitative immunoblot. Here we describe the development and validation of a new method to measure proNGF in the CSF of living patients. This method, based on molecular size separation by capillary electrophoresis, is automated and shows a 40-fold increase in sensitivity with respect to the proNGF immunoblot, largely used in literature, and is robust, specific, and scalable to high-throughput. We have measured proNGF in the cerebrospinal fluid of 84 living patients with AD, 13 controls, and 15 subjective memory complaints (SMC) subjects. By comparing the proNGF levels in the three groups, we found a very significant difference between proNGF levels in AD samples compared with both controls and SMC subjects, while no significant difference was found between SMC and controls. Because of the development of this new immunoassay, we are ready to explore the potentiality of proNGF as a new biomarker for AD or subgroups thereof, as well as for other neurodegenerative diseases.

Functional Evaluation and Clinical Implications of FLT3 J uxtamembrane Domain Short Variants in Pediatric AML

Introduction: Somatic FLT3 activating mutations are common in AML and occur as internal tandem duplications (FLT3 /ITD) within the juxtamembrane domain (JMD) or in the activating loop of the tyrosine kinase domain (FLT3 /ALM). There is considerable variability in the length of the ITD, varying from 6 to >300 bp. We, and others, have previously demonstrated that ITD length is associated with outcome, where those with longer ITDs based on median ITD length (>48bp) had adverse outcome. However, the underlying etiology of this association has not been established. We hypothesized that longer ITDs are more likely to disrupt the auto-inhibitory properties of the JMD, leading to enhanced receptor phosphorylation and autonomous activation. We aimed to define a biologic threshold for ITD-associated relapse risk based on the length of ITD that causes maximal activation. We also evaluated the functional properties of newly identified single nucleotide variants (SNVs) within the FLT3 JMD.Methods: To evaluate the impact of FLT3 /ITD length on receptor auto-phosphorylation, FLT3 /ITD clones with a variety of ITD lengths (6, 12, 18, 24, 30, 36, 42, 48, 54 bps) were engineered and expressed in HEK293 cells. Semi-quantitative immunoblotting was used to evaluate the level of phosphorylated FLT3 (pFLT3), and comparison of pFLT3 to non-pFLT3 was calculated and presented as fold change (FC) of pFLT3:FLT3 for the different ITD lengths. We also assessed the functionality of newly discovered JMDSNVs in a similar manner, and further evaluated their response to FLT3 inhibitors. For evaluation of the clinical impact of ITD length, of the 1,022 patients treated on AAML0531, FLT3 /ITD data including ITD length as determined by fragment length analysis and allelic ratio was available from 144 patients and was used for clinical correlation with ITD length.Results: Initial evaluation of the activation potential of the different ITD lengths showed a correlation between ITD length and FLT3 phosphorylation (Fig 1A). A modest autonomous FLT3 phosphorylation was observed at 6 and 12 bp, with incremental increase in 18bp and 24bp. Maximal pFLT3 of FC was observed at 30 bp, with no significant increases detected with longer ITDs. Therefore, a biologic threshold of 30 bp was used to evaluate the clinical outcomes of patients with short ITDs (S-ITD; <30 bps) and long ITDs (L-ITD; ≥30 bps). Among the 144 FLT3 /ITD+ patients, n=37 had S-ITDs and n=107 had L-ITDs. Overall, based on cooperating cytogenetic and molecular features, a higher number of patients with S-ITDs were considered as low risk as compared to L-ITDs (36% vs. 13%; p=0.003), with a higher incidence of t(8;21) in the S-ITD cohort. There was no correlation observed between ITD length and allelic ratio (Pearson correlation r=0.54). Evaluation of clinical outcome based on ITD length as defined above demonstrated that those with L-ITD had a 5-year overall survival of 47±13% compared to 83±16% in those with S-ITD (p=0.004; Fig1B).We additionally evaluated the functional significance of newly discovered FLT3 SNVs in the JMD. A total of 20 unique mutations affecting 13 distinct codons were identified. We evaluated the effect of each amino acid variant on FLT3 activation as measured by aberrant pFLT3. Six of the 20 mutations resulted in varying degrees of autonomous pFLT3 (E573D, L576R, V592A, F594Y, F594C, Y559C). Notably, the V592A mutation resulted in potent autonomous receptor activation, with a pFLT3 level greater than that observed the D835 FLT3/ ALM mutation. All variants resulting in autonomous pFLT3 were found to be highly sensitive to the FLT3 inhibitor crenolanib, with IC50s for inhibition of pFLT3 ranging between 1.3-5.6 nMConclusion: The JMD is a critical region of FLT3, and disruption through ITDs or SNVs can result in profound dysregulation of the receptor and downstream signaling, thus serving as oncogenic events. We show that longer ITDs result in more potent FLT3 receptor activation and are associated with inferior clinical outcome. FLT3 /ITD and ALMs have recently been shown to be excellent targets for therapeutic intervention with FLT3 inhibitors. Further investigation on the use FLT3 inhibitor therapy should focus on those patients who may derive the most therapeutic benefit from these agents, which may include those with highly damaging JMD variants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SVEGFR1 Is Enriched in Hepatic Vein Blood-Evidence for a Provisional Hepatic Factor Candidate?

Background: Pulmonary arteriovenous malformations (PAVMs) are common sequelae of palliated univentricular congenital heart disease, yet their pathogenesis remain poorly defined. In this preliminary study, we used paired patient blood samples to identify potential hepatic factor candidates enriched in hepatic vein blood.Methods: Paired venous blood samples were collected from the hepatic vein (HV) and superior vena cava (SVC) from children 0 to 10 years with univentricular and biventricular congenital heart disease (n = 40). We used three independent protein analyses to identify proteomic differences between HV and SVC blood. Subsequently, we investigated the relevance of our quantified protein differences with human lung microvascular endothelial assays.Results: Two independent protein arrays (semi-quantitative immunoblot and quantitative array) identified that soluble vascular endothelial growth factor receptor 1 (sVEGFR1) is significantly elevated in HV serum compared to SVC serum. Using ELISA, we confirmed the previous findings that sVEGFR1 is enriched in HV serum (n = 24, p < 0.0001). Finally, we studied the quantified HV and SVC serum levels of sVEGFR1 in vitro. HV levels of sVEGFR1 decreased tip cell selection (p = 0.0482) and tube formation (fewer tubes [p = 0.0246], shorter tube length [p = 0.0300]) in vitro compared to SVC levels of sVEGFR1.Conclusions: Based on a small heterogenous cohort, sVEGFR1 is elevated in HV serum compared to paired SVC samples, and the mean sVEGFR1 concentrations in these two systemic veins cause pulmonary endothelial phenotypic differences in vitro. Further research is needed to determine whether sVEGFR1 has a direct role in pulmonary microvascular remodeling and PAVMs in patients with palliated univentricular congenital heart disease.

An approach for quantifying western blots: the case of signal intensity and the statistical analysis

Article Details: Received: 2020-04-07 | Accepted: 2021-01-25 | Available online: 2021-06-30 https://doi.org/10.15414/afz.2021.24.02.141-146 In biological sciences, western blotting technique is widely used to quantify the expression of proteins in a given sample. However, there is no unified method for quantifying the expression of proteins. As a consequence, quantitative analysis of expression of protein through western blotting often suffers from data inconsistency. At the same time, extraction of the poor sample size (n=3/5/7) turns such analysis non-Gaussian and less robust to statistical errors. In present study, we attempt  a noble approach while analyzing an image from western blotting using Gaussian blur as filter and thereby generating data in order to perform meaningful statistical analysis. The differences among various blots that correspond to the expressed target proteins are tested viably using appropriate statistical tools. This procedure of quantifying western blotting is comprehensive, simple and can be applied to collect data in compliance with statistical norms.  Furthermore, repeating western blotting on a set of particular proteins may improve the analysis part as well. Keywords:  blotting, statistical analysis, non-parametric, densitometry References Aldridge, G.M. et al. (2008). The use of total protein stains as loading controls: An alternative to high-abundance singleprotein controls in semiquantitative immunoblotting. Journal of Neuroscience Methods, 172(2), 250–254. Butler, T.A.J. et al. (2019). Misleading westerns: common quantification mistakes in western blot densitometry and proposed corrective measures. BioMedical Research, ID 5214821. https://doi.org/10.1155/2019/5214821 Eaton, S.L. et al. (2013). Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting. PLoS ONE, 8(8), e72457. https://doi.org/10.1371/journal.pone.0072457 Ghosh, R., Gilda, J.E. &amp; Gomes, A.V. (2014). The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review of Proteomics, 11, 549–560. Graham, B. (2016). Quantifying western blots: none more black. BMC Biology, 14, 116. https://doi.org/10.1186/s12915-016-0339-1 Kreutz, C. et al. (2007). An error model for protein quantification. Bioinformatics, 23 (20), 2747–2753, https://doi.org/10.1093/bioinformatics/btm397 Maleki, F. et al. (2019). Size matters: how sample size affects the reproducibility and specificity of gene set analysis. Hum Genomics, 13, 42. https://doi.org/10.1186/s40246-019-0226-2 Taylor, S.C. et al. (2013). A defined methodology for reliable quantification of Western blot data. Molecular Biotechnology, 55(3), 217–226. https://doi.org/10.1007/s12033-013-9672-6 Towbin, H., Staehelin, T. &amp; Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76(9), 4350–4354. Welinder, C. &amp; Ekblad, L. (2011). Coomassie staining as loading control in Western blot analysis. Journal of Proteome Research, 10(3), 1416–1419

Molecular and Immunological Identification of Low Allergenic Fruits among Old and New Apple Varieties.

About 50–70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdańska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

Evaluation of the diagnostic performance of an immunoblot for ANCA and anti-GBM antibody detection

The use of high-quality antigen-specific immunoassays for detecting anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (GBM) autoantibodies is recommended in patients with suspected ANCA vasculitis and/or anti-GBM disease. We analysed the diagnostic performance of a semi-quantitative and rapid immunoblot (EUROIMMUN AG, Lübeck, Germany) in two settings. Patient sera from different cohorts (ANCA vasculitis n = 187, anti-GBM disease n = 19, and disease controls n = 51) were used. The diagnostic performance of the immunoblot was assessed when used as a confirmatory test for the presence of ANCA in suspected ANCA vasculitis and when evaluating the presence of ANCA and/or anti-GBM antibodies in AAV and/or anti-GBM disease patients with a rapidly progressive glomerulonephritis (RPGN). In a confirmatory test setting, the immunoblot had an optimal sensitivity and specificity of 97.4 and 98.1% for PR3-ANCA and 98.5 and 96.4% for MPO-ANCA, respectively. With increasing test result ranges, a higher interval likelihood ratio (LR) was found for both ANCA entities. When evaluating for ANCA in patients with RPGN, the highest diagnostic accuracy (sensitivity 92.9% and specificity 100%) was obtained by using different cut-off values of positivity for PR3- (>5) and MPO-ANCA (>10). Also, the diagnostic performance for detecting anti-GBM was good (sensitivity 100% and specificity 100%). There are advantages over other assays in terms of time, costs, and interpretation of results. The immunoblot is a useful addition to current guidelines, particularly when a rapid diagnosis is necessary.

P0013MAP KINASES-REGULATED PROTEINS IN SIGNALING PATHWAYS OF VASOPRESSIN IN KIDNEY COLLECTING DUCT

Abstract Background and Aims Activation of G protein-coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R. However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2 expression and phosphorylation, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R. Tripartite motif-containing protein 28 (TRIM28) was identified as a substrate of ERK1 as well as a vasopressin-responsive protein via bioinformatic tools. We further evaluated whether TRIM28 plays a role in vasopressin-mediated regulation of AQP2 in the kidney CD. Method To identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on high-throughput profiles of transcriptome and proteome (http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); and 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate protein (TRIM28) was examined by immunohistochemistry. The role of TRIM28 in vasopressin-mediated AQP2 regulation was examined by quantitative real-time PCR (qRT-PCR) and semiquantitative immunoblotting after RNA interference of TRIM28 in mpkCCDc11 cells. Results Immunoblotting of mpkCCDc11 cells revealed that both p-ERK1/2 and pS261-AQP2 expression was decreased in response to dDAVP (10-9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase-substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed TRIM28, as the substrate of ERK1, has the only one phosphorylation site which was down-regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin-protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP-induced AQP2 mRNA and protein up-regulation was significantly attenuated in mpkCCDc11 cells with siRNA-mediated knockdown of TRIM28. Conclusion We identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.