Search Results (15)

Ovarian cancer (OC) is usually diagnosed at an advanced stage, contributing to its high mortality rate. The presence of concurrent bacterial infections in these patients is a common clinical observation, and the mechanisms by which this coinfection influences tumor progression are still not fully understood. This study investigates the role of polydisperse extracellular vesicles (PEVs) secreted by OC cells in response to bacterial components, aiming to elucidate a potential communication pathway between OC and the bacterial microenvironment. We stimulated a human OC cell line in vitro with a fraction of E. coli. Our results show that this bacterial stimulation significantly increases the secretion of PEVs by cancer cells. A subsequent proteomic analysis of these PEVs revealed an enrichment of proteins, including filamin A, filamin B, alpha-enolase, and heat shock cognate 71 kDa protein. In addition, the PEVs displayed protease activity (on fibronectin and gelatin) and phosphatase activity against para-nitrophenyl phosphate, indicating their capacity to alter cellular signaling. This represents a novel mechanism through which bacterial coinfection may influence the biological behavior of OC if bacteria interact with tumor cells, potentially contributing to their aggressiveness and the challenges associated with their treatment. Our work highlights the importance of studying the interplay between the tumor and its associated microbiota to better understand ovarian cancer progression and identify new therapeutic targets. Full article

Background: Using fish collagen supplements in daily nutrition may positively influence health and healthy aging. However, their systemic, molecular-level effects on humans are not well characterized. Therefore, given the scarcity of proteomic data, this study aimed to assess the serum proteomic changes during the fish collagen supplementation in healthy women. Methods: This was a crossover interventional study. Thirty healthy women received either 5 mL of fish gel collagen (from silver carp: Hypophthalmichthys molitrix) supplementation with 200 mL of pure water for 40 days or 200 mL of pure water for 40 days only. The washout between the fish collagen and pure water supplementation was 40 days. The nutritional status and dietary intake were assessed. Proteome analyses were conducted using a MALDI-TOF mass spectrometer in a positive linear mode in the m/z 1000–10,000 range. Results: The diet of the women in this study was not well-balanced. Supplementation did not affect nutritional status. Only water content significantly increased. During the fish collagen supplementation, the following discriminative proteins were identified: Filamin-A, Filamin-B, actin, Vimentin, Tropomyosin beta chain, 40S ribosomal protein S8, ATP-dependent RNA helicase DHX8, and FERM domain-containing protein 4A. Conclusions: Changes in serum proteins may reflect broader cytoskeletal remodeling and cellular adaptation resulting from collagen intake. Full article

We investigated the interaction of HspB7 and its α-crystallin domain with the wild-type (WT) C-terminal fragment of human filamin C (FLNC), containing immunoglobulin-like domains 22–24 and its three mutants associated with cardio- and myopathies. The physicochemical properties of the WT FLNC fragment and its three mutants, p.Glu2472_Asn2473delinsAsp (EN/D) located in the 22nd domain, p.P2643_L2645del (ΔPGL), and p.W2710X (Wmut) both located in the 24th immunoglobulin-like domain were analyzed. Although all FLNC fragments had similar secondary structures, WT FLNC and its EN/D and ΔPGL mutants formed dimers, whereas Wmut formed either monomers or aggregates. The surface hydrophobicity of EN/D, ΔPGL, and especially Wmut mutants was larger than that of the WT fragment. Size exclusion chromatography, native gel electrophoresis, and chemical crosslinking indicated that the efficiency of interaction with HspB7 or its α-crystallin domain decreased in the order WT~EN/D > ΔPGL. Wmut was unable to interact with either HspB7 or its α-crystallin domain. Modeling via Alphafold 3 indicated that EN/D mutation affected the orientation of two loops connecting β-strands in the 22nd domain, while the ΔPGL and Wmut mutations exposed a hydrophobic groove in the 24th domain thereby reducing their interaction with HspB7. These findings reveal the molecular mechanisms underlying filaminopathies associated with three mutations in the C-terminal region of filamin C. Full article

Background: Facial features are the first basic sign of medical knowledge of children and adults with congenital malformations. Children born with multiple contractures almost always receive the misdiagnosis of arthrogryposis multiplex. Larsen syndrome can easily be diagnosed at birth via the proper interpretations of its characteristic facial features and multiple dislocations. Comprehensive clinical diagnosis can facilitate an orthopedic strategy for early treatment and can enhance the recognition of unreported craniocervical malformation complexes. Material and Methods: Six children (four boys and two girls, with ages ranging from a few months to 7 years old) were referred to our department for diagnosis and treatment. All children received their first misdiagnosis by the pediatricians as manifesting arthrogryposis multiplex congenita. The clinical phenotype was our first decisive tool for diagnosis. All children exhibited the classical phenotype of dish-like facies associated with multiple joint dislocations. Radiological phenotypic characteristics confirmed our clinical diagnosis of Larsen syndrome. Three children out of six showed unpleasant cervical spine deformities. The first child, a 2-year-old, became tetraplegic after minor trauma. One child presented with progressive rigid cervical kyphosis. The third child was a product of a first-relative marriage and was born with congenital tetraplegia. A genotype was carried out for confirmation. Results: Three children underwent open reduction for congenital hip and knee dislocations. One child underwent spinal fusion CO-C7 because of tetraplegia. A 3D-reformatted and reconstruction CT scan of the craniocervical junction showed two forms of unusual dys-segmentation, firstly along C2-3 effectively causing the development of acute-angle cervical kyphosis. Secondly, an infant with congenital tetraplegia showed a serious previously undescribed atlanto–axial malformation complex. Namely, atlanto–axial maldevelopment (dys-segmentation) of (C1/C2) was associated with hypoplasia of the anterior and the posterior rings of the atlas. Genetic tests of these children were compatible with the autosomal dominant type of Larsen syndrome and manifested a heterozygous mutation in FLNB mapped 3p14.3, encoding an actin-binding protein, filamin B. The child with congenital tetraplegia showed no mutations in FLNB, though his clinical and radiological phenotype and his family history of first-relative marriage were totally compatible with the diagnosis of the autosomal recessive type of Larsen syndrome. Conclusions: Our strategy was and still is based on a coherent clinical and radiological diagnosis, which is based on comprehensive clinical and radiological phenotypic characterizations. We implemented a 3D-reformatted CT scan to further understand the craniocervical junction pathology in three children. Strikingly, prenatal onset of lethal maldevelopment (dys-segmentation) of the atlanto–axial spine segments has been diagnosed in an infant with congenital tetraplagia. A less serious cervical spine malformation was detected in two children who presented with progressive acute-angle cervico and cervico-thoracic kyphosis. Our clinical strategy can form the basis for a thorough clinical assessment for infants and children born with multiple malformation complexes and can lead to recognition of novel understandings. Full article

Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions. Full article

Filamin C-related disorders include myopathies and cardiomyopathies linked to variants in the FLNC gene. Filamin C belongs to a family of actin-binding proteins involved in sarcomere stability. This study investigates the pathogenic impact of the FLNC c.3557C > T (p.Ala1186Val) pathogenic variant associated with an early-onset cytoplasmic body myopathy and cardiomyopathy in three unrelated patients. We performed clinical imaging and myopathologic and genetic characterization of three patients with an early-onset myopathy and cardiomyopathy. Bioinformatics analysis, variant interpretation, and protein structure analysis were performed to validate and assess the effects of the filamin C variant. All patients presented with a homogeneous clinical phenotype marked by a severe contractural myopathy, leading to loss of gait. There was prominent respiratory involvement and restrictive or hypertrophic cardiomyopathies. The Ala1186Val variant is located in the interstrand loop involved in intradomain stabilization and/or interdomain interactions with neighbor Ig-like domains. 3D modeling highlights local structural changes involving nearby residues and probably impacts the protein stability, causing protein aggregation in the form of cytoplasmic bodies. Myopathologic studies have disclosed the prominent aggregation and upregulation of the aggrephagy-associated proteins LC3B and p62. As a whole, the Ala1186Val variant in the FLNC gene provokes a severe myopathy with contractures, respiratory involvement, and cardiomyopathy due to protein aggregation in patients’ muscles. Full article

The history of arrhythmogenic cardiomyopathy (AC) as a genetically determined desmosomal disease started since the original discovery by Lancisi in a four-generation family, published in 1728. Contemporary history at the University of Padua started with Dalla Volta, who haemodynamically investigated patients with “auricularization” of the right ventricle, and with Nava, who confirmed familiarity. The contemporary knowledge advances consisted of (a) AC as a heart muscle disease with peculiar electrical instability of the right ventricle; (b) the finding of pathological substrates, in keeping with a myocardial dystrophy; (c) the inclusion of AC in the cardiomyopathies classification; (d) AC as the main cause of sudden death in athletes; (e) the discovery of the culprit genes coding proteins of the intercalated disc (desmosome); (f) progression in clinical diagnosis with specific ECG abnormalities, angiocardiography, endomyocardial biopsy, 2D echocardiography, electron anatomic mapping and cardiac magnetic resonance; (g) the discovery of left ventricular AC; (h) prevention of SCD with the invention and application of the lifesaving implantable cardioverter defibrillator and external defibrillator scattered in public places and playgrounds as well as the ineligibility for competitive sport activity for AC patients; (i) genetic screening of the proband family to unmask asymptomatic carriers. Nondesmosomal ACs, with a phenotype overlapping desmosomal AC, are also treated, including genetics: Transmembrane protein 43, SCN5A, Desmin, Phospholamban, Lamin A/C, Filamin C, Cadherin 2, Tight junction protein 1. Full article

Intense exercise can cause inflammation and oxidative stress due to the production of reactive oxygen species. These pathophysiological processes are interdependent, and each one can induce the other, creating a vicious circle. A placebo-controlled blind study was carried out in show jumping horses (n. 16) to evaluate the effects of a commercial dietary supplement (Dolhorse^® N.B.F. Lanes srl, Milan, Italy) containing Verbascum thapsus leaf powder (1.42%), Curcuma longa (14.280 mg/kg), and Boswellia serrata (Roxb ex Colebr) (14.280 mg/kg) extracts. Before and after 10 days of dietary supplementation, blood samples were collected to evaluate the protein levels, antioxidants, and inflammatory responses by proteomic analysis or real-time Reverse Transcriptase-Polymerase Chain Reaction (real-time RT-PCR). A total of 36 protein spots, connected to 29 proteins, were modulated by dietary supplementation, whereas real-time RT-PCR revealed a significant downregulation of proinflammatory cytokines (interleukin 1α (p < 0.05) and interleukin-6 (0.005), toll-like receptor 4 (p < 0.05), and IKBKB (p < 0.05) in supplemented sport horses. Immunoglobulin chains, gelsolin, plasminogen, vitamin D binding protein, apolipoprotein AIV, and filamin B were overexpressed, whereas haptoglobin, α-2-HS-glycoprotein, α2-macroglobulin, afamin, amine oxidase, 60S acidic ribosomal protein, and complement fragments 3, 4, and 7 were reduced. No effect was observed on the antioxidant defense systems. The present results suggest this phytotherapy may reinforce the innate immune responses, thus representing a valid adjuvant to alleviate inflammation, which is a pathophysiological process in sport horses. Full article

(1) Objective: The aim of this dynamic LC-MS (liquid chromatography and mass spectrometry) human platelet proteomic study was to identify the potential proteins candidates for biomarkers of acute ischemic stroke (AIS), their changes during the acute phase of stroke and to define potential novel drug targets. (2) Methods: A total of 32 patients (18–80 years old) were investigated that presented symptoms of AIS lasting less than 24 h from the onset, confirmed by neurological examination and/or new cerebral ischemia visualized in the CT (computed-tomography) scans. The analysis of platelet proteome was performed using LC-MS at baseline, and then on the third and seventh day from the onset of symptoms. The control group was demographically matched without any clinical signs of acute brain injury. (3) Results: The differences between platelets, at 24 h after first symptoms of stroke subjects and the control group included: β-amyloid A4 and amyloid-like protein 2, coactosin-like protein, thymidine phosphorylase 4 (TYMP-4), interferon regulatory factor 7 (IRF7), vitamin K-dependent protein S, histone proteins (H2A type 1 and 1-A, H2A types 2B and J, H2Av, -z, and -x), and platelet basic protein. The dynamic changes in the platelet protein concentration involved thrombospondin-1, thrombospondin-2, filamin A, B, and C. (4) Conclusions: This is the first human dynamic LC-MS proteomic study that differentiates platelet proteome in the acute phase of ischemic stroke in time series and compares the results with healthy controls. Identified proteins may be considered as future markers of ischemic stroke or therapeutic drug targets. Thymidine phosphorylase 4 (TYMP-4) holds promise as an interesting drug target in the management or prevention of ischemic stroke. Full article

The proper function of cardiomyocytes (CMs) is highly related to the Z-disc, which has a pivotal role in orchestrating the sarcomeric cytoskeletal function. To better understand Z-disc related cardiomyopathies, novel models of Z-disc damage have to be developed. Human pluripotent stem cell (hPSC)-derived CMs can serve as an in vitro model to better understand the sarcomeric cytoskeleton. A femtosecond laser system can be applied for localized and defined damage application within cells as single Z-discs can be removed. We have investigated the changes in force generation via traction force microscopy, and in gene expression after Z-disc manipulation in hPSC-derived CMs. We observed a significant weakening of force generation after removal of a Z-disc. However, no significant changes of the number of contractions after manipulation were detected. The stress related gene NF-kB was significantly upregulated. Additionally, α-actinin (ACTN2) and filamin-C (FLNc) were upregulated, pointing to remodeling of the Z-disc and the sarcomeric cytoskeleton. Ultimately, cardiac troponin I (TNNI3) and cardiac muscle troponin T (TNNT2) were significantly downregulated. Our results allow a better understanding of transcriptional coupling of Z-disc damage and the relation of damage to force generation and can therefore finally pave the way to novel therapies of sarcomeric disorders. Full article

Spondylocarpotarsal synostosis syndrome (SCT) is characterized by vertebral fusions, a disproportionately short stature, and synostosis of carpal and tarsal bones. Pathogenic variants in FLNB, MYH3, and possibly in RFLNA, have been reported to be responsible for this condition. Here, we present two unrelated individuals presenting with features typical of SCT in which Sanger sequencing combined with whole genome sequencing identified novel, homozygous intragenic deletions in FLNB (c.1346-1372_1941+389del and c.3127-353_4223-1836del). Both deletions remove several consecutive exons and are predicted to result in a frameshift. To our knowledge, this is the first time that large structural variants in FLNB have been reported in SCT, and thus our findings add to the classes of variation that can lead to this disorder. These cases highlight the need for copy number sensitive methods to be utilized in order to be comprehensive in the search for a molecular diagnosis in individuals with a clinical diagnosis of SCT. Full article

Venom immunotherapy (VIT) is administered to allergic patients to reduce the risk of dangerous systemic reactions following an insect sting. To better understand the mechanism of this treatment and its impact on the human organism, we analysed serum proteomic patterns obtained at five time-points from Hymenoptera-venom-allergic patients undergoing VIT. For statistical analyses, patients were additionally divided into two groups (high responders and low responders) according to serum sIgG4 levels. VIT was found to be associated with changes in seven proteins: the fibrinogen alpha chain, complement C4-A, complement C3, filamin-B, kininogen-1, myosin-9 and inter-alpha-trypsin inhibitor heavy chain H1. The number of discriminative m/z (mass-to-charge ratio) features increased up to the 90th day of VIT, which may be associated with the development of immunity after the administration of increased venom doses. It may also suggest that during VIT, there may occur processes involved not only in protein synthesis but also in protein degradation (caused by proteolytic venom components). The results are consistent with measured serum sIgG4 levels, which increased from 2.04 mgA/I at baseline to 7.25 mgA/I at 90 days. Moreover, the major proteomic changes were detected separately in the high responder group. This may suggest that changes in protein–peptide profiles reflect the actual response to VIT. Full article

Background: Genetic hypomyelinating diseases are a heterogeneous group of disorders involving the white matter. One infantile hypomyelinating leukoencephalopathy is associated with the homozygous variant (Cys4-to-Ser (C4S)) of the c11orf73 gene. Methods: We observed that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins but not the wild type ones of C11orf73 are microscopically localized in the lysosome. And, they downregulate lysosome-related signaling in an immunoblotting technique. Results: The C4S mutant proteins specifically interact with Filamin A, which is known to anchor transmembrane proteins to the actin cytoskeleton; the C4S mutant proteins and Filamin A are also observed in the lysosome fraction. While parental FBD-102b cells and cells harboring the wild type constructs exhibit morphological differentiation, cells harboring C4S mutant constructs do not. It may be that morphological differentiation is inhibited because expression of these C4S mutant proteins leads to defects in the actin cytoskeletal network involving Filamin A. Conclusions: The findings that leukoencephalopathy-associated C11ORF73 mutant proteins specifically interact with Filamin A, are localized in the lysosome, and inhibit morphological differentiation shed light on the molecular and cellular pathological mechanisms that underlie infantile hypomyelinating leukoencephalopathy. Full article

Filamins (FLN) are a family of actin-binding proteins involved in regulating the cytoskeleton and signaling phenomenon by developing a network with F-actin and FLN-binding partners. The FLN family comprises three conserved isoforms in mammals: FLNA, FLNB, and FLNC. FLNB is a multidomain monomer protein with domains containing an actin-binding N-terminal domain (ABD 1–242), encompassing two calponin-homology domains (assigned CH1 and CH2). Primary variants in FLNB mostly occur in the domain (CH2) and surrounding the hinge-1 region. The four autosomal dominant disorders that are associated with FLNB variants are Larsen syndrome, atelosteogenesis type I (AOI), atelosteogenesis type III (AOIII), and boomerang dysplasia (BD). Despite the intense clustering of FLNB variants contributing to the LS-AO-BD disorders, the genotype-phenotype correlation is still enigmatic. In silico prediction tools and molecular dynamics simulation (MDS) approaches have offered the potential for variant classification and pathogenicity predictions. We retrieved 285 FLNB missense variants from the UniProt, ClinVar, and HGMD databases in the current study. Of these, five and 39 variants were located in the CH1 and CH2 domains, respectively. These variants were subjected to various pathogenicity and stability prediction tools, evolutionary and conservation analyses, and biophysical and physicochemical properties analyses. Molecular dynamics simulation (MDS) was performed on the three candidate variants in the CH2 domain (W148R, F161C, and L171R) that were predicted to be the most pathogenic. The MDS analysis results showed that these three variants are highly compact compared to the native protein, suggesting that they could affect the protein on the structural and functional levels. The computational approach demonstrates the differences between the FLNB mutants and the wild type in a structural and functional context. Our findings expand our knowledge on the genotype-phenotype correlation in FLNB-related LS-AO-BD disorders on the molecular level, which may pave the way for optimizing drug therapy by integrating precision medicine. Full article

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction. Full article