Changes In Specific Proteins Research Articles

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 μg); or MIF mAb (15 μg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Histopathological and Biochemical Assessment of Annona muricata and Allium sativum Pooled Extract on Testosterone Induced Prostate Hyperplasia in Albino Rats

Aims: To investigate the effect of pooled extract of Annona muricata (A. muricata) and Allium sativum (A. sativum) on testosterone-induced prostate hyperplasia in male albino rats.
 Experimental Procedure: Male albino rats for the study were divided into seven groups identified as A-G groups. Group A and G were used as negative and positive controls with 12 and 5 rats respectively. Male rats in group B-G were induced with 3mg/kg body weight of testosterone propionate injection subcutaneously for 28 days. Animals in group B and C received 100mg/kg body weight of individual Annona muricata and Allium sativum extract respectively, while animals in group D-F received 100mg/kg, 300mg/kg and 600mg/kg body weight of the pooled extracts. We evaluated relative prostate weight(PI),serum prostate specific antigen(PSA),interleukins-6(IL-6), total protein, and histological changes including KI 67 and BCL2 expressions.
 Results and Conclusion: The study revealed that pooled extracts of A. muricata and A. sativum significantly inhibited prostatic hyperplasia; decreased PI,serum PSA,IL-6, total protein, and expression of KI 67, far better than the any of the single plant extract in this study. This suggests that pooled extract of A, muricata and A, sativum may be used as a therapeutic agent for prostatic hyperplasia, as a result of the influence of the phytochemical constituents.

A Meta-Analysis on Presynaptic Changes in Alzheimer's Disease.

A key aspect of synaptic dysfunction in Alzheimer's disease (AD) is loss of synaptic proteins. Previous publications showed that the presynaptic machinery is more strongly affected than postsynaptic proteins. However, it has also been reported that presynaptic protein loss is highly variable and shows region- and protein-specificity. The objective of this meta-analysis was to provide an update on the available literature and to further characterize patterns of presynaptic protein loss in AD. Systematic literature search was conducted for studies published between 2015-2022 which quantified presynaptic proteins in postmortem tissue from AD patients and healthy controls. Three-level random effects meta-analyses of twenty-two identified studies was performed to characterize overall presynaptic protein loss and changes in specific regions, proteins, protein families, and functional categories. Meta-analysis confirmed overall loss of presynaptic proteins in AD patients. Subgroup analysis revealed region specificity of protein loss, with largest effects in temporal and frontal cortex. Results concerning different groups of proteins were also highly variable. Strongest and most consistently affected was the family of synaptosome associated proteins, especially SNAP25. Among the most severely affected were proteins regulating dense core vesicle exocytosis and the synaptic vesicle cycle. Results confirm previous literature related to presynaptic protein loss in AD patients and provide further in-depth characterization of most affected proteins and presynaptic functions.

Proteomic Changes in the Hippocampus after Repeated Explosive-Driven Blasts.

Repeated blast-traumatic brain injury (blast-TBI) has been hypothesized to cause persistent and unusual neurological and psychiatric symptoms in service members returning from war zones. Blast-wave primary effects have been supposed to induce damage and molecular alterations in the brain. However, the mechanisms through which the primary effect of an explosive-driven blast wave generate brain lesions and induce brain consequences are incompletely known. Prior findings from rat brains exposed to two consecutive explosive-driven blasts showed molecular changes (hyperphosphorylated-Tau, AQP4, S100β, PDGF, and DNA-polymerase-β) that varied in magnitude and direction across different brain regions. We aimed to compare, in an unbiased manner, the proteomic profile in the hippocampus of double blast vs sham rats using mass spectrometry (MS). Data showed differences in up- and down-regulation for protein abundances in the hippocampus of double blast vs sham rats. Tandem mass tag (TMT)-MS results showed 136 up-regulated and 94 down-regulated proteins between the two groups (10.25345/C52B8VP0X). These TMT-MS findings revealed changes never described before in blast studies, such as increases in MAGI3, a scaffolding protein at cell-cell junctions, which were confirmed by Western blotting analyses. Due to the absence of behavioral and obvious histopathological changes as described in our previous publications, these proteomic data further support the existence of an asymptomatic blast-induced molecular altered status (ABIMAS) associated with specific protein changes in the hippocampus of rats repeatedly expsosed to blast waves generated by explosive-driven detonations.

Biochemical analyses of tau and other neuronal markers in the submandibular gland and frontal cortex across stages of Alzheimer disease

Hyperphosphorylation of the microtubule-associated protein tau is hypothesized to lead to the development of neurofibrillary tangles in select brain regions during normal aging and in Alzheimer disease (AD). The distribution of neurofibrillary tangles is staged by its involvement starting in the transentorhinal regions of the brain and in final stages progress to neocortices. However, it has also been determined neurofibrillary tangles can extend into the spinal cord and select tau species are found in peripheral tissues and this may be depended on AD disease stage. To further understand the relationships of peripheral tissues to AD, we utilized biochemical methods to evaluate protein levels of total tau and phosphorylated tau (p-tau) as well as other neuronal proteins (i.e., tyrosine hydroxylase (TH), neurofilament heavy chain (NF-H), and microtubule-associated protein 2 (MAP2)) in the submandibular gland and frontal cortex of human cases across different clinicopathological stages of AD (n = 3 criteria not met or low, n = 6 intermediate, and n = 9 high likelihood that dementia is due to AD based on National Institute on Aging-Reagan criteria). We report differential protein levels based on the stage of AD, anatomic specific tau species, as well as differences in TH and NF-H. In addition, exploratory findings were made of the high molecular weight tau species big tau that is unique to peripheral tissues. Although sample sizes were small, these findings are, to our knowledge, the first comparison of these specific protein changes in these tissues.

Proteomic Landscape of Human Sperm in Patients with Different Spermatogenic Impairments

Although the proteome of sperm has been characterized, there is still a lack of high-throughput studies on dysregulated proteins in sperm from subfertile men, with only a few studies on the sperm proteome in asthenozoospermic and oligoasthenozoospermic men. Using liquid chromatography-mass spectrometry (LC-MS/MS) along with bioinformatics analyses, we investigated the proteomic landscape of sperm collected from subfertile men (n = 22), i.e., asthenozoospermic men (n = 13), oligoasthenozoospermic men (n = 9) and normozoospermic controls (n = 31). We identified 4412 proteins in human sperm. Out of these, 1336 differentially abundant proteins were identified in 70% of the samples. In subfertile men, 32 proteins showed a lower abundance level and 34 showed a higher abundance level when compared with normozoospermic men. Compared to normozoospermic controls, 95 and 8 proteins showed a lower abundance level, and 86 and 1 proteins showed a higher abundance level in asthenozoospermic and oligoasthenozoospermic men, respectively. Sperm motility and count were negatively correlated with 13 and 35 and positively correlated with 37 and 20 differentially abundant proteins in asthenozoospermic and oligoasthenozoospermic men, respectively. The combination of the proteins APCS, APOE, and FLOT1 discriminates subfertile males from normozoospermic controls with an AUC value of 0.95. Combined APOE and FN1 proteins discriminate asthenozoospermic men form controls with an AUC of 1, and combined RUVBL1 and TFKC oligoasthenozoospermic men with an AUC of 0.93. Using a proteomic approach, we revealed the proteomic landscape of sperm collected from asthenozoospermic or oligoasthenozoospermic men. Identified abundance changes of several specific proteins are likely to impact sperm function leading to subfertility. The data also provide evidence for the usefulness of specific proteins or protein combinations to support future diagnosis of male subfertility.

Amino acid substitutions in specific proteins correlate with farnesol unresponsiveness in Candida albicans

BackgroundThe quorum-sensing molecule farnesol, in opportunistic yeast Candida albicans, modulates its dimorphic switch between yeast and hyphal forms, and biofilm formation. Although there is an increasing interest in farnesol as a potential antifungal drug, the molecular mechanism by which C. albicans responds to this molecule is still not fully understood.ResultsA comparative genomic analysis between C. albicans strains that are naturally unresponsive to 30 µM of farnesol on TYE plates at 37 °C versus responsive strains uncovered new molecular determinants involved in the response to farnesol. While no signature gene was identified, amino acid changes in specific proteins were shown to correlate with the unresponsiveness to farnesol, particularly with substitutions in proteins known to be involved in the farnesol response. Although amino acid changes occur primarily in disordered regions of proteins, some amino acid changes were also found in known domains. Finally, the genomic investigation of intermediate-response strains showed that the non-response to farnesol occurs gradually following the successive accumulation of amino acid changes at specific positions.ConclusionIt is known that large genomic changes, such as recombinations and gene flow (losses and gains), can cause major phenotypic changes in pathogens. However, it is still not well known or documented how more subtle changes, such as amino acid substitutions, play a role in the adaptation of pathogens. The present study shows that amino acid changes can modulate C. albicans yeast’s response to farnesol. This study also improves our understanding of the network of proteins involved in the response to farnesol, and of the involvement of amino acid substitutions in cellular behavior.

Alteration in Levels of Specific miRNAs and Their Potential Protein Targets between Human Pancreatic Cancer Samples, Adjacent Normal Tissue, and Xenografts Derived from These Tumors.

Herein, we describe the global comparison of miRNAs in human pancreatic cancer tumors, adjacent normal tissue, and matched patient-derived xenograft models using microarray screening. RNA was extracted from seven tumor, five adjacent normal, and eight FI PDX tumor samples and analyzed by Affymetrix GeneChip miRNA 4.0 array. A transcriptome analysis console (TAC) was used to generate comparative lists of up- and downregulated miRNAs for the comparisons, tumor vs. normal and F1 PDX vs. tumor. Particular attention was paid to miRNAs that were changed in the same direction in both comparisons. We identified the involvement in pancreatic tumor tissue of several miRNAs, including miR4534, miR3154, and miR4742, not previously highlighted as being involved in this type of cancer. Investigation in the parallel mRNA and protein lists from the same samples allowed the elimination of proteins where altered expression correlated with corresponding mRNA levels and was thus less likely to be miRNA regulated. Using the remaining differential expression protein lists for proteins predicted to be targeted for differentially expressed miRNA on our list, we were able to tentatively ascribe specific protein changes to individual miRNA. Particularly interesting target proteins for miRs 615-3p, 2467-3p, 4742-5p, 509-5p, and 605-3p were identified. Prominent among the protein targets are enzymes involved in aldehyde metabolism and membrane transport and trafficking. These results may help to uncover vulnerabilities that could enable novel approaches to treating pancreatic cancer.

Prenatal methadone exposure selectively alters protein expression in primary motor cortex: Implications for synaptic function.

As problematic opioid use has reached epidemic levels over the past 2decades, the annual prevalence of opioid use disorder (OUD) in pregnant women has also increased 333%. Yet, how opioids affect the developing brain of offspring from mothers experiencing OUD remains understudied and not fully understood. Animal models of prenatal opioid exposure have discovered many deficits in the offspring of prenatal opioid exposed mothers, such as delays in the development of sensorimotor function and long-term locomotive hyperactivity. In attempt to further understand these deficits and link them with protein changes driven by prenatal opioid exposure, we used a mouse model of prenatal methadone exposure (PME) and preformed an unbiased multi-omic analysis across many sensoriomotor brain regions known to interact with opioid exposure. The effects of PME exposure on the primary motor cortex (M1), primary somatosensory cortex (S1), the dorsomedial striatum (DMS), and dorsolateral striatum (DLS) were assessed using quantitative proteomics and phosphoproteomics. PME drove many changes in protein and phosphopeptide abundance across all brain regions sampled. Gene and gene ontology enrichments were used to assess how protein and phosphopeptide changes in each brain region were altered. Our findings showed that M1 was uniquely affected by PME in comparison to other brain regions. PME uniquely drove changes in M1 glutamatergic synapses and synaptic function. Immunohistochemical analysis also identified anatomical differences in M1 for upregulating the density of glutamatergic and downregulating the density of GABAergic synapses due to PME. Lastly, comparisons between M1 and non-M1 multi-omics revealed conserved brain wide changes in phosphopeptides associated with synaptic activity and assembly, but only specific protein changes in synapse activity and assembly were represented in M1. Together, our studies show that lasting changes in synaptic function driven by PME are largely represented by protein and anatomical changes in M1, which may serve as a starting point for future experimental and translational interventions that aim to reverse the adverse effects of PME on offspring.

ESM-1 Mediates Cell Progression in Clear Cell Renal Cell Carcinoma by Affecting Wnt/β-Catenin Signalling Pathway.

Overexpressed endothelial cell specific molecule-1 (ESM-1) has been identified in various human malignancies, but its expression and function in clear cell renal cell carcinoma (ccRCC) progression are still uncovered. This study explored the critical roles as well as molecular mechanism of ESM-1 in ccRCC progression. The ESM-1 expression in ccRCC tissues and cells was measured using Western blot assay. The function of ESM-1 knockdown in ccRCC cell viability, invasion as well as migration was analysed. Changes in specific proteins were also detected by Western blot analysis. The ESM-1 expression increased in ccRCC tissue samples and cells, which indicated poor prognosis. Moreover, ESM-1 silencing considerably inhibited ccRCC cell growth, invasion and migration in vitro. ESM-1 partially promoted ccRCC development through wingless-type mouse mammary tumour integration site family/beta-catenin (Wnt/β-catenin signalling). ESM-1 acted as an oncogene by influencing the Wnt/β-catenin pathway in ccRCC.

Proteomic Identification of Saliva Proteins as Noninvasive Diagnostic Biomarkers.

Many biomedically relevant biomarkers are proteins with characteristic biochemical properties and a relatively restricted subcellular distribution. The comparative and mass spectrometry-based proteomic analysis of body fluids can be particularly instrumental for the targeted identification of novel protein biomarkers with pathological relevance. In this respect, new research efforts in biomarker discovery focus on the systematic mapping of the human saliva proteome, as well as the pathobiochemical identification of disease-related modifications or concentration changes in specific saliva proteins. As a product of exocrine secretion, saliva can be considered an ideal source for the biochemical identification of new disease indicators. Importantly, saliva represents a body fluid that is continuously available for diagnostic and prognostic assessments. This chapter gives an overview of saliva proteomics, including a discussion of the usefulness of both liquid chromatography and two-dimensional gel electrophoresis for efficient protein separation in saliva proteomics.

621 Validated method for assessing the effects of blue light on human skin

The COVID pandemic caused an increase in virtual meetings & work from home scenarios that resulted in people spending increased time in front of computer screens & electronic devices. Studies have shown that blue light can produce cytotoxic effects, primarily through the production of reactive oxygen species & increased inflammation. However, the topic has been controversial with some studies claiming no adverse effects of blue light on the skin. Methods for testing the effects of blue light using in vitro testing models are lacking. Our work was conducted in order to develop a reproducible, validated testing method for assessing the effects of blue light on the skin. We designed a custom blue-light box that can be used to generate blue light at 460 nm wavelength. We performed a series of studies to optimize the dose and timing of the exposure & skin-culture conditions. Our work demonstrates that 6 hours of daily blue light for 5 consecutive days (total 30 J/cm2) produced a dose-and-time dependent decrease in skin health in 3D full thickness in vitro skin tissues. In addition, gene expression data showed an increase in the expression of genes that regulate inflammation and oxidative stress pathways (IL1A, IL6, CXCL8, COX2, CYP1B1, & NQO1) & a decrease in the expression of genes that maintain skin barrier and integrity (KRT1, KRT10, LOR, DSC and Collagen). Genes regulating skin aging & hydration including MMP1 & FLG were also regulated by exposure to blue light. Enzyme-linked immunoassays were performed to confirm changes in specific proteins. Exposure to blue light significantly increased 8-hydroxy-2' -deoxyguanosine, a marker for oxidative stress, & MMP1, markers for photoaging. Immunohistochemistry staining was performed to confirm changes in Collagen, Filaggrin & NQO1 protein expression in skin tissue. Our results showed that consistent blue light exposure produced skin damage via alterations in key biological pathways. This work provides a new, reproducible in vitro testing method for assessing the effects of blue light on human skin using gene expression, protein ELISA and IHC staining.

Development of a Novel Rat Intervertebral Disc Degeneration Model by Surgical Multifidus Resection–Induced Instability

This study aimed to investigate whether surgical resection of multifidus in rats could generate a reliable model of intervertebral disc degeneration (IVDD). Instability of the lumbar spine in Sprague-Dawley rats was induced by multifidus resection. Longissimus changes were examined by hematoxylin and eosin staining and immunohistochemistry. Specific protein and mRNA changes in the nucleus pulposus (NP) were quantified by Western blot and reverse transcription-polymerase chain reaction. Bone alterations were assessed using X-ray imaging, and disc changes were evaluated by hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry. Fat infiltration and increased tumor necrosis factor-α expression in the longissimus were detected following surgery. Reverse transcription-polymerase chain reaction and Western blot results demonstrated that the inflammation and catabolism in the NP were increased after the surgical intervention. Moreover, X-ray imaging showed that the disc height had decreased and bone spurs had formed at the vertebral rims. Histological analyses further revealed degeneration of the annulus fibrosus, endplate, and NP. Furthermore, in contrast to the sham group, the collagen II expression was reduced, while matrix metalloproteinase-13 was increased in the surgery group. Surgical resection of the multifidus in rats resulted in a reproducible IVDD model. Because the present procedure does not impart direct injury to the intervertebral disc, it can better imitate the pathological states in humans. Therefore, our rat multifidus resection model might help us further understand the intrinsic pathophysiology of IVDD.

An Integrative Multiomics Approach to Characterize Prebiotic Inulin Effects on Faecalibacterium prausnitzii.

Faecalibacterium prausnitzii, a major commensal bacterium in the human gut, is well known for its anti-inflammatory effects, which improve host intestinal health. Although several studies have reported that inulin, a well-known prebiotic, increases the abundance of F. prausnitzii in the intestine, the mechanism underlying this effect remains unclear. In this study, we applied liquid chromatography tandem mass spectrometry (LC-MS/MS)-based multiomics approaches to identify biological and enzymatic mechanisms of F. prausnitzii involved in the selective digestion of inulin. First, to determine the preference for dietary carbohydrates, we compared the growth of F. prausnitzii in several carbon sources and observed selective growth in inulin. In addition, an LC-MS/MS-based intracellular proteomic and metabolic profiling was performed to determine the quantitative changes in specific proteins and metabolites of F. prausnitzii when grown on inulin. Interestingly, proteomic analysis revealed that the putative proteins involved in inulin-type fructan utilization by F. prausnitzii, particularly β-fructosidase and amylosucrase were upregulated in the presence of inulin. To investigate the function of these proteins, we overexpressed bfrA and ams, genes encoding β-fructosidase and amylosucrase, respectively, in Escherichia coli, and observed their ability to degrade fructan. In addition, the enzyme activity assay demonstrated that intracellular fructan hydrolases degrade the inulin-type fructans taken up by fructan ATP-binding cassette transporters. Furthermore, we showed that the fructose uptake activity of F. prausnitzii was enhanced by the fructose phosphotransferase system transporter when inulin was used as a carbon source. Intracellular metabolomic analysis indicated that F. prausnitzii could use fructose, the product of inulin-type fructan degradation, as an energy source for inulin utilization. Taken together, this study provided molecular insights regarding the metabolism of F. prauznitzii for inulin, which stimulates the growth and activity of the beneficial bacterium in the intestine.

Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels.

Endometriosis is a benign disease affecting one in ten women of reproductive age worldwide. Although the pain level is not correlated to the extent of the disease, it is still one of the cardinal symptoms strongly affecting the patients’ quality of life. Yet, a molecular mechanism of this pathology, including the formation of pain, remains to be defined. Recent studies have indicated a close interaction between newly generated nerve cells and macrophages, leading to neurogenic inflammation in the pelvic area. In this context, the responsiveness of an endometriotic cell culture model was characterized upon inflammatory stimulation by employing a multi-omics approach, including proteomics, metabolomics and eicosanoid analysis. Differential proteomic profiling of the 12-Z endometriotic cell line treated with TNFα and IL1β unexpectedly showed that the inflammatory stimulation was able to induce a protein signature associated with neuroangiogenesis, specifically including neuropilins (NRP1/2). Untargeted metabolomic profiling in the same setup further revealed that the endometriotic cells were capable of the autonomous production of 7,8-dihydrobiopterin (BH2), 7,8-dihydroneopterin, normetanephrine and epinephrine. These metabolites are related to the development of neuropathic pain and the former three were found up-regulated upon inflammatory stimulation. Additionally, 12-Z cells were found to secrete the mono-oxygenated oxylipin 16-HETE, a known inhibitor of neutrophil aggregation and adhesion. Thus, inflammatory stimulation of endometriotic 12-Z cells led to specific protein and metabolite expression changes suggesting a direct involvement of these epithelial-like cells in endometriosis pain development.

P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time

Abstract Study question Does metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines? Summary answer The expression of three different genes and proteins was investigated, of which all displayed changes over time. What is known already Endometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes. Study design, size, duration The EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls. Participants/materials, setting, methods The study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter. Main results and the role of chance The expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well. Limitations, reasons for caution The results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time. Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin over time, suggesting differentially pathways in long term metformin treatment using physiologically achievable metformin levels. Trial registration number Not applicable

Proteotoxicity in Cardiac Amyloidosis: Amyloidogenic Light Chains Affect the Levels of Iintracellular Proteins in Human Heart Cells

Introduction. AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. In addition to the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at the cellular level are complex and largely undefined. Herein we sought to characterize the molecular changes occurring in primary human cells (cardiac fibroblasts, hCF) exposed in vitro to soluble amyloidogenic cardiotropic LCs, and control LC from patients with multiple myeloma, by evaluating their proteome.Methods. Monoclonal LCs were purified from patients with AL amyloidosis and severe heart involvement (CardioLCs), and from multiple myeloma patients without amyloidosis (MMLCs, used as controls). The effects of various LCs (4 CardioLCs and 3 MMLCs) on hCF physiology were explored regarding viability and cytotoxicity. A combination of gel-based and label-free shotgun analyses, followed by bioinformatics and data validation studies, was used to characterize the proteome alterations of cultured hCF exposed for 24h to a representative cardiotropic LC (CardioLC1), by comparison with untreated cells and with cells incubated with a representative MMLC (MMLC2). In order to assess the generalizability of the results, the quantitative changes in a subset of proteins have been further assessed by immunoblotting in hCF exposed to each of the LCs employed in the study.Results. Incubation of hCF with the various cardiotropic LCs translates into impaired viability, with apoptosis activation and oxidative stress. These alterations are associated with significant proteome remodeling: combining the results of the two proteomic methodologies, 85 differentially represented proteins were found overall in the comparison between CardioLC1-treated and untreated hCF, and 51 in the CardioLC1-treated versus the MMLC2-treated comparison. The differential proteins are involved in key cellular processes, including cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. Among the proteins identified as differential by proteomics, 8 species (Talin, Heat shock protein 27, Transgelin, Porin 31HM, Proteasome subunit beta type-2, Cysteine and glycine-rich protein 2, Cullin-associated NEDD8-dissociated protein 1 and Vimentin) have been selected overall for independent validation by western blotting. Selection was based on their fold change, biological role and/or availability of validated antibodies. Changes are being confirmed to occur also in hCF treated with the other cardiotropic LCs.Conclusions. These results support and expand the concept that soluble species of amyloidogenic cardiotropic LCs possess toxicity towards cardiac cells. Overall, our investigation has allowed characterizing the molecular alterations in target cells exposed to an extrinsic proteotoxic stimulus, represented by monoclonal cardiotropic LCs. Quantitative changes in specific proteins may in the future be explored as potential markers of cardiac proteotoxic stress. Dysregulation of species involved in crucial cellular functions as cytoskeletal remodeling, mitochondrial activity and metabolism, protein synthesis, quality control and degradation, may play a causative role in the cellular alterations that lead to the clinical picture, and targeting these altered pathways may be beneficial for counteracting organ damage in AL amyloidosis. DisclosuresPalladini:Prothena: Honoraria, Other: Travel grant; Celgene: Other: Travel grants; Jannsen-Cilag: Membership on an entity's Board of Directors or advisory committees.

Abstract P797: Sex Specific Plasma Protein Changes During Thrombectomy

Introduction: Sex differences in stroke have been apparent with premenopausal females having a lower incidence of stroke with better outcomes than postmenopausal females and males. We examined sex-specific outcomes and changes in plasma proteins following emergent large vessel occlusions. The previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC), clinicaltrials.gov NCT03153683, allows for analysis of plasma proteins both systemically and distal to the thrombus. Methods: Plasma samples, processed in accordance with the BACTRAC protocol, were sent to Olink to run cardiometabolic and inflammatory panels. Demographics are reported as mean±SEM. Significance determined in Prism with Mann-Whitney, t-test, or pair mixed-effect analysis. Results: We evaluated 34 subjects, >18 yrs old (20 females, 14 males) enrolled in BACTRAC. There was no significant difference in age (68.9±2.7, 65.4±4.5 yrs, respectively) or comorbidities (hypertension, diabetes, cholesterolemia). Interestingly, males had a larger (p<0.1) change in Modified Rankin Scale (mRS, premorbid-discharge, 3.4±1.8, 2.2±1.6, respectively) with larger infarcts (86,666±30,889 mm 3 , 36,228±10,943 mm 3 , respectively). This coincided with a lower (p<0.05) CTA collateral scores for males compared to females (0.64±0.67, 1.1±0.13, respectively). 12 proteins were significantly (p<0.1) higher in females, compared to males (5 proteins upregulated in both the systemic and intracranial, 3 systemic specific, and 4 intracranial specific). Males had 15 proteins significantly higher than females (3 proteins upregulated in both the systemic and intracranial, 12 systemic specific, and 0 intracranial specific). The most significant intracranial protein for females is coagulation factor XI (F11) and males is transforming growth factor beta-1 (TGFB1). Analysis of an additional 16 subjects has begun to validate the sex specific proteins. Conclusions: Unexpectedly, males have larger infarcts and less independence following large vessel occlusions in BACTRAC. We hypothesize this is due to fewer collaterals which leads to sex specific signaling patterns. Additional analysis of the plasma and subjects in BACTRAC are needed to target sex specific therapeutic.

Protein Microarrays for Ocular Diseases.

The eye is a multifaceted organ organized in several compartments with particular properties that reflect their diverse functions. The prevalence of ocular diseases is increasing, mainly because of its relationship with aging and of generalized lifestyle changes. However, the pathogenic molecular mechanisms of many common eye pathologies remain poorly understood. Considering the unquestionable importance of proteins in cellular processes and disease progression, proteomic techniques, such as protein microarrays, represent a valuable approach to analyze pathophysiological protein changes in the ocular environment. This technology enables to perform multiplex high-throughput protein expression profiling with minimal sample volume requirements broadening our knowledge of ocular proteome network in eye diseases.In this review, we present a brief summary of the main types of protein microarrays (antibody microarrays, reverse-phase protein microarrays, and protein microarrays) and their application for protein change detection in chronic ocular diseases such as dry eye, age-related macular degeneration, diabetic retinopathy, and glaucoma. The validation of these specific protein changes in eye pathologies may lead to the identification of new biomarkers, depiction of ocular disease pathways, and assistance in the diagnosis, prognosis, and development of new therapeutic options for eye pathologies.

Thiol switches in membrane proteins - Extracellular redox regulation in cell biology.

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g., cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications.