Search

Oropharyngeal, airway, intestinal, and genital mucosal epithelia are the main portals of entry for the majority of human pathogenic viruses. To initiate systemic infection, viruses must first be transmitted across the mucosal epithelium and then spread across the body. However, mucosal epithelia have well-developed tight junctions, which have a strong barrier function that plays a critical role in preventing the spread and dissemination of viral pathogens. Viruses can overcome these barriers by disrupting the tight junctions of mucosal epithelia, which facilitate paracellular viral penetration and initiate systemic disease. Disruption of tight and adherens junctions may also release the sequestered viral receptors within the junctional areas, and liberation of hidden receptors may facilitate viral infection of mucosal epithelia. This review focuses on possible molecular mechanisms of virus-associated disruption of mucosal epithelial junctions and its role in transmucosal viral transmission and spread. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of the pandemic coronavirus disease 2019 (COVID‐19) that exhibits an overwhelming contagious capacity over other human coronaviruses (HCoVs). This structural snapshot describes the structural bases underlying the pandemic capacity of SARS‐CoV‐2 and explains its fast motion over respiratory epithelia that allow its rapid cellular entry. Based on notable viral spike (S) protein features, we propose that the flat sialic acid‐binding domain at the N‐terminal domain (NTD) of the S1 subunit leads to more effective first contact and interaction with the sialic acid layer over the epithelium, and this, in turn, allows faster viral 'surfing' of the epithelium and receptor scanning by SARS‐CoV‐2. Angiotensin‐converting enzyme 2 (ACE‐2) protein on the epithelial surface is the primary entry receptor for SARS‐CoV‐2, and protein–protein interaction assays demonstrate high‐affinity binding of the spike protein (S protein) to ACE‐2. To date, no high‐frequency mutations were detected at the C‐terminal domain of the S1 subunit in the S protein, where the receptor‐binding domain (RBD) is located. Tight binding to ACE‐2 by a conserved viral RBD suggests the ACE2‐RBD interaction is likely optimal. Moreover, the viral S subunit contains a cleavage site for furin and other proteases, which accelerates cell entry by SARS‐CoV‐2. The model proposed here describes a structural basis for the accelerated host cell entry by SARS‐CoV‐2 relative to other HCoVs and also discusses emerging hypotheses that are likely to contribute to the development of antiviral strategies to combat the pandemic capacity of SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Abbreviations AADC Aromatic l -amino acid decarboxylase AAV Adeno-associated virus ASO Antisense oligonucleotide MPS Mucopolysaccharidosis rAAV Recombinant adeno-associated virus The impact of genome and exome sequencing has led to an unprecedented rise in diagnoses of genetic paediatric neurological diseases.1 This provides an opportunity to understand disease mechanisms and to develop novel therapeutic approaches for significant unmet clinical needs. Managed access agreements for access to Spinraza are available on public healthcare in more than 40 countries as of August 2019.29 This success stimulated the development of a personalized ASO (Milasen) for a patient with Batten disease.30 Viral gene therapy for spinal muscular atrophy type 1 has resulted in a fundamental change in rAAV gene therapy. The first was ornithine transcarbamylase deficiency gene therapy delivered by intrahepatic infusion of wild-type adenovirus that resulted in vector-related immune response, multi-organ failure, and death.4 The second was gamma retroviral correction of X-linked severe combined immunodeficiency, which reconstituted immunity but vector-related leukaemia consequently developed.5 These highlighted an inadequate understanding of vector properties, and gene therapists have worked tirelessly to engineer recombinant viral vectors to improve safety and efficiency. Lentivirus-based gene therapies are used for X-linked adrenoleukodystrophy and metachromatic leukodystrophy.26,27 In 2018, Lenti-D, an investigational gene therapy lentivirus, was granted US Food and Drug Administration "breakthrough therapy" designation and used to treat 17 patients with X-linked adrenoleukodystrophy. [Extracted from the article]

Novel coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2. The virus causes an exaggerated immune response, resulting in a cytokine storm and acute respiratory distress syndrome, the leading cause of COVID-19-related mortality and morbidity. So far, no therapies have succeeded in circumventing the exacerbated immune response or cytokine storm associated with COVID-19. Mesenchymal stem cells (MSCs), through their immunomodulatory and regenerative activities, mostly mediated by their paracrine effect and extracellular vesicle production, have therapeutic potential in many autoimmune, inflammatory, and degenerative diseases. In this paper, we review clinical studies on the use of MSCs for COVID-19 treatment, including the salutary effects of MSCs on the pathophysiology of COVID-19 and the immunomodulation of the cytokine storm. Ongoing clinical trial designs, cell sources, dose and administration, and populations are summarized, and the paracrine mode of benefit is discussed. We also offer suggestions for optimizing MSC-based therapies, including genetic engineering, strategies for cell surface modification, nanotechnology applications, and combination therapies. [ABSTRACT FROM AUTHOR]

The heparan sulfate 6- O -endosulfatases, Sulfatase 1 (Sulf1), and Sulfatase 2 (Sulf2), are extracellular enzymes that regulate cellular signaling by removing 6- O -sulfate from the heparan sulfate chain. Although previous studies have revealed that Sulfs are essential for normal development, their functions in the adult brain remain largely unknown. To gain insight into their neural functions, we used in situ hybridization to systematically examine Sulf1/2 mRNA expression in the adult mouse brain. Sulf1 and Sulf2 mRNAs showed distinct expression patterns, which is in contrast to their overlapping expression in the embryonic brain. In addition, we found that Sulf1 was distinctly expressed in the nucleus accumbens shell, the posterior tail of the striatum, layer 6 of the cerebral cortex, and the paraventricular nucleus of the thalamus, all of which are target areas of dopaminergic projections. Using double-labeling techniques, we showed that Sulf1 -expressing cells in the above regions coincided with cells expressing the dopamine D1 and/or D2 receptor. These findings implicate possible roles of Sulf1 in modulation of dopaminergic transmission and dopamine-mediated behaviors. [ABSTRACT FROM AUTHOR]

B-cell lymphoma is a group of hematological malignancies with high clinical and biological heterogeneity. The pathogenesis of B-cell lymphoma involves a complex interaction between tumor cells and the tumor microenvironment (TME), which is composed of stromal cells and extracellular matrix. Although the roles of the TME have not been fully elucidated, accumulating evidence implies that TME is closely relevant to the origination, invasion and metastasis of B-cell lymphoma. Explorations of the TME provide distinctive insights for cancer therapy. Here, we epitomize the recent advances of TME in B-cell lymphoma and discuss its function in tumor progression and immune escape. In addition, the potential clinical value of targeting TME in B-cell lymphoma is highlighted, which is expected to pave the way for novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) as a possible infectious etiology in Alzheimer's disease (AD) has been proposed since the 1980s. The accumulating research thus far continues to support the association and a possible causal role of HSV-1 in the development of AD. HSV-1 has been shown to induce neuropathological and behavioral changes of AD, such as amyloid-beta accumulation, tau hyperphosphorylation, as well as memory and learning impairments in experimental settings. However, a neuroanatomical standpoint of HSV-1 tropism in the brain has not been emphasized in detail. In this review, we propose that the hippocampal vulnerability to HSV-1 infection plays a part in the development of AD and amnestic mild cognitive impairment (aMCI). Henceforth, this review draws on human studies to bridge HSV-1 to hippocampal-related brain disorders, namely AD and aMCI/MCI. Next, experimental models and clinical observations supporting the neurotropism or predilection of HSV-1 to infect the hippocampus are examined. Following this, factors and mechanisms predisposing the hippocampus to HSV-1 infection are discussed. In brief, the hippocampus has high levels of viral cellular receptors, neural stem or progenitor cells (NSCs/NPCs), glucocorticoid receptors (GRs) and amyloid precursor protein (APP) that support HSV-1 infectivity, as well as inadequate antiviral immunity against HSV-1. Currently, the established diseases HSV-1 causes are mucocutaneous lesions and encephalitis; however, this review revises that HSV-1 may also induce and/or contribute to hippocampal-related brain disorders, especially AD and aMCI/MCI. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disease that currently cannot be cured by any drug or intervention, due to its complicated pathogenesis. Current animal and cellular models of AD are unable to meet research needs for AD. However, recent three-dimensional (3D) cerebral organoid models derived from human stem cells have provided a new tool to study molecular mechanisms and pharmaceutical developments of AD. In this review, we discuss the advantages and key limitations of the AD cerebral organoid system in comparison to the commonly used AD models, and propose possible solutions, in order to improve their application in AD research. Ethical concerns associated with human cerebral organoids are also discussed. We also summarize future directions of studies that will improve the cerebral organoid system to better model the pathological events observed in AD brains. [ABSTRACT FROM AUTHOR]

Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today. [ABSTRACT FROM AUTHOR]

During the current formidable COVID-19 pandemic, it is appealing to address ideas that may invoke therapeutic interventions. Clotting disorders are well recognized in patients infected with severe acute respiratory syndrome (SARS) caused by a novel coronavirus (SARS-CoV-2), which lead to severe complications that worsen the prognosis in these subjects. Increasing evidence implicate Heparan sulfate proteoglycans (HSPGs) and Heparanase in various diseases and pathologies, including hypercoagulability states. Moreover, HSPGs and Heparanase are involved in several viral infections, in which they enhance cell entry and release of the viruses. Herein we discuss the molecular involvement of HSPGs and heparanase in SARS-CoV-2 infection, namely cell entry and release, and the accompanied coagulopathy complications, which assumedly could be blocked by heparanase inhibitors such as Heparin and Pixatimod. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) remains one of the leading causes of death and disability worldwide; more than 10 million people are hospitalized for TBI every year around the globe. While the primary injury remains unavoidable and not accessible to treatment, the secondary injury which includes oxidative stress, inflammation, excitotoxicity, but also complicating coagulation abnormalities, is potentially avoidable and profoundly affects the therapeutic process and prognosis of TBI patients. The endothelial glycocalyx, the first line of defense against endothelial injury, plays a vital role in maintaining the delicate balance between blood coagulation and anticoagulation. However, this component is highly vulnerable to damage and also difficult to examine. Recent advances in analytical techniques have enabled biochemical, visual, and computational investigation of this vascular component. In this review, we summarize the current knowledge on (i) structure and function of the endothelial glycocalyx, (ii) its potential role in the development of TBI associated coagulopathy, and (iii) the options available at present for detecting and protecting the endothelial glycocalyx. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPGs) consist of a core protein with side chains of the glycosaminoglycan heparan sulfate (HS). We have previously identified (i) the HSPGs syndecan-1 (SDC1), and collagen type XVIII (COL18) inside mouse and human islet beta cells, and (ii) a critical role for HS in beta cell survival and protection from reactive oxygen species (ROS). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress contributes to oxidative stress and type 2 diabetes (T2D) by depleting beta cell HSPGs/HS. A rapid loss of intra-islet/beta cell HSPGs, HS and heparanase (HPSE, an HS-degrading enzyme) accompanied upregulation of islet ER stress gene expression in both young T2D-prone db/db and Akita Ins2WT/C96Y mice. In MIN6 beta cells, HSPGs, HS and HPSE were reduced following treatment with pharmacological inducers of ER stress (thapsigargin or tunicamycin). Treatment of young db/db mice with Tauroursodeoxycholic acid (TUDCA), a chemical protein folding chaperone that relieves ER stress, improved glycemic control and increased intra-islet HSPG/HS. In vitro, HS replacement with heparin (a highly sulfated HS analogue) significantly increased the survival of wild-type and db/db beta cells and restored their resistance to hydrogen peroxide-induced death. We conclude that ER stress inhibits the synthesis/maturation of HSPG core proteins which are essential for HS assembly, thereby exacerbating oxidative stress and promoting beta cell failure. Diminished intracellular HSPGs/HS represent a previously unrecognized critical link bridging ER stress, oxidative stress and beta cell failure in T2D. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome–related coronavirus-2 (SARS-CoV-2), a β-coronavirus, is the cause of the recently emerged pandemic and worldwide outbreak of respiratory disease. Researchers exchange information on COVID-19 to enable collaborative searches. Although there is as yet no effective antiviral agent, like tamiflu against influenza, to block SARS-CoV-2 infection to its host cells, various candidates to mitigate or treat the disease are currently being investigated. Several drugs are being screened for the ability to block virus entry on cell surfaces and/or block intracellular replication in host cells. Vaccine development is being pursued, invoking a better elucidation of the life cycle of the virus. SARS-CoV-2 recognizes O-acetylated neuraminic acids and also several membrane proteins, such as ACE2, as the result of evolutionary switches of O-Ac SA recognition specificities. To provide information related to the current development of possible anti–SARS-COV-2 viral agents, the current review deals with the known inhibitory compounds with low molecular weight. The molecules are mainly derived from natural products of plant sources by screening or chemical synthesis via molecular simulations. Artificial intelligence–based computational simulation for drug designation and large-scale inhibitor screening have recently been performed. Structure–activity relationship of the anti–SARS-CoV-2 natural compounds is discussed. [ABSTRACT FROM AUTHOR]

Various anticoagulant properties have been associated with hydroxyethyl starch (HES). However, the mechanism remains unclear and it has not been fully considered whether these properties are beyond the dilutional effect itself. The aim of this study was to reproduce the coagulopathy induced by HES and to test the hypothesis that the coagulopathy is caused by endothelial or glycocalyx damage due to localization of HES on the endothelium, which is caused by the high shear viscosity of dilutional blood. Using a rat model, we compared blood coagulability measured by Sonoclot, levels of endothelial and glycocalyx damage markers and coagulation factors, and blood shear viscosity when hemodilution was performed with physiological saline (PS), 6% HES 130/0.4 in PS, and 10% HES 200/0.5 in PS. We also evaluated the localization rates of fluorescently labeled HES on endothelium in the isolated aorta. HES decreased the fibrin gel formation rate more than did PS. HES was shown to cover the endothelium, possibly due to its high shear viscosity, and this mechanism potentially acted to protect, rather than damage, the endothelium and glycocalyx. However, this covering effect may be the cause of coagulopathy due to inhibition of von Willebrand factor secretion from the endothelium. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer occurs through multiple steps until advanced metastasis. Signaling pathways studies can result in the identification of targets to interrupt cancer progression. Glypicans are cell surface proteoglycans linked to the membrane through glycosylphosphatidylinositol. Their interaction with specific ligands has been reported to trigger diverse signaling, including Wnt. In this study, prostate cancer cell lines PC-3, DU-145, and LNCaP were compared to normal prostate RWPE-1 cell line to investigate glypican family members and the activation of the Wnt signaling pathway. Results: Glypican-1 (GPC1) was highly expressed in all the examined cell lines, except for LNCaP, which expressed glypican-5 (GPC5). The subcellular localization of GPC1 was detected on the cell surface of RWPE-1, PC-3, and DU-145 cell lines, while GPC5 suggested cytoplasm localization in LNCaP cells. Besides glypican, flow cytometry analysis in these prostate cell lines confirmed the expression of Wnt-3a and unphosphorylated β-catenin. The co-immunoprecipitation assay revealed increased levels of binding between Wnt-3a and glypicans in cancer cells, suggesting a relationship between these proteoglycans in this pathway. A marked increase in nuclear β-catenin was observed in tumor cells. However, only PC-3 cells demonstrated activation of canonical Wnt signaling, according to the TOPFLASH assay. Conclusions: GPC1 was the majorly expressed gene in all the studied cell lines, except for LNCaP, which expressed GPC5. We assessed by co-immunoprecipitation that these GPCs could interact with Wnt-3a. However, even though nuclear β-catenin was found increased in the prostate cancer cells (i.e., PC-3, DU-145 and LNCaP), activation of Wnt pathway was only found in PC-3 cells. In these PC-3 cells, GPC1 and Wnt-3a revealed high levels of colocalization, as assessed by confocal microscopy studies. This suggests a localization at the cellular surface, where Frizzled receptor is required for downstream activation. The interaction of Wnt-3a with GPCs in DU-145 and LNCaP cells, which occurs in absence of Wnt signaling activation, requires further studies. Once non-TCF-LEF proteins can also bind β-catenin, another signaling pathway may be involved in these cells with regulatory function. [ABSTRACT FROM AUTHOR]

Background: Despite recent advances in the understanding of the molecular landscape of biliary tract cancer (BTC), advanced disease continues to carry a poor prognosis, and the benefit from systemic treatments remains modest. However, BTCs have emerged as malignancies harboring specific potentially druggable aberrations, and thus, several molecularly targeted treatments have been recently tested. Among these, fibroblast growth factor receptor (FGFR) inhibitors have shown interesting results in previously treated BTC patients with advanced disease Areas covered: In this review, we aimed to provide an overview of available evidence on FGFR inhibitors in elderly patients with metastatic BTC, especially focusing on subgroup analyses of recently published trials exploring this novel therapeutic approach in these aggressive malignancies. Expert opinion: The FGFR1, FGFR2, and FGFR3 inhibitor pemigatinib has been recently approved by the United States Food and Drug Administration (FDA) in metastatic BTCs harboring FGFR2 fusion or other rearrangement. However, few data are available regarding the use of FGFR inhibitors in elderly BTCs, a patient population that remains seriously under-represented in clinical trials. [ABSTRACT FROM AUTHOR]

We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-initiated outbreak of COVID-19 has spread rapidly around the world, posing a huge threat to public health. Natural oligo- and polysaccharides with low toxicity, good sustainability, high biocompatibility, respectable safety, immune regulation, and antiviral activity can be employed as promising candidates for the prevention and inhibition of viral infections, especially COVID-19. Glycosaminoglycans, marine polysaccharides, terrestrial plant polysaccharides, and some others have exhibited potential antiviral activity against pathogenic viruses, in the format of polysaccharide-centered vaccine adjuvants, nano-based structures, drug conveyance platforms, etc. In this review, significant recent advancements pertaining to the antiviral applications of oligo- and polysaccharides against SARS-CoV-2 are highlighted, including important challenges and future perspectives. [ABSTRACT FROM AUTHOR]

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N -glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N -glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (FGFs) are a family of proteins possessing paracrine, autocrine, or endocrine functions in a variety of biological processes, including embryonic development, angiogenesis, tissue homeostasis, wound repair, and cancer. Canonical FGFs bind and activate tyrosine kinase FGF receptors (FGFRs), triggering intracellular signaling cascades that mediate their biological activity. Experimental evidence indicates that FGFs play a complex role in the physiopathology of the prostate gland that ranges from essential functions during embryonic development to modulation of neoplastic transformation. The use of ligand- and receptor-deleted mouse models has highlighted the requirement for FGF signaling in the normal development of the prostate gland. In adult prostate, the maintenance of a functional FGF/FGFR signaling axis is critical for organ homeostasis and function, as its disruption leads to prostate hyperplasia and may contribute to cancer progression and metastatic dissemination. Dissection of the molecular landscape modulated by the FGF family will facilitate ongoing translational efforts directed toward prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Heparan sulfate is a glycosaminoglycan present in nearly all mammalian tissues. Heparan sulfate moieties are attached to the cell surface via heparan sulfate proteoglycans (HSPGs) which are composed of a protein core bound to multiple heparan sulfate chains. HSPGs contribute to the structural integrity of the extracellular matrix and participate in cell signaling by releasing bound cytokines and chemokines once cleaved by an enzyme, heparanase. HSPGs are often exploited by viruses during infection, particularly during attachment and egress. Loss or inhibition of HSPGs initially during infection can yield significant decreases in viral entry and infectivity. In this review, we provide an overview of HSPGs in the lifecycle of multiple viruses, including herpesviruses, human immunodeficiency virus, dengue virus, human papillomavirus, and coronaviruses. [ABSTRACT FROM AUTHOR]

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan‐1 (Sdc‐1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc‐1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor‐initiating cell phenotype. Sdc‐1 small‐interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT‐29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex‐determining region Y)‐box 2, KLF2, and TCF4/TCF7L2. Sdc‐1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial‐to‐mesenchymal transition‐related gene expression. Sdc‐1‐depleted HT‐29 xenograft growth was increased compared to controls. Decreased Sdc‐1 expression was associated with an increased activation of β1‐integrins, focal adhesion kinase (FAK), and wingless‐type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc‐1‐depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc‐1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness‐associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. Databases: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751. [ABSTRACT FROM AUTHOR]

The pathological hallmark of Parkinson's disease (PD) is Lewy bodies that form within the brain from aggregated forms of α‐synuclein (α‐syn). These toxic α‐syn aggregates are transferred from cell to cell by release of fibrils from dying neurons into the extracellular environment, followed by their subsequent uptake by neighboring cells. This process leads to spreading of the pathology throughout the brain in a prion‐like manner. Identifying new pathways that hinder the internalization of such α‐syn fibrils is of high interest for their downstream potential exploitation as a way to create disease‐modifying therapeutics for PD. Here, we show that Thiamet‐G, a highly selective pharmacological agent that inhibits the glycoside hydrolase O‐GlcNAcase (OGA), blunts the cellular uptake of α‐syn fibrils. This effect correlates with increased nucleocytoplasmic levels of O‐linked N‐acetylglucosamine (O‐GlcNAc)‐modified proteins, and genetic knockdown of OGA expression closely phenocopies both these effects. These reductions in the uptake of α‐syn fibrils caused by inhibition of OGA are both concentration‐ and time‐dependent and are observed in multiple cell lines including mouse primary cortical neurons. Moreover, treatment of cells with the OGT inhibitor, 5SGlcNHex, increases the level of uptake of α‐syn PFFs, further supporting O‐GlcNAcylation of proteins driving these effects. Notably, this effect is mediated through an unknown mechanism that does not involve well‐characterized endocytotic pathways. These data suggest one mechanism by which OGA inhibitors might exert their protective effects in prion‐like neuropathologies and support exploration of OGA inhibitors as a potential disease‐modifying approach to treat PD. [ABSTRACT FROM AUTHOR]

Although very different in etiology and symptoms, numerous neurodegenerative diseases can be classified as proteinopathies. More so, evidence indicates that the key misfolded proteins at the basis of different neuropathies might share common mechanisms of propagation. As such, the prion-like spreading of protein aggregates through the neural network is subject of intensive research focus and requires adequate models. Here, we made use of the well-defined architecture and large accessibility of the visual system, of which the retinotopic connections represent a simple route of anterograde signaling and an elegant model to investigate transsynaptic, prion-like spreading. In two independent studies, uptake and seeding of alpha-synuclein and tau were examined after intravitreal injection of preformed fibrils. However, extracellular matrix components in the vitreous space and at the vitreoretinal surface appeared to act as a barrier for the entry of both fibrils into the retina. These results show that further experimental refinement is needed to fully realize the potential of the visual system as a model for studying the molecular and cellular mechanisms of anterograde, transsynaptic spreading of prion-like proteins. [ABSTRACT FROM AUTHOR]

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region. [ABSTRACT FROM AUTHOR]

Copyright of Advances in Hygiene & Experimental Medicine / Postepy Higieny i Medycyny Doswiadczalnej is the property of Sciendo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Extracellular signaling molecules, among them the fibroblast growth factors (FGFs), enable cells to communicate with neighboring cells. Such signaling molecules that receive and transmit a signal require specific tyrosine kinase receptors located at the cell surface (fibroblast growth factor receptors, FGFRs). The binding of a signaling molecule to its specific receptor results in receptor dimerization and conformational changes in the cytoplasmic part of the receptor. The conformational changes lead to trans-autophosphorylation of the tyrosine kinase domains of the receptors and subsequently to induction of several downstream signaling pathways and expression of appropriate genes. The signaling pathways activated by FGFs control and coordinate cell behaviors such as cell division, migration, differentiation, and cell death. FGFs and their transmembrane receptors are widely distributed in different tissues and participate in fundamental processes during embryonic, fetal, and adult human life. The human FGF/FGFR family comprises 22 ligands and 4 high affinity receptors. In addition, FGFs bind to low affinity receptors, heparan sulfate proteoglycans at the cell surface. The availability of appropriate ligand/receptor pair, combined with the co-receptor, initiates signaling. Inappropriate FGF/FGFR signaling can cause skeletal disorders, primarily dwarfism, craniofacial malformation syndromes, mood disorders, metabolic disorders, and Kallman syndrome. In addition, aberrations in FGF/FGFR signaling have already been reported in several types of malignant diseases. Knowledge about the molecular mechanisms of FGF/FGFR activation and signaling is necessary to understand the basis of these diseases. [ABSTRACT FROM AUTHOR]

Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. We have previously shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. Here we demonstrate that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, our results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception. A stem cell niche is the specialized micro-environment that provides the signal to the resident stem cells to support their undifferentiated, self-renewing state. This study shows that the cells that compose the niche do not only provide the signal, but also take up the receptor of stem cells for subsequent lysosomal degradation; this mechanism is essential for restriction of niche signal range. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers. Collectively, HNSCC ranks sixth in incidence rate worldwide. Apart from classical risk factors like tobacco and alcohol, infection of human papillomavirus (HPV) is emerging as a discrete risk factor for HNSCC. HPV-positive HNSCC represent a distinct group of diseases that differ in their clinical presentation. These lesions are well-differentiated, occur at an early age, and have better prognosis. Epidemiological studies have demonstrated a specific increase in the proportions of the HPV-positive HNSCC. HPV-positive and HPV-negative HNSCC lesions display different disease progression and clinical response. For tumorigenic-transformation, HPV essentially requires a permissive cellular environment and host cell factors for induction of viral transcription. As the spectrum of host factors is independent of HPV infection at the time of viral entry, presumably entry of HPV only selects host cells that are permissive to establishment of HPV infection. Growing evidence suggest that HPV plays a more active role in a subset of HNSCC, where they are transcriptionally-active. A variety of factors provide a favorable environment for HPV to become transcriptionally-active. The most notable are the set of transcription factors that have direct binding sites on the viral genome. As HPV does not have its own transcription machinery, it is fully dependent on host transcription factors to complete the life cycle. Here, we review and evaluate the current evidence on level of a subset of host transcription factors that influence viral genome, directly or indirectly, in HNSCC. Since many of these transcription factors can independently promote carcinogenesis, the composition of HPV permissive transcription factors in a tumor can serve as a surrogate marker of a separate molecularly-distinct class of HNSCC lesions including those cases, where HPV could not get a chance to infect but may manifest better prognosis. [ABSTRACT FROM AUTHOR]

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV. Here the authors report the construction of a genome-scale porcine CRISPR/Cas9 library, called PigGeCKO, for screening and analyses of host resistance genes and factors associated with Japanese encephalitis virus replication. [ABSTRACT FROM AUTHOR]

Impairments of vision and hearing are highly prevalent conditions limiting the quality of life and presenting a major socioeconomic burden. For a long time, retinal and cochlear disorders have remained intractable for causal therapies, with sensory rehabilitation limited to glasses, hearing aids, and electrical cochlear or retinal implants. Recently, the application of gene therapy and optogenetics to eye and ear has generated hope for a fundamental improvement of vision and hearing restoration. To date, one gene therapy for the restoration of vision has been approved, and ongoing clinical trials will broaden its application including gene replacement, genome editing, and regenerative approaches. Moreover, optogenetics, i.e., controlling the activity of cells by light, offers a more general alternative strategy. Over little more than a decade, optogenetic approaches have been developed and applied to better understand the function of biological systems, while protein engineers have identified and designed new opsin variants with desired physiological features. Considering potential clinical applications of optogenetics, the spotlight is on the sensory systems, particularly the eye and ear. Multiple efforts have been undertaken to restore lost or hampered function in the eye and ear. Optogenetic stimulation promises to overcome fundamental shortcomings of electrical stimulation, namely, poor spatial resolution and cellular specificity, and accordingly to deliver more detailed sensory information. This review aims to provide a comprehensive reference on current gene therapeutic and optogenetic research relevant to the restoration of hearing and vision. We will introduce gene-therapeutic approaches and discuss the biotechnological and optoelectronic aspects of optogenetic hearing and vision restoration. [ABSTRACT FROM AUTHOR]

Background and Purpose: Intra-islet heparan sulfate (HS) plays an important role in the maintenance of pancreatic islet function. The aim of this study was to investigate the effect mechanism of HS loss on the functioning of islets in diabetic mice.Experimental Approach: The hypoglycaemic effect of a heparanase inhibitor, OGT2115, was tested in a streptozotocin-induced diabetic mouse model. The islets in pancreatic sections were also stained to reveal their morphology. An insulinoma cell line (MIN6) and primary isolated murine islets were used to investigate the effect of OGT2115 in vitro.Key Results: Intra-islet HS was clearly lost in streptozotocin-induced diabetic mice due to the increased heparanase expression in damaged islets. OGT2115 prevented intra-islet HS loss and improved the glucose profile and insulin secretion in streptozotocin-treated mice. The apoptosis of pancreatic beta cells and the infiltration of mononuclear macrophages, CD4- and CD8-positive T-cells in islets was reduced by OGT2115 in streptozotocin-treated mice, but OGT2115 did not alter the direct streptozotocin-induced damage in vitro. The expression of heparanase was increased in high glucose-treated isolated islets but not in response to direct streptozotocin stimulation. Further experiments showed that high glucose stimuli could decreased expression of PPARγ in cultured islets, thereby relieving the PPARγ-induced inhibition of heparanase gene expression.Conclusion and Implications: Hyperglycaemia could cause intra-islet HS loss by elevating the expression of heparanase, thereby aggravating inflammatory cell infiltration and islet damage. Inhibition of heparanase might provide benefit for pancreatic beta cell protection in Type 1 diabetes. [ABSTRACT FROM AUTHOR]

Although the main causative genes for hereditary multiple exostoses (HME) are exostosin (EXT)-1 and EXT-2, there are numerous patients with HME without EXT-1 and EXT-2 mutations. The present study aimed to identify novel candidate genes for the development of HME in patients without EXT-1 and EXT-2 mutations. Whole-exome sequencing was performed in a Chinese family with HME and without EXT-1 and EXT-2 mutations, followed by a combined bioinformatics pipeline including annotation and filtering processes to identify candidate variants. Candidate variants were then validated using Sanger sequencing. A total of 1,830 original variants were revealed to be heterozygous mutations in three patients with HME which were not present in healthy controls. Two mutations [c.C1849T in solute carrier family 20 member 2 (SLC20A2) and c.G506A in leucine zipper and EF-hand containing transmembrane protein 1 (LETM1)] were identified as possible causative variants for HME through a bioinformatics filtering procedure and harmful prediction. Sanger sequencing results confirmed these two mutations in all patients with HME. A mutation in SLC20A2 (c.C1849T) led to a change in an amino acid (p.R617C), which may be involved in the development of HME by inducing metabolic disorders of phosphate and abnormal proliferation and differentiation in chondrocytes. In conclusion, the present study revealed two mutations [SLC20A2 (c.C1849T) and LETM1 (c.G506A) in a Chinese family with HME. The mutation in SLC20A2 (c.C1849T)] was more likely to be involved in the development of HME. [ABSTRACT FROM AUTHOR]

The mammary gland is a secretory organ, which develops as a network of growing epithelial ducts composed of luminal and basal cells that invade the surrounding adipose tissue through a series of developmental cycles. Mammary stem cells (MaSCs) maintain an accurate tissue homeostasis, and their proliferation and cell fate determination are regulated by multiple hormones and local factors. The WNT pathway plays a critical role in controlling the enormous tissue expansion and remodeling during mammary gland development through the maintenance and differentiation of MaSCs, and its deregulation has been implicated in breast cancer (BC) initiation and progression. The R‐spondins (RSPOs) are four secreted proteins that strongly enhance target cell sensitivity to WNT ligands. Moreover, leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGRs) 4–6 are considered obligate high‐affinity receptors for RSPOs and have been described as stem cell markers. Importantly, elevated RSPO expression has been recently identified in several tumor types from patients, including BC, and it has been reported that they play a significant role in mammary tumor progression in experimental models. In this review, exploring our present knowledge, we summarize the role of the RSPO‐LGR axis as a WNT‐enhancing signaling cascade in the MaSC compartment and during the normal and neoplastic mammary gland development. In addition, we include an updated expression profile of the RSPOs and their action mediators at the cell membrane, the LGRs, and the ubiquitin‐ligases ZNRF3/RNF43, in different BC subtypes. Finally and based on these data, we discuss the significance of tumor‐associated alterations of these proteins and their potential use as molecular targets for detection and treatment of BC. [ABSTRACT FROM AUTHOR]

Neutrophil elastase (NE) is a major protease in the airways of patients with cystic fibrosis (CF) that activates airway inflammation by several mechanisms. NE stimulates epithelial toll like receptors (TLR) resulting in cytokine upregulation and release, upregulates MUC5AC, a major airway mucin, degrades both phagocytic receptors and opsonins resulting in both neutrophil and macrophage phagocytic failure, generates oxidative stress via extracellular generation and uptake of heme free iron, and activates other proteases. Altogether, these mechanisms create a significant inflammatory challenge that impairs innate immune function and results in airway remodeling. Currently, a major gap in our therapeutic approach to CF lung disease is the lack of an effective therapeutic strategy targeting active NE and its downstream pro-inflammatory sequelae. Polysulfated glycosaminoglycans (GAGs) are potent anti-elastase drugs that have additional anti-inflammatory properties. Heparin is a prototype of a glycosaminoglycan with both anti-elastase and anti-inflammatory properties. Heparin inhibits NE in an allosteric manner with high potency. Heparin also inhibits cathepsin G, blocks P-selectin and L-selectin, hinders ligand binding to the receptor for advanced glycation endproducts, and impedes histone acetyltransferase activity which dampens cytokine transcription and High Mobility Group Box 1 release. Furthermore, nebulized heparin treatment improves outcomes for patients with chronic obstructive pulmonary disease (COPD), asthma, acute lung injury and smoke inhalation. However, the anticoagulant activity of heparin is a potential contraindication for this therapy to be developed for CF lung disease. Therefore, modified heparins and other GAGs are being developed that retain the anti-elastase and anti-inflammatory qualities of heparin with minimal to no anticoagulant activity. The modified heparin, 2-O, 3-O desulfated heparin (ODSH), maintains anti-elastase and anti-inflammatory activities in vitro and in vivo , and has little residual anticoagulant activity. Heparan sulfate with O-sulfate residues but not N-sulfate residues blocks allergic asthmatic inflammation in a murine model. Polysulfated hyaluronic acid abrogates allergen- triggered rhinosinusitis in a murine model. Finally, nonsaccharide glycosaminoglycan mimetics with specific sulfate modifications can be designed to inhibit NE activity. Altogether, these novel GAGs or GAG mimetics hold significant promise to address the unmet need for inhaled anti-elastase and anti-inflammatory therapy for patients with CF. [ABSTRACT FROM AUTHOR]

Background: Munchkin cats were founded on a naturally occurring mutation segregating into long-legged and short-legged types. Short-legged cats showed disproportionate dwarfism (chondrodysplasia) in which all four legs are short and are referred as standard Munchkin cats. Long-legged animals are referred as non-standard Munchkin cats. A previous study using genome-wide single nucleotide polymorphisms (SNPs) for genome-wide association analysis identified a significantly associated region at 168–184 Mb on feline chromosome (FCA) B1. Results: In this study, we validated the critical region on FCA B1 using a case-control study with 89 cats and 14 FCA B1-SNPs. A structural variant within UGDH (NC_018726.2:g.173294289_173297592delins108, Felis catus 8.0, equivalent to NC_018726.3:g.174882895_174886198delins108, Felis catus 9.0) on FCA B1 was perfectly associated with the phenotype of short-legged standard Munchkin cats. Conclusion: This UGDH structural variant very likely causes the chondrodysplastic (standard) phenotype in Munchkin cats. The lack of homozygous mutant phenotypes and reduced litter sizes in standard Munchkin cats suggest an autosomal recessive lethal trait in the homozygote state. We propose an autosomal dominant mode of inheritance for the chondrodysplastic condition in Munchkin cats. [ABSTRACT FROM AUTHOR]

Post‐translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O‐glycosylation is among the most abundant and diverse PTMs. Initiation of O‐GalNAc glycosylation is regulated by 20 distinct GalNAc‐transferases (GalNAc‐Ts), and deficiencies in individual GalNAc‐Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc‐Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc‐Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc‐T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc‐T1 targets are associated with components of the endomembrane system, GalNAc‐T2 targets with cell–ECM adhesion, and GalNAc‐T3 targets with epithelial differentiation. Thus, GalNAc‐T isoforms serve specific roles during human epithelial tissue formation. Synopsis: GalNAc‐T isoform knock out in human skin organoids reveals selective phenotypes in tissue organization and differentiation. By using mass spectrometry‐based phospho‐ and O‐glycoproteomics, the identified phenotypes are correlated with unique signaling networks and isoform‐selective protein substrates. GalNAc‐T isoform knock out in human keratinocytes perturb the formation of 3D organoids.Combination of RNA_Seq, differential O‐glycoproteomics and phosphoproteomics allow to correlate tissue phenotypes with biological pathways.GalNAc‐T2 is important for cell‐to‐matrix adhesion, whereas GalNAc‐T3 affects epidermal differentiation. [ABSTRACT FROM AUTHOR]

Synucleinopathies are a group of disorders characterized by the accumulation of inclusions rich in the a‐synuclein (aSyn) protein. This group of disorders includes Parkinson's disease, dementia with Lewy bodies (DLB), multiple systems atrophy, and pure autonomic failure (PAF). In addition, genetic alterations (point mutations and multiplications) in the gene encoding for aSyn (SNCA) are associated with familial forms of Parkinson's disease, the most common synucleinopathy. The Synuclein Meetings are a series that has been taking place every 2 years for about 12 years. The Synuclein Meetings bring together leading experts in the field of Synuclein and related human conditions with the goal of discussing and advancing the research. In 2019, the Synuclein meeting took place in Ofir, a city in the outskirts of Porto, Portugal. The meeting, entitled "Synuclein Meeting 2019: Where we are and where we need to go", brought together >300 scientists studying both clinical and molecular aspects of synucleinopathies. The meeting covered a many of the open questions in the field, in a format that prompted open discussions between the participants, and underscored the need for additional research that, hopefully, will lead to future therapies for a group of as of yet incurable disorders. Here, we provide a summary of the topics discussed in each session and highlight what we know, what we do not know, and what progress needs to be made in order to enable the field to continue to advance. We are confident this systematic assessment of where we stand will be useful to steer the field and contribute to filling knowledge gaps that may form the foundations for future therapeutic strategies, which is where we need to go. [ABSTRACT FROM AUTHOR]