The prognosis of metastatic melanoma (MM) patients has remained poor for a long time. However, the recent introduction of effective target therapies (BRAF and MEK inhibitors for BRAFV600-mutated MM) and immunotherapies (anti-CTLA-4 and anti-PD-1) has significantly improved the survival of MM patients. Notably, all these responses are highly dependent on the fitness of the host immune system, including the innate compartment. Among immune cells involved in cancer immunity, properly activated plasmacytoid dendritic cells (pDCs) exert an important role, bridging the innate and adaptive immune responses and directly eliminating cancer cells. A distinctive feature of pDCs is the production of high amount of type I Interferon (I-IFN), through the Toll-like receptor (TLR) 7 and 9 signaling pathway activation. However, published data indicate that melanoma-associated escape mechanisms are in place to hijack pDC functions. We have recently reported that pDC recruitment is recurrent in the early phases of melanoma, but the entire pDC compartment collapses over melanoma progression. Here, we summarize recent advances on pDC biology and function within the context of melanoma immunity. The role of plasmacytoid dendritic cells (pDCs) in human pathology has been largely explored, mainly in autoimmune diseases . Tumor-aPlasmacytoid dendritic cells have been described, for the first time, by Karl Lennert  and subs− CD123+ CD303+ dendritic cells [+CD80− cells retain a plasmacytoid morphology and are specialized in I-IFN production; ii) PD-L1-CD80+ cells adopt a dendritic morphology and promote T cell activation with Th2 polarization; iii) PD-L1+CD80+ double positive pDCs have both innate and adaptive functions and an intermediate morphology [+ and CXCL10− subsets are defined by a distinct transcriptional program. This finding likely substantiates a diverse contribution of anti-viral responses and interferon-dependent inflammation [Circulating pDCs are a rare subset, corresponding to 0.2–0.8% of the total peripheral blood mononuclear cells (PBMCs). pDCs lack expression of the lineage markers specific for B cells, T cells, natural killer cells and myelo-monocytic cells. Human pDCs result negative for the myeloid dendritic cell (mDC) marker CD11c, as well. They can be identified based on their selective expression of surface antigens, such as the blood DC antigen 2  and the leukocyte immunoglobulin-like receptor subfamily A member 4  . Human pic cells . Human pic cells ,25,26. Rrphology . Further+ HSPCs into the three major subsets of human DCs, combining Flt3L with mouse bone marrow stromal cells (MS5) [The initial development of pDCs takes place in the bone marrow, from hematopoietic stem progenitor cells (HSPCs), and requires Fms-like tyrosine kinase 3 ligand (Flt3L) ,28,29. Tls (MS5) . Using tAlthough many in vivo studies have focused on the pDC development within the myeloid lineage, pDCs can also be obtained from lymphoid progenitors. Recently, it has been demonstrated that human pDCs could arise from multipotent lymphoid progenitors (MLPs) and, even more, MLPs show better potential for pDC generation, compared to common myeloid progenitors (CMPs) . InteresThe molecular basis of tissue migration of pDCs have been largely studied and reviewed elsewhere . BrieflypDCs represent a unique cell population within the immune system bridging the innate and adaptive immune responses , with doThe sensing of viruses or self-nucleic acids by pDCs is mainly mediated by endosomal Toll-like Receptor (TLR) 7 and 9 that rapidly induce the secretion of high levels of I-IFN, especially IFN-α ,49. It hThe TLR7 recognizes ssRNA viruses, endogenous RNA and synthetic oligoribonucleotides or imidazoquinoline compounds. Whereas, TLR9 detects DNA viruses containing unmethylated CpG-rich DNA sequences, endogenous DNA and synthetic CpG DNA. Viruses and endogenous nucleic acids reach the endosomal compartments in pDCs through autophagy or Fc receptors-mediated endocytosis as complexes with antibodies ,55,56. AHuman pDCs express a set of surface activating (FcγIIa and CD300a/c) and inhibitory  receptors that fine-tune the amplitude of I-IFN response and pDC activation state in response to TLR7/9 ligands. The counter regulation of TLR signaling is essential to prevent ongoing cytokine production implicated in autoimmune diseases. BDCA-2 cross-linking triggers the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway suppressing the ability of pDCs to produce I-IFN in response to TLR7/9 ligands ,64,65. TNKp44 is expressed on pDCs in tonsils and is induced on blood pDCs after in vitro culture with IL-3. NKp44 associates with DAP12 and NKp44 cross-linking inhibits I-IFN production by CpG DNA-activated pDCs . NKp44 rAlthough TLRs are the main innate receptors, involved in pDC activation, other cytoplasmic nucleic acid sensors are expressed by pDCs. RIG-I is expressed at low level in pDCs under steady-state conditions, but its expression can be greatly enhanced by endosomal TLR stimulation in I-IFN-independent manner . RIG-I aHuman type III IFNs include four subtypes: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B) and the more recently discovered IFN-λ4 ,81,82. IThe expression of functional receptor, IFNLR, is restricted to epithelial cells and leukocyte subsets, including pDCs . IFN-λ t-, a small subset of circulating pDC-like cells has been found to express CD56, a marker specific for NK cells and associated with cytolytic effector function [In addition to their central role in interferon production, pDCs may exert direct effector functions. In particular, the activation of pDCs via TLR7/9 stimulation induces the expression of TNF-related apoptosis-inducing ligand (TRAIL) ,92, whicfunction ,99,100.+ T cells [+ T cells [+ T cells [The major function of DCs is to capture antigens in the peripheral tissues and transport them to lymph nodes, where they are presented to T cells for efficient initiation of T cell-dependent immune responses . pDCs di T cells ,102 and  T cells ,104,105  T cells ,107. Est T cells ,72,108.  T cells ,64,72. UUnstimulated or alternatively activated pDCs are able to induce T cell tolerance to tumor cells, harmless antigens and alloantigens, primarily through the induction of regulatory T cells (Tregs) ,111,112.A better understanding of the role of pDCs in cancer has been limited by the absence of appropriate murine models lacking this cell type. It is well-recognized that I-IFN is a critical component of the cancer immunoediting process in mice . The endThe first attempts to deplete pDCs by using antibodies  have been reviewed by Swiecki and Colonna . These aA transgenic mouse that expresses the diphtheria toxin receptor (DTR) under the control of pDC-specific gene promoter   has beenIn conclusion, the role of pDCs in cancer immunoediting is still poorly investigated, mainly due to the lack of appropriate model system for stable pDC manipulation.Tumor associated-pDCs (TA-pDCs) have been detected in a wide variety of human neoplasms such as primary carcinomas from different primary sites , cutaneoThe recruitment of pDCs to tumor tissues is regulated by CKs secreted by neoplastic cells ,46,130. The pDC recruitment has been documented in human primary breast cancer , and theData on the frequency of circulating pDCs have been reported from cohorts of different cancer types. In general, although with some level of heterogeneity, circulating pDCs are decreased in human cancer patients compared to healthy donors, particularly in advanced stage diseases, with a single relevant exception in a study of non-small-cell lung cancer (NSCLC) . SpecifiThe clinical significance of the density of the tumour-infiltrating pDCs and the frequency of circulating pDCs in human cancer has been analyzed in few studies. An increased pDC infiltration has been associated with poor outcome in breast cancer, ovarian cancer, NSCLC, OSCC and melanoma ,152,153.+ pDCs has been reported in breast cancers, ovarian cancers, melanoma and liver tumors [+ Tregs are strong predictors of disease progression and early relapse in ovarian cancer [Finally, the induction of Tregs by ICOSLr tumors ,156,157.n cancer ,138,151.Properly activated pDCs are endowed with anti-tumor activity by inducing the apoptosis of neoplastic cells and secreting type I and type III IFN. However, the tumor microenvironment can subvert the anti-tumor function of pDCs. Tumor cells produce immunosuppressive cytokines , recruit Tregs, activate negative regulatory pathways  or express immunomodulatory molecules , thus establishing an immunosuppressive microenvironment. Furthermore, TA-pDCs themselves can induce immune tolerance in many types of cancers, supporting the immunosuppressive milieu. For instance, TA-pDCs are mostly defective in their effector functions  and immaSeveral studies have demonstrated that TA-pDCs display an immature phenotype, as shown by the lack of expression of the maturation marker CD83 and the co-stimulatory molecules CD80 and CD86 ,158,159.pDCs isolated from HNSCC tissues or exposed to HNSCC supernatants show an impaired IFN-α production upon CpG stimulation compared to blood-derived pDCs or pDCs exposed to control medium ,140,173.+ T cells and ICOSL-expressing TA-pDCs sustain the expansion of ICOS+ FoxP3+ Tregs in the tumor microenvironment and promote the secretion of IL-10 and TGF-β from Tregs, which support an immunosuppressive microenvironment and tumor progression [+ Tregs recruitment in metastatic TDLNs of CRC patients, further supporting that pDCs might contribute to Tregs development in the tumor milieu [+ Tregs [Several studies have demonstrated that TA-pDCs might exert a tolerogenic function. For instance, pDCs induce the expansion and suppressive function of Tregs through the ICOS/ICOSL pathway or IDO expression  executingression ,176,177.r milieu . It shou[+ Tregs ,180,181.+ and CD8+ T cell proliferation [Besides the induction of Tregs, pDCs might fulfill tumor-promoting functions through additional mechanisms. For instance, in ovarian cancer, pDCs modulate neo-angiogenesis via TNF-α and IL-8 production upon CD40L activation . Recentlferation ,119; on feration .+ pDC infiltration is observed in basal cell carcinoma lesions treated with the topical TLR7 agonist IMQ, suggesting that TLR-activated pDCs contribute to rejection of cutaneous tumors [+ cytotoxic T cells [In contrast to their pro-tumor functions, pDCs are endowed with an anti-tumorigenic capacity. Primarily, IFN-α production upon TLR-activation has direct and indirect effects on cancer cells and induce tumor regression, as shown in mouse model of orthotopic mammary tumor ,184,185,s tumors ,188. Fin T cells . NotablyCutaneous Melanoma (CM) is a neoplasm originated from skin melanocytes that progressively develop a malignant phenotype . Several+ and CD8+ T cell reactivity can be detected within tumor-infiltrating lymphocytes.Across human cancer types, CM is characterized by the highest prevalence of somatic mutations associated to UV signature ,195,196.Melanoma is considered an immunogenic “hot” tumor and its immune-mediated spontaneous regression has been documented . A largeV600E PCM and distant metastasis exhibited an increased pDC numbers compared with BRAF-negative [+ and NF-1+ PCM, particularly when compared with the BRAF+ subgroup, whereas no differences were observed in CD8+ T-cell infiltration between TCGA subgroups [+ T cell response. Analysis of the melanoma TCGA data indicates a strong correlation between CD8 gene transcripts and DC markers suggesting that lack of T-cell infiltration and activation is associated to failed recruitment and activation of Batf3 DCs [Contexture of the innate immune components is emerging as relevant in shaping the adaptive immune responses in PCM subtypes. Notably, BRAFnegative . Moreoveubgroups ,209. Furatf3 DCs .+ tumor-infiltrating lymphocytes expressing PDCD1 (encoding PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) correlate with response to checkpoint blockade therapy and survival in melanoma patients [By applying CYBERSORT method , tumor-ipatients .Remarkably, also melanomas responding to lymphocyte adoptive transfer or to checkpoint blockade display an increased mutational load and T cells specific for neoantigens .The therapeutic scenario of patients with inoperable or metastatic melanoma (MM) has been dramatically improved by the development of new treatment strategies. Before this modern era, historical dacarbazine was associated with a limited impact on patients’ prognosis and the median overall survival (OS) was about 6-9 months . The intApproximately one-half of melanoma harbors a BRAF mutation, mostly at codon 600: this molecular activation occurs early during melanoma evolution, driving both cancer growth and dissemination ,218. As Advances in the tumor immunology field has led to the development of inhibitors of immune-checkpoint molecules such as CTLA-4 and programmed cell death 1 (PD-1) . Cancer Lately, the role of combination of checkpoint inhibitors has also been explored, revealing a synergic effect in term of survival and response. The phase III randomized trial checkmate 067 confirmed a statistically significant benefit of anti-PD-1 and anti-CTLA-4 (nivolumab plus ipilimumab) combination in comparison to ipilimumab and nivolumab alone ,236; speFollowing these important advances in the treatment of MM, a new adjuvant therapy setting has emerged for high-risk stage III patients . Target Future challenges might involve the role of neo-adjuvant approach . Both imThe oncogenic BRAF can lead to immunomodulation and immune escape in melanoma, by i) the secretion of immunosuppressive cytokines in the microenvironment; ii) eliciting immune suppressive phenotype of the immune cells in the microenvironment; iii) modulating the MHC level in tumor cells .V600E mutant melanoma cells produce immunosuppressive factors such as IL-10, IL-6 or VEGF [V600E melanoma cells release IL-1 that up-regulates the transcription of several genes such as the co-inhibitory molecules PD-L1 and PD-L2 [The constitutive activation of MAPK pathway along with STAT3 pathway plays an essential role inducing the immune evasion by melanoma cells . BRAFV60 or VEGF ,247. Thend PD-L2 .V600E mutant proteins is related to the down-regulation of MHC-I molecules, via a rapid and constitutive internalization of MHC-I from the melanoma cell surface and subsequent sequestration within the endocytic compartments, thus diminishing CD8+ T-cell recognition of tumor cells and facilitating tumor evasion [+ T-cell recognition and effector function [Another immunosuppressive effect of BRAF evasion . Inhibitfunction .Growing evidence suggest that for their therapeutic efficacy BRAFi and MEKi rely on factors that affect tumor-host interaction including the enhancement of melanoma antigen expression and the increase in immune response against tumor cells ,251. TheClinicaltrials.gov study identifiers: NCT01400451, NCT01656642, NCT01767454, NCT02027961, NCT02130466). The scientific rationale for such strategies is based on the interplay between the MAPK pathway and the immune response in the tumor microenvironment. Pre-clinical and clinical observations indicate that inhibition of the MAPK pathway may have a favorable effect on the melanoma-specific immune response also at the level of T cells, DCs and tumor cells. The inhibition of BRAF and MEK leads to up-regulation of melanoma differentiation antigens (MDA), such as MART-1, gp-100 and tyrosinase, [Giving the high response rates of kinase inhibition and the response durability of immune checkpoint blockade, combinatorial approaches could be promising, boosting the immune responses and overcoming immune mediated mechanisms of resistance. These combinations have been tested in both preclinical models ,255 and osinase,  and MHC osinase,  along wiosinase, . Notablyosinase, ,256,257.osinase,  likely o+ T-cells [Recent observations indicate that MEKi could negatively affect the human immune repertoire. In details, DC viability, cytokines secretion, expression of co-stimulatory molecules  and MHC-I are impaired by direct exposure of these cells to MEKi . In addi T-cells . On post T-cells . All theIn conclusion, MAPK inhibition might overcome the immune evasion of melanoma cells, enhancing DC function by several mechanisms.Different studies have provided evidence that pDCs are part of the PCM immune contexture. While pDCs are almost absent in normal skin and melanocytic nevi, they are identified in PCM, predominantly located in the peritumoral areas admixed to lymphoid cells; more rarely, pDCs are found within the tumor nests in close association to melanoma cells ,261. A p+ hematopoietic progenitor cells (HPC) and of terminally differentiated pDCs to soluble melanoma components reduces the generation of terminally differentiated pDCs and induces pDC apoptosis [In line with the previous reports ,261, we poptosis . These fIt has been recently reported that the occurrence of BRAF mutation is associated to an increased pDC infiltration compared with BRAF-wild type melanomas . Our fin+ and GrB+ lymphocytes [A small set of retrospective analyses has provided data on the prognostic significance of circulating and melanoma-infiltrating pDCs ,265,266.phocytes .By analyzing the levels of circulating pDCs in melanoma patients at different disease stages, a lower frequency of pDCs has been measured in patients with systemic spread (stage IV AJCC) compared to loco-regional disease. This decrease has been detected also in patients with active disease at the time of inclusion . A decli+ fraction a higher proportion of pDCs infiltrates cutaneous tumors compared with LN metastasis. In PCM, pDCs were positively correlated with the Breslow index and significantly associated with a poor clinical outcome [+ pDCs and Th2 T cells compared with patients at an early stage of the disease [Aspord et al. found that within the CD45 outcome . On the  disease .The differences observed in the clinical significance of the pDCs compartment found between studies are reported in As aforementioned, proper pDC activation induces anti-tumor immunity, whereas pDCs conditioning by the tumor microenvironment mediate immune suppression. By using the human pDC cell line GEN2.2, it has been demonstrated that TLR7 and TLR9 stimulation can induce a TRAIL-mediated cytotoxic activity in pDCs that could participate to the tumor cell clearance . In a mu+ pDCs are enriched at the melanoma sites and polarize toward an immune-suppressive environment [The function of pDCs obtained from melanoma patients has been rarely investigated and the large majority of functional studies on PCM-infiltrating pDCs adopted surrogates of the main pDC functions. The expression of the IFN-α inducible protein MxA in PCM is very limited in the majority of the cases  and the ironment . In summAlthough pDCs represent a minor population among circulating immune cells and in the tumor microenvironment, they represent a promising target for cancer immunotherapy , based oClinicaltrials.gov study identifier: NCT02339324), to overcome PD-L1-mediated escape [The administration of recombinant IFN-α was an approved cancer immunotherapeutic approach, as monotherapy or combinational therapy . Howeverd escape . Of noted escape .Clinicaltrials.gov study identifiers: NCT02644967, NCT03445533, NCT03052205, NCT03084640, NCT03618641, NCT02680184, NCT03831295 and NCT02521870), even for patients with MM refractory to PD-1 blockade [It has been proposed that pDC functions are hijacked by the tumor microenvironment, but upon appropriate re-activation, they can be reprogrammed to anti-tumor functions. One of the possible approaches is the therapeutic activation of tumor-associated pDCs using TLR7 and TLR9 agonists. These compounds have shown some level of clinical benefit in antitumor immunity, through the production of I-IFN and cytotoxic molecules by pDCs ,277,278.blockade . Antitumblockade . A phaseblockade . Clinicablockade .Imiquimod (IMQ) is a synthetic TLR7 agonist approved by the FDA for the treatment of non-melanoma skin cancers . Aspord et al. investigated the IMQ effects using an innovative melanoma-bearing humanized mouse model. IMQ strongly inhibits melanoma tumor growth by i) prompt mobilization of pDCs; ii) triggering the pDCs cytotoxic functions and iii) up-regulation of expression of I-IFN-inducible genes . In humaAntigen-pulsed activated pDCs generate an antigen-specific CTL response against melanoma , encouraIn conclusion, properly activated pDCs could represent an addition on the existing and prospective treatments for melanomas.


Atherosclerosis and its complications, including acute coronary syndromes, are the major cause of death worldwide. The two most important pathophysiological mechanisms underlying atherosclerosis include increased platelet activation and increased low-density lipoproteins (LDL) concentration. In contrast to LDL, oxidized (ox)-LDL have direct pro-thrombotic properties by functional interactions with platelets, leading to platelet activation and favoring thrombus formation. In this review, we summarize the currently available evidence on the interactions between LDL-cholesterol and platelets, which are based on (i) the presence of ox-LDL-binding sites on platelets, (ii) generation of ox-LDL by platelets and (iii) the role of activated platelets and ox-LDL in atherosclerosis. In addition, we elaborate on the clinical implications of these interactions, including development of the new therapeutic possibilities. The ability to understand and modulate mechanisms governing interactions between LDL-cholesterol and platelets may offer new treatment strategies for atherosclerosis prevention. Atherosclerosis and its complications, including acute coronary syndromes, are the major cause of death worldwide . New insOx-LDL are defined as “circulating LDL-derived particle which have peroxides or their degradation products associated with the LDL particle” . In contOx-LDL activate platelets via interaction with two types of platelet receptors: cluster of differentiation (CD) 36 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) .CD36 belongs to the class B scavenger receptors (SR) family . SR compIIbβ3 (receptor for fibrinogen). Hence, ox-LDL binding to CD36 enables the formation of platelet-leukocyte complexes via P-selectin and cross-linking of the adjacent platelets via fibrinogen [CD36, an 88-kDa transmembrane glycoprotein receptor, is constitutively expressed on various cell types, including platelets . Expressbrinogen . Furtherbrinogen ,23.The interaction between platelets and ox-LDL via CD36 is reciprocal. Not only ox-LDL activate platelets, but platelets bound to ox-LDL exert pro-atherogenic effects, including the release of atherosclerosis-promoting chemokines such as monocyte chemotactic protein-1 and interleukin 1β precursor ,21. FurtLOX-1 is a 50 kDa type 2 transmembrane glycoprotein that mediates the uptake of ox-LDL by endothelial cells and platelets ,25. UnliIIbβ3 and α2β1, which are receptors for fibrinogen and collagen, respectively, crucial in thrombus formation [LOX-1 effect on platelets resembles the effect of the well-established platelet activator ADP, which decreases the intracellular concentration of cyclic adenosine monophosphate (cAMP) . Bindingormation . The proormation . Anti-LOormation . Furtherormation . Finallyormation . Altogetormation .Reactive oxygen species (ROS) are highly reactive molecules, generated in response to both endogenous and exogenous stimuli, which promotes protein, lipid and DNA damage. Increased local and systemic levels of ROS induce endothelial cell activation, dysfunction, apoptosis and impaired vasorelaxation, and contribute to atherosclerosis development and progression . Traditi2O2) [2O2 induces platelet aggregation mediated by fibrinogen binding to integrin αIIbβ3, which is independent of ADP secreted from platelet dense granules [2O2 also enhances aggregation induced by phospholipid-derived arachidonate [ROS are physiologically produced by a cell membrane enzymatic complex, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and by mitochondria in the electron transport chain . The piv2O2) . H2O2 ingranules . H2O2 alhidonate . Altogethidonate .NOX isoforms are the main sources of ROS in platelets, followed by cyclooxygenase (COX), xanthine oxidase (XO) and mitochondrial respiration . In humaResting platelets take up ox-LDL at different concentrations . When plWhereas native LDL does not affect platelet activation , bindingFollowing ox-LDL intake and adherence to collagen, platelets lead to immune cells accumulation at the sites vascular injury . MoreoveAltogether, following activation by ox-LDL, platelets seem to have increased prothrombotic, pro-inflammatory and proatherogenic properties. However, some controversies regarding the effect of different ox-LDL phenotypes on platelets remain to be elucidated.The atherosclerotic process can either be clinically silent, or result in plaque expansion and vessel constriction, causing anginal symptoms. More dangerously, plaques may rupture, leading to a clinically manifest acute coronary syndrome, stroke or acute limb ischemia, depending on their location. There are various factors that determine plaque stability, including platelets function and their interaction with LDL.Platelets are the first circulating blood cells that adhere to the sites of vascular injury . Beyond Both activated platelets and ox-LDL exert proinflammatory and procoagulant effects on several vascular cells , thus supporting that atherosclerosis is a chronic inflammatory disease ,49.Activated platelets interact with endothelium and change the chemotactic and adhesive properties of endothelial cells, a critical initial step in atherogenesis . In the + progenitor cells transformation into macrophages or foam cells, depending on the microenvironment [Foam cell generation from macrophages with subsequent fatty streak formation plays a key role in the early atherogenesis . Ox-LDL-ironment . A two-fironment . In factironment .The role of activated platelets and ox-LDL in atherosclerosis development and progression is presented in The mechanisms underlying platelet activation in atherosclerosis are still incompletely understood. Platelet-derived extracellular vesicles (PEVs) disseminate the procoagulant and proinflammatory effects of activated platelets, thus contributing to platelet interactions with other cells and with ox-LDL particles .EVs are a diverse group of membrane-enclosed structures, released from cells to bloodstream in physiological and pathological conditions . EVs actPEVs are secreted during platelets activation and share many functional features with activated platelets, this having a crucial role in platelet aggregation and thrombus formation . For exaSimilarly to platelets, PEVs may carry the CD36 receptor for LDL, resulting in generation of a PEV–LDL complexes . It was Altogether, the release of PEVs during platelet activation may not only have direct, but also indirect prothrombotic effect via interactions with ox-LDL, and both antiplatelet and lipid-lowering therapies may modulate PEV concentrations.In patients with hypercholesterolemia, the abnormalities in platelet composition and function indicate that circulating lipoproteins in blood influence platelet properties ,66. HypeNOX-2 dependent production of ROS is increased in platelets in presence of ox-LDL. ROS play an important role in platelet activation process. Inhibition of NOX2-derived ROS may represent a novel pathway to inhibit platelet aggregation and eventually thrombus growth . At the LOX-1 play an important role in ADP-induced activation of fibrinogen receptors, contributing to platelet aggregation and thrombus formation . There aCalcium channel blockers (CCB) and angiotensin II receptor blockers (ARB) decrease the progression of atherosclerosis and decrease the incidence of cardiovascular events . CCB witγ (PPAR-γ) agonists used in patients with diabetes mellitus were demonstrated to decrease LOX-1 expression independent of their hypoglycemic or insulin-sensitizing actions [In chronic hyperglycemia, glucose reacts with proteins to form AGE, which on one hand are ligands for LOX-1, and on the other hand upregulate LOX-1 . Sulfony actions , thus deSodium-glucose co-transporter-2 (SGLT2) inhibitors have recently emerged as a breakthrough therapy in diabetic patients due to substantial improvements in overall and cardiovascular outcomes . SGLT2 i+ progenitor cells [Anti-inflammatory pleiotropic effects of statins are increasingly recognized as being responsible for the improved outcomes in atherosclerotic patients, including patients after myocardial infarction, stroke and those with peripheral artery disease . Statinsor cells , and  kinase from mitogen-activated protein kinase (MAPK) kinase family . ERK5 waThe PEV–CD36 complex also activates MAPK signals and contributes to platelet activation. In addition, PEVs aggravate oxidative stress in a CD36- and phosphatidylserine-dependent manner. It was observed that PEV concentrations are reduced in patients treated with P2Y12 antagonists, statins and eicosapentaenoic acid ,83. HencHigh concentration of ox-LDL in vascular wall may be associated with higher risk of future plaque rupture, potentially making ox-LDL an important marker of the ‘vulnerable plaque’, as well as offering opportunities for diagnostic imaging and the targeted delivery of therapeutic agents . LDL conPlatelets and ox-LDL interactions play a key role in atherosclerosis development and progression due to (i) the presence of LDL-binding sites on platelets, (ii) generation of ox-LDL by platelets and (iii) targeting platelet by ox-LDL. The ability to understand and modulate mechanisms governing these interactions may offer new treatment strategies for atherosclerosis prevention. At the moment, the interactions between ox-LDL and platelets may be alleviated using three major groups of drugs: antihypertensive agents, antihyperglycemic agents and statins. However, since CD36 and LOX-1 are molecules in atherosclerosis development and progression, their pharmacological inhibition would be an exciting and promising avenue in developing therapeutic agents to alleviate or even reverse the atherosclerotic plaque formation. Modulation of proinflammatory and prothrombotic properties of PEVs-ox-LDL interactions are another potential future therapeutic option. Further research on the interactions between platelets and ox-LDL is urgently needed to optimize primary and secondary prevention of atherosclerosis.


Combining diagnosis with therapy, magnetic iron oxide nanoparticles (INOPs) act as an important vehicle for drug delivery. However, poor biocompatibility of INOPs limits their application. To improve the shortcomings, various surface modifications have been developed, including small molecules coatings, polymers coatings, lipid coatings and lipopolymer coatings. These surface modifications facilitate iron nanoparticles to cross the blood-brain-barrier, which is essential for diagnosis and treatments of brain diseases. Here we focus on the characteristics of different coated INOPs and their application in brain disease, particularly gliomas, Alzheimer’s disease (AD) and Parkinson’s disease (PD). Moreover, we summarize the current progress and expect to provide help for future researches. There have been two generations of iron contrast agents (CAs) for MRI, the first one has diagnostic capability only while the new one, combining diagnosis with therapy, has multiple functions. The new one loaded with therapeutic agents after surface coating to facilitate MRI guided drug delivery, gene delivery, photothermal therapy (PTT), photodynamic therapy (PDT) or magnetic hyperthermia has gained attention . H. H11]. HBrain diseases are hard to treat because of BBB, and many iron nanoparticles are designed to overcome this difficulty ,48,49. OTo change the surface charges and hydrophobicity it is possible to increase biocompatibility, endow INOPs with the ability to target and release on demand . Differe− on the surface of INOPs provides a chance for the attachment of small molecules and surfactants, which can maintain a small hydrodynamic radius, retain the original magnetic properties and improve hydrophilicity at the same time [The presence of numerous OHame time . The ligame time  have str3 or –CH2–CH3) with hydroxyl group of INOPs [2, Stöber method, microemulsion, aerosol pyrolysis, and methods based on sodium silicate solution [2, –SH and –COOH groups to naked iron oxide NPs, respectively [3O4 while MPTES modification resulted in a slight decrease in saturation magnetization [Silicon is one of the most widely studied small molecule coatings. Silane can be covalently bound to the surface of INOPs using the reaction of alkoxysilane functions  was used as MRI probe for glioma. Its positive charge was favorable for BBB crossing, increasing the accumulation in the tumor region [The result of thermotherapy using dextran and amino-silane coated INOPs revealed that amino-silane coated INOPs prolonged the survival time by 4.5-fold over dextran-INOPs in malignant glioma rat . PEG witr region . Gold anr region .2O3 nanoparticles by bidentate chelating interaction [Inspired by natural phenomena, catechol-derived ligands for INOPs have been developed. Dopamine and its modifications are important catechol. Rajh et al. believed dopamine assembled on Feeraction . In weakeraction . Moreove2) relaxivities of nanoparticles (101.9 mM−1s−1) was slightly lower than that of FDA approved dextran-coated Feridex (111.5 mM−1s−1). Nanoparticles exhibited a higher cellular uptake, leading to higher cytotoxicity [Caffeic acid , with positive biological effects such as anti-oxidant, anti-inflammatory, anti-HIV, anti-tumor, and anti-metastatic effects was used as coating of INOPs due to its strong affinity for INOPs, which could make anti-oxidant drugs keep for at least 45 min in the brain . It playtoxicity .INOPs coating can be obtained through lots of approaches, such as in situ coating, ligand exchange, post-synthesis adsorption, post-synthesis end grafting , and so To combine the advantages of different polymers, copolymers are developed. For example, copolymers of PEG and PEI have both the ability to load gene and prolong half-life in blood . CopolymDextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) were described in 1982 by in-situ technology and have been approved by Food and Drug Administration (FDA) . CarboxyDextran and its modifications are used in the study of cerebral disease. Stem cell therapeutics for PD focus on the differentiation of mesenchymal stem cells (MSCs) to dopaminergic (DA) producing cells, replacing damaged DA neurons in PD. Dextran-coated or dextran-derivatives-coated iron oxide (IO) nanoparticles (NPs) (Dex-IO NPs) can activate the tropism of human MSCs (hMSCs) from bone marrow toward tumor . A combi3O4 was developed, improving the problem of chitosan’s bad solubility [Chitosan (CS) is another important polysaccharides coating, providing positive charge for cell adsorption and opening tight junctions between epithelial cells, thus facilitating transportation . Stericalubility . Trimethlubility . This malubility , indicatA combinatorial nano-system was made to treat glioblastoma multiforme (GBM). With tumor-specific ligand-transferrin (Tf) as target, CS-INOPs containing antitumor drug doxorubicin (DOX) for cancer cell killing and fluorescent dye Rhodamine B isothiocyanate (RBITC) for simultaneously intracellular fluorescent showed improved cell uptake and cell killing through a concurrence of cell apoptosis and autophagy in the treated tumor U251 cells. The saturation magnetization of INOPs was estimated to be 56.06 emu/g while CS/INOPs and Tf-CS/INOPs were 35.34 and 20.94 emu/g respectively, demonstrating that the saturation magnetization decreased after coating. The apoptosis of U251 MG cells treated with Tf-CS/INOPs were about 70% and the cell viability came down to about 55% . AnotherPEG, as a amphiphilic molecule at the size lower than 100 kD , is synt3O4 nanoparticles via a bifunctional PEG with the end of silane and trifluoroethylester (TFEE). Intracellular uptake of INOPs-PEG-MTX was higher than NP-dextran in a concentration-dependent manner in glioma [50 of INOPs-Dox in C6-ADR glioma was approximately 3- to 5-times lower than that of free DOX in cells, demonstrating that preventing drug efflux from drug-resistant cells caused enhanced cell death [A MGMT inhibitors O6-benzyl guanosine (BGS) covalently attached to PEG-INOPs in a pH-response manner to improve cell sensitivity to TMZ. The combination reduced the cell resistance by approximately 40-fold while BGS reduced resistance by 19-fold, demonstrating BGS-PEG-INOPs’ greater sensitivity to TMZ in human GBM cells . Methotrn glioma . Moreovell death . PEI-PEGll death .PEI with highly positive charge is used for gene delivery ,118. It PEI-INOP was made to determine its applicability in gene therapy of brain cancer. The result exhibited no difference in cell toxicity compared with slightly anionic G100 but lower blood concentration . After fPCB has both cationic and anionic groups, endowing it with the charge-reversible ability in different pH. It is neutral in physiological condition, resisting nonspecific protein adsorption, which changes to positive charge via protonation in acidic environment, helping escape from lysosome . These cSimvastatin covalently connected ROS-response PCB (Sim-se-se-PCB) was constructed to load hydrophobic SPIONs and absorb negative let-7b antisense oligonucleotide, reducing the differentiation of exogenous neural stem cells (NSCs) and enhancing the secretion of brain-derived neurotrophic factor (BDNF) synergistically. The transfection efficiency of PCB-Se-Se-Sim/let-7b NPs was comparable to that of Lipofectamine 2000 and significantly (170.8%) increased the BDNF level in the culture medium, which further induced the proliferation of NSCs and rescued the memory deficits in 2× Tg-AD mice .Based on PCB, other copolymers were designed to form INOPs-loaded micelles. PCB-poly (2-hydroxyethyl methacrylate)-poly (carboxybetaine) (PHEMA-PCB) polymers were constructed to coat INOPs and delivery retinoic acid (RA) and siRNA (SOX-9), reducing the inhibition the effect of retinoic acid. NPs caused knock-down of approximately 52.3% of SOX-9 mRNA compared with negative controls and significantly improved the learning and memory of AD mice  Figure .PLGA is a copolymer of poly lactic acid (PLA) with poly glycolic acid (PGA). It is highly biocompatible and has been approved by FDA as drug delivery vehicles . VariousPolymers provide a promising future in the application of INOPs in cerebral disease after targeting and drugs loading. However, the coating materials may cause low magnetic properties. And the blood concentration of NPs is low, which may cause disassembly of micelle before reaching the target.Lipid, as the constituent of cellular membranes, provides a biocompatible protective barrier for nanoparticles. It can form a closed double layer structure in aqueous solution containing hydrophilic and hydrophobic space. There are two embedding methods for iron nanoparticles, in the phospholipid bilayer or in the core . Researc+Cu2+ cells improved to 91% from 63% and had a greater contrast-to-noise ratio of MRI. NPs could significantly rescue memory deficits in AD transgenic mice [Numerous studies have been done on the brain application of DSPE-PEG based iron liposome. DSPE-PEG-B6, high affinity for transferrin receptor (TfR), DSPE-PEG-Mazindol (MA), a high affinity dopamine transporter, DSPE-PEG-phenylboronic acid, loading epigallocatechin gallate (EGCG), and INOPs were self-assembled to treat PD. With the help of MA, NPs were internalized into dopaminergic neurons after crossing BBB with help of B6. In the ROS environment of dopaminergic neurons, EGCG played the role of anti α-synclein(αS) aggregation and promoting neurotransmitter circulation. INOPs functioned as MR imaging. NPs reduced αS aggregation compared to cells treated with only EGCG and obtained good therapeutic efficacy in PD . Testingnic mice . Differenic mice .Lipid coating provides more biocompatible modification for INOPs. Besides, it can encapsulate lots of INOPs and deliver them to the target site, avoiding dilution like micelle, resulting in a high concentration of drugs for therapy and INOPs for imaging.INOPs in the core of liposomes need to be hydrophilic. Polymers are one of the methods to change INOPs from hydrophilicity to hydrophobicity, which provide a chance for the combination of lipid and polymers. Moreover, different from supported lipid bilayers (SLB), the inner and the outer leaflet being composed of the same molecules, hybrid lipid bilayer (HLB) is composed of different molecules where the composition of the two leaflets are not identical and only the outer leaflet actually contains lipid molecules, adapting a lipid-bilayer-like arrangement . The comThree drugs, Dox, combretastatin A4 (CA4), and all-trans retinoic acid (ATRA) were combined through pH-sensitive poly (β-amino ester) (PAE), hypoxic-response azobenzene (AZO) and hydrophobic interaction together with IONPs delivered in the core of DSPE-PEG and PAE constituted micelle to release in order. CA4 was released in GBM acid environment to destroy the angiogenesis after B6 mediated BBB crossing. Then ATRA and Dox played their roles of enhancing GSCs differentiating to glioblastomas and killing them after PAE-mediated lysosomal escape. NPs extended the circulation time of the drugs and maintained survival rate at 60% after treating compared with dying in other groups . After tOther materials are also used to improve iron nanoparticles performance . Au coat3O4 was coated with positively charged p53 plasmid-loaded poly (2-dimethyl amino) ethyl methacrylate (PDM)-coated Au through consecutive electrostatic assembly. The C6 glioma cell viability decreased to 24.1% by combining PTT with gene therapy in vitro and produced an effective inhibition of high-proliferation-rate C6 glioma in vivo [The negatively charged polydopamine (PDA) coated Fe in vivo   encapsulate multiple therapy molecules, like gene, protein, chemical drugs to achieve multi-drug cooperation therapy; (2) deliver drugs to target sites to increase drug concentration in disease site; (3) release loaded drugs according to various stimuli conditions of disease site. There have been FDA-approved INOPs ,150. Forn adults , which sINOPs approved by FDA are in the first generation-imaging. Later, NanoTherm was approved to treat recurrent glioblastoma multiforme in 2010 . There a


The COVID-19 pandemic has led to environmental recovery in some ecosystems from a global “anthropause,” yet such evidence for natural resources with extraction or production value  is limited. This brief report provides a data-driven global snapshot of expert-perceived impacts of COVID-19 on inland fisheries. We distributed an online survey assessing perceptions of inland fishery pressures in June and July 2020 to basin-level inland fishery experts ; 437 respondents from 79 countries addressed 93 unique hydrological basins, accounting for 82.1% of global inland fish catch. Based on the responses analyzed against extrinsic fish catch and human development index data, pandemic impacts on inland fisheries 1) add gradation to the largely positive environmental narrative of the global pandemic and 2) identify that basins of higher provisioning value are perceived to experience greater fishery pressures but may have limited compensatory capacity to mitigate COVID-19 impacts along with negative pressures already present. Inland fisheries are important global contributors to food security, livelihoods, and ecosystem services  while suWe received 437 survey responses from 79 countries and 93 hydrological basins [HydroBASINS Level-3 ]. These Clear clusters of like responses emerged . Notablyn = 232, 50 basins), prominent patterns emerged: perceived increased pressure in areas of relatively lower catch and lower HDI (46.0%); decreased pressure in higher-catch, lower-HDI areas (38%); no change in higher-catch, higher-HDI areas (48%); and evenly distributed pressure responses (±4%) in lower-catch, higher-HDI areas  preexisting, persistent, or confounded environmental stressors; 2) fishery composition , type, and access; 3) consumer behavior; 4) fishery management/enforcement; and 5) regional employment opportunities. Possible sources of respondent variation include 1) virus and lockdown onset, severity, and extent; 2) institutional adaptive capacity; and 3) temporal interpretation of pressures.Respondents indicated decreased fishery pressure may arise from reduced market demand and stay-at-home orders preventing travel to and within fishing areas. In some tropical regions where the crisis coincided with peak fishing season, reduced access and fishing effort may also increase brood fish survival and replenish fish stocks . No percLessened pressure in higher-catch, lower-HDI areas is supported by decreased commercial fishing or preexisting institutional oversight; no change or increased pressure in higher-catch, higher-HDI areas by continued or increased commercial fishing or counteracting short- and long-term impacts; and greatest response uniformity in lower-catch, higher-HDI areas by shifting consumer demands or societal buffers absorbing rapid-onset shocks A. Notabl1)Inland fisheries, as a provisioning ecosystem service, add gradation to the predominately positive narrative of COVID-19 environmental impacts .2)Inland fisheries with higher provisioning value for upholding livelihoods and nutrition are perceived at higher risk of increased pressures due to COVID-19.COVID-19 effects on inland fisheries reflect the complexity of the pandemic. Crisis mitigation drives short-term compensatory impacts. Economic recovery efforts compounded by preexisting ecological stressors dictate longer-term fishery impacts. Based on the results of this study, which help improve the global understanding of COVID-19 impacts on natural resource use, we can conclude the following:SI Appendix for extended methods.We distributed an electronic survey to ∼1,900 fishery professionals (basin-level experts identified by the Food and Agriculture Organization of the United Nations [FAO]) assessing perceived inland fishery pressures. Source groups  represented management, research, and other sectors approximately equally. We asked, “In light of the current COVID-19 pandemic, what impact do you predict it may have on your fishery?” with the following available responses: 1) lessened pressure, 2) no change in pressure, or 3) increased pressure. No temporal constraints were specified. Respondents self-identified a fishery of expertise, indicated its location (selected geographic coordinates and/or region/basin), and could provide comments. We collected responses from 16 June to 15 July 2020. The University of Florida Institutional Review Board approved anonymous data collection and informed consent waivers (IRB 202000533). Data are available through the Consortium of Universities for the Advancement of Hydrologic Science, Inc. HydroShare data repository . We subsSupplementary File


Adductor-type spasmodic dysphonia (ADSD) manifests in effortful speech temporarily relievable by botulinum neurotoxin type A (BoNT-A). Previously, abnormal structure, phonation-related and resting-state sensorimotor abnormalities as well as peripheral tactile thresholds in ADSD were described. This study aimed at assessing abnormal central tactile processing patterns, their spatial relation with dysfunctional resting-state connectivity, and their BoNT-A responsiveness. Functional MRI in 14/12 ADSD patients before/under BoNT-A effect and 15 controls was performed (i) during automatized tactile stimulus application to face/hand, and (ii) at rest. Between-group differential stimulation-induced activation and resting-state connectivity  networks), as well as within-patient BoNT-A effects on these differences were investigated. Contralateral-to-stimulation overactivity in ADSD before BoNT-A involved primary and secondary somatosensory representations, along with abnormalities in higher-order parietal, insular, temporal or premotor cortices. Dysphonic impairment in ADSD positively associated with left-hemispheric temporal activity. Connectivity was increased within right premotor (sensorimotor network), left primary auditory cortex (auditory network), and regionally reduced at the temporoparietal junction. Activation/connectivity before/after BoNT-A within-patients did not significantly differ. Abnormal ADSD central somatosensory processing supports its significance as common pathophysiologic focal dystonia trait. Abnormal temporal cortex tactile processing and resting-state connectivity might hint at abnormal cross-modal sensory interactions. In the most frequent phenotype, adductor-FLD , laryngeal muscle spasms during vocalisation lead to effortful, jerky speech patterns2. Previous studies have outlined several functional and structural cortical abnormalities in the disease involving cortices of the phonation network3, including altered phonation-related activity using functional MRI (fMRI)6 or transcranial magnetic stimulation7, resting-state connectivity8 and grey matter structure9 in primary and higher-order  sensorimotor cortices in ADSD. Subcortically, abnormal putaminal structure and connectivity9 as well as thalamic and cerebellar structure and phonation-related activity10 have been shown.Focal laryngeal dystonia (FLD) is a debilitating task-specific focal dystonia (TSFD) affecting the laryngeal muscles, resulting in phonation impairment that can be classified into a number of overlapping clinical phenotypes12. Botulinum neurotoxin type A (BoNT-A) injection into the vocal cords yields temporary symptomatic relief in ADSD by attenuating the symptom-producing muscle spasms (primarily of the thyroarytenoid muscles), yet its possible central role is not well understood13. Reduction of phonation-induced electromyographic activity in the bilateral thyroarytenoid muscles in ADSD after unilateral injection with BoNT-A hinted at a possible role of altered sensory feedback from the treated muscle, leading to adapted motor drive also to the non-treated muscle14. If one postulates that this represents a central modulation due to peripheral BoNT-A application, it remains to date unresolved if this is guided by modulation of sensory feedback following BoNT-A-induced changes of muscle activity in the injected muscle during phonation, or if there is a direct central BoNT-A effect also present in absence of phonatory activity13. BoNT-A-associated alterations of cortical activity have been reported in orofacial non-TSFD during both motor task and somatosensory processing16, but conflicting observations have been made during phonation in spasmodic dysphonia5. Further clarification of the relationship of BoNT-A injections and sensory processing in ADSD may inform the development of sensory  modulation strategies to complementarily enhance benefit gained from BoNT-A treatment.Phonation and speech production in the healthy rely on both somatosensory and auditory feedback mechanisms17. Yet the central patterns of somatosensory stimulus processing abnormalities in the absence of motor task have not been investigated in ADSD, complicated by anatomic constraints in this TSFD. While the principle feasibility of direct laryngeal stimulation was described during magnetencephalography (MEG) in a small group of healthy subjects, several challenges such as its moderate reproducibility, sensitivity to noise/artefacts and considerable efforts regarding both technique and subject training have not yet been sufficiently addressed18. Findings in other types of FD suggest that abnormal somatosensory processing may constitute a form of endophenotypic trait19. This provides the opportunity to investigate the somatosensory system in ADSD through somatosensory stimulation to more accessible asymptomatic body regions.Abnormal somatosensory processing and dysfunctional interaction with the motor system is one central aspect in pathophysiologic theories in focal dystonia (FD)This work aimed at characterizing (i) patterns of abnormal central tactile stimulus processing in ADSD, (ii) possible spatial relations between areas of altered stimulation-related activity and dysfunctional  connectivity at rest, and (iii) possible effects of BoNT-A treatment on the above findings, avoiding confounds by motor execution, BoNT-A-modulated muscle function, or acute compensation mechanisms.2. Twelve patients  received regular abobotulinumtoxin injections  in 3–9-month intervals  with good clinical benefit (as certified by patients‘ subjective reports and the expert impression of the treating phoniatrist), two were BoNT-A-naïve (i.e. have never recieved BoNT-A treatment). Except for the BoNT-A-naïve patients that were only scanned once, patients were scanned twice in pseudo-randomized order: 34.9 ± 7.3d after BoNT-A treatment (PATpost) when their voice was at its best and post-injection breathiness had recovered, and prior to the next BoNT-A treatment  when the BoNT-A effect had clinically waned. The voice handicap index (VHI) was collected at both timepoints to ascertain subjective impairment by dysphonia. The institutional ethics board  approved of the study and all participants gave written informed consent according to the Declaration of Helsinki. All methods were carried out in accordance with relevant guidelines and regulations. All participants had a normal structural MRI scan and, as certified by medical history and a detailed clinical examination by a senior neurologist and movement disorders specialist (C.D.), no additional neuropsychiatric disorders (including no other movement disorders), no sensory deficits, and no neuroleptic drug use; patients had no dystonic symptoms in body regions other than the laryngeal muscles and did not receive voice therapy or medication for dystonia beyond BoNT-A.Fourteen right-handed patients with idiopathic isolated ADSD  were recruited from the hospital movement disorders outpatient clinic in cooperation with a specialized outpatient phoniatric center and compared to a control group of fifteen age- and sex-matched healthy volunteers . Diagnosis of ADSD was established by an experienced phoniatrist, with additional clinical neurological evaluation by a movement disorders-specialised neurologist. Procedures included evaluation of the medical history, phoniatric voice analysis for typical auditive voice/speech abnormalities , voice acoustic analysis and electroglottography , microlaryngoscopy and microstroboscopy. We limited our study to ADSD, as it is far more common than the abductor typeMRI acquisition parameters and software for data analysis are detailed in the supplement.20. In brief, trains of punctate tactile stimuli  were pseudo-randomly applied to forehead , upper lip  and hand  on either side by von Frey-monofilaments during three fMRI runs using an automated MR-compatible simulation device. A stimulation-induced painless feeling of touch was additionally ensured by mechanical and pain threshold testing in all participants21. Preprocessing was performed as in earlier studies employing this stimulation paradigm20, with an additional inclusion of six head motion parameters as regressors in the first-level general linear model contrasting the individual regressors  for the six stimulation conditions  to the implicit baseline (rest) separately for each condition. One patient was excluded due to suspicion of corrupted stimulation paradigm. Population-based inferences were drawn by introducing the first-level individual contrast images to a second-level flexible factorial model with factors subject, condition (see above) and group  accounting for possible non-sphericity of the error term, with post-hoc planned longitudinal contrasts (PATpre vs. PATpost) applied in case of a significant difference to healthy subjects pre-BoNT-A. A comparison of healthy subjects with patients post-BoNT-A was not primarily performed as no ancillary information was expected compared to the direct within patient group longitudinal analysis. Considering the subtlety of short-time punctate tactile stimuli and the interindividual anatomical variability of sensory representations, significance was determined at a cluster-forming (peak-level) threshold of p < 0.001 with cluster-level multiple comparison-correction at pFWEc < 0.05 , adjusted for the six conditions (p < 0.0083 (0.05/6)).The tactile stimulation experiment was performed as previously outlined3dDespike22. One control was excluded from further analysis due to high frame-to-frame motion. The sICA approach reliably separates non-grey matter signal and noise from physiologically meaningful components23. For short-range FC analysis, we removed five white matter and cerebrospinal fluid signal principal components in an additional step using the CompCor (component-based noise correction) method, and bandpass filtering (0.01–0.08 Hz) was applied. Image smoothing for regional FC analysis was performed after connectivity measure calculation as recommended24.Spatial independent component analysis (sICA) and regional homogeneity (ReHo) analysis were performed on resting-state data to investigate both changes in (i) long-range and (ii) regional functional connectivity (FC). Basic preprocessing of resting-state fMRI (rs-fMRI) scans involved realignment, slice-timing correction, normalization to Montreal Neurological Institute (MNI) space after coregistration with the anatomical scan, followed by linear detrending, regression of six head motion parameters and despiking using AFNI’s  25. Data across participants and conditions were concatenated and stepwise principal component analysis data reduction was performed guided by a prior dimensionality estimation using the minimum description length algorithm, retaining 39 subject-level and 28 group-level components. Data were variance-normalized and z-transformed. The Infomax algorithm was run 100 times in ICASSO26 and cluster quality quantified using the index Iq mirroring the difference between intra- and extracluster similarity (range 0–1). Direct data back-reconstruction from the aggregate spatiotemporal dataset was performed using GICA I, which is robust for lower model orders25. Following the study goal, we then selected three robust (Iq > 0.95) lower-model order cortical resting-state networks of interest guided by the spatial pattern of tactile stimulation-induced cerebral activity . To ensure that only highly connected network regions were analysed, a combined binary mask representing the effect of the within-group analysis (pFWE < 0.05) was applied. Results were peak-level-corrected for multiple comparisons at pFWE < 0.05, adjusted for three investigated networks (p < 0.017 (0.05/3)).sICA extracts maximally independent spatial resting-state networks comprising distributed, functionally related  brain regions by decomposing their linearly mixed signals contained in the spatiotemporal fMRI datasety figure  for furty figure . Between24. After z-transformation to increase normality and data smoothing, between-group analysis was performed as above using a flexible factorial random effects model . Following the aim of the study, the statistical analysis was constrained to a combined binary mask of voxels with a robust within-group response to tactile stimulation (pFWE < 0.05) across conditions. Results were peak-level-corrected for multiple comparisons at pFWE < 0.05.ReHo characterizes the local temporal coherence/synchronization of the regional blood oxygen-level dependent signal. The similarity of the time series of each voxel with those of its 26 nearest neighbours (voxels) was calculated in a whole-brain approach, using Kendall’s coefficient of concordanceSignificant associations of severity by VHI and duration of dysphonia with functional activity/connectivity changes were investigated within PAT post-hoc by multiple regression analysis for those analyses showing significant differential between-group activity/connectivity patterns. FWE-multiple comparison corrections were applied as for the respective between-group task/rest analyses and the significance level adjusted for the number of regressors (p < 0.025 (0.05/2)).group×condition], ps > 0.05). Average frame-to-frame motion (rotation and translation) across analysed subject scanning sessions during rest/task fMRI was 0.12 ± 0.046 mm/0.12 ± 0.054 mm and not significantly different between groups .All patients reported a subjective voice improvement after BoNT-A treatment. Accordingly, the VHI improved significantly post BoNT-A . Mechanical detection and pain thresholds and mean perceived intensities did not significantly differ between groups  [Tactile stimulation in patients (pre-/post-BoNT-A) and controls yielded activity in a network involving primary somatosensory cortex (S1), secondary somatosensory cortex (S2), superior parietal cortex, precuneus, intraparietal sulcus, supramarginal gyrus, supplementary motor area, dorsal and ventral premotor cortex, inferior and middle frontal gyrus, insular cortex, superior and middle temporal cortex, anterior cingulate cortices, and thalamus figure .Between-group analysis of PATpre against CONTR revealed significantly increased functional activity in the contralateral S1 during left hand/face  and right face  stimulation, and in the contralateral S2 during right face  stimulation  within the SMN in PATpre compared to CONTR, increased FC in the left primary auditory cortex  within the AN in PATpre compared to CONTR and no significant abnormalities in the CEN. We further observed significantly reduced regional homogeneity at the right parieto-temporal junction  caudal to A1 during right face (V1) stimulation in PATpre  and distant (hand) non-dystonic S1 representations and in both hemispheres, fitting the concept of an underlying endophenotype. Demonstration of such endophenotypic abnormality in cutaneous surrogate areas in ADSD may encourage further research in sensory modulation strategies in the disease. Modulation of sensory input from mostly proximate body regions (i.e. sensory tricks) is known to ameliorate dystonic symptoms in some FD patients, and sensory retraining was able to improve motor function in TSFD of the hand in the past29. Quite recent work in FLD already made steps in this direction, indicating positive modulatory effects on voice function through ventral cervical cutaneous vibration30, hence the investigation of effects of sensory retraining strategies applied to nondystonic/surrogate sensory areas might be of interest for future work.This work provides evidence for abnormal primary and higher-order somatosensory input processing in ADSD in absence of motor tasks, amending observations in other cranial FDs18. Increase and spread of somatosensory activation affecting symptomatic and non-symptomatic body parts have been pathophysiologically attributed to deficient intracortical inhibition and dysfunctional plasticity resulting in dedifferentiation and topographic shifts of otherwise concise cortical activation33. Such cortex-level dedifferentiation in FDs has been suggested to manifest peripherally in altered tactile discrimination thresholds29, and altered somatosensory temporal discrimination has also been reported for ADSD34. In patients with cervical dystonia, peripheral temporal discrimination thresholds and central correlates of intracortical inhibition were abnormally reduced and further deteriorated by high-frequency somatosensory stimulation of a non-dystonic body part, whereas the opposite effect was seen in healthy controls35. As observed for a body part not affected by dystonic posturing, this observation had been discussed in support of a primary rather than adaptive nature of abnormal somatosensory processing and underlines the vulnerability of such predisposing abnormality to intensive somatosensory input as it occurs during high-proficiency motor tasks, as are affected in TSFDs like ADSD.Altered cortical activity during left hand stimulation spread lateral into the somatotopic face representations, while increased activity during face stimulation was rather limited to its expected somatotopic representation located superior and posterior to the putative S1 larynx representation reported for the healthy36. Disordered information transfer from the sensory to the motor cortices (i.e sensorimotor integration) is discussed as a possible mechanism of dystonic posturing17, occurring either at the cortico-cortical or subcortico-cortical level involving the basal ganglia or cerebellum38. In support of a possible top-down process , recent work in spasmodic dysphonia suggested abnormal premotor-parietal-putaminal circuitry with abnormal excitatory left inferior parietal projections to the putamen and interhemispheric information transfer from the right to left premotor cortex36. Yet, the lack of differentiability of primary and adaptive changes by fMRI ultimately limited the interpretation of the findings’ origins. Past studies in other FDs have attempted to modulate sensorimotor higher-order axis processing through transcranial stimulation paradigms (mostly of the premotor cortex), showing mixed and mostly short-term results on motor function; yet such studies are to date lacking in FLD39.Beyond S1, higher-order somatosensory cortices encompassing the left-hemispheric S2 as well as left- and right-hemispheric superior and inferior parietal cortex showed increased tactile-stimulation induced activity in ADSD. Further, right-hemispheric PMv activity was enhanced during left face stimulation (with respective contrahemispheric trends observed during stimulation of other body parts), and medial PMd connectivity was increased at rest. Other rs-fMRI studies in spasmodic dysphonia described reduced connectivity in either the supplementary motor area or the PMd within the SMN. Yet, cluster locations differed compared to the present study, and the lack of dimensionality information in previous studies impairs comparability40. Given that patients in the present study did not undergo a complementary external rating of their disease-related voice impairment , this association warrants confirmation in future studies. Additionally, FC of the left A1 cortex within the auditory network was increased at rest, and regional FC in the parieto-temporal junction at rest was disturbed in the right hemisphere with a trend for the left hemisphere (possibly owed to right-hemispheric dominance of processing at the TPJ11). Associations of disease duration with functional connectivity, hinted at by previous work in FLD reporting inverse correlation of disease duration with SMN parietal connectivity41, were not observed in this work. The above primary/associative (superior) temporal regions have been suggested to be part of primary areas within the phonation network besides the larynx motor cortex, associated PMv, supplementary motor area and cerebellum3, that guides speech processing modulated by somatosensory/auditory feedback loops12 which have been shown to interact e.g. during stabilisation of the fundamental frequency42. Cross-modal interplays within the sensory system have recently gained increasing attention in the healthy. Multisensory processing may occur at different hierarchical levels of the respective sensory system and/or may modulate the sensory processing in classical (primary) stimulus-specific areas via feedback mechanisms44. Especially a role of regions in and around (posterior to) the auditory cortex has been suggested of relevance in this regard using event-related potentials in humans and intracranial recording in macaques44. With regard to audiotactile processing, fMRI studies have shown considerable spatial overlap of tactile and auditory stimulation-induced cortical networks with possible frequency-specific components46. A MEG study described modulation of somatosensory processing speed in the healthy in the presence of auditory stimuli47. Cross-modal convergence may occur at the cortico-cortical or subcortical (e.g. superior colliculi) level43. Disturbed neuronal synchrony by regional homogeneity in the TPJ may hint at disturbed intracortical processing in this multimodal area. Impaired sensory processing beyond the somatosensory system is yet little researched in FD. Nevertheless, impaired visuo-cerebellar (seed-based/ between-network) connectivity profiles have been described in cranial FDs, and for musician’s embouchure dystonia auditory network abnormalities at rest have previously been shown48, hinting at a possible subtype-associated component. Elucidating the effects of possible cross-modal dysfunctional interactions may hence be of interest in future studies aiming at further elucidating FD pathophysiology or between-subtype differences. Further, in analogy to somatosensory retraining approaches, this might incent the development of auditory (re)training approaches to ameliorate FLD symptoms, e.g. based on auditory perceptual observations such as an abnormal Lombard effect previously observed in ADSD patients49.An interesting aspect of this study was the observation of temporal cortex and temporoparietal junction abnormalities in absence of phonation. Cortical activity after right-sided face stimulation was increased in the left posterior STG. In parts of the left posterior STG, the impairment (by VHI) further predicted the degree of right face stimulation-induced activity in patients, amending a prior structural analysis that observed a positive relationship of left superior temporal cortical thickness with the mean number of voice breaks in sporadic spasmodic dysphonia50, the degree to which (parts of) certain intrinsic connectivity networks are recruited for51 and during52 a task or intervention may be varying and undergo dynamic change. Hence, beyond methodological aspects  these observations may point to potential context-related dynamics in the disease whose relevance needs to be better understood to gain further insights into the complex focal dystonia pathophysiology, and may warrant cautious study design in interventional studies aiming at focal non-invasive  modulation of cortical activity to ameliorate motor function as discussed above.While both connectivity and stimulation-induced activation abnormalities affected the premotor cortex and upper parts of the temporal cortex, we did not observe spatial overlap. Premotor cortices were abnormally active in the ventral domain during tactile processing, while task-free connectivity was altered dorsally. In the upper temporal cortex, changes differed in their anterior-posterior location Fig. . While s14 might indeed rather be closely linked to its modulation of muscle activity. Prior investigations of BoNT-A effects on phonation-induced brain activity were equivocal in spasmodic dysphonia5. Other studies investigating BoNT-A effects focused on craniocervical non-TSFDs and reported varying sensorimotor system effects13.In the present study we did not find evidence for BoNT-A-induced modulation of abnormal cortical processing. Methodological aspects such as small sample size, small applied average doses (Ø 15 units Abobotulinumtoxin) and the fact that the larynx representation was not directly tested may have resulted in reduced sensitivity to detect those changes. Alternatively, the probably endophenotypic changes in non-symptomatic representations might mirror underlying predisposing pathophysiology and hence not be responsive to BoNT-A, indicating that modulation of sensory input through BoNT-A observed in earlier clinical workThe present study provides evidence for abnormally organized somatosensory processing in ADSD amending previous observations of abnormal peripheral tactile discrimination thresholds in the disease. Beyond supporting the notion of dysfunctional sensory processing as common pathophysiologic trait across FD subtypes, evidence of abnormal central somatosensory processing in cutaneous surrogate areas may serve as working point regarding sensory modulation strategies in the disease. Temporal cortex abnormalities during rest and tactile stimulus processing might hint at abnormal cross-modal sensory interactions warranting further research.


Infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily lead to upper respiratory tract infection and its sequelae frequently dominate the clinical course of COVID-19 , 25. In Here, we report on autoptic neuropathological findings for a study cohort of seven COVID-19 patients, all of whom were positive for SARS-CoV-2 by nasopharyngeal swab testing, and compare our observations with those made in a SARS-CoV-2 negative control autopsy cohort comprising individuals with non-septic and systemic inflammatory/septic clinical courses (Suppl. Tables 1—3). All patients of our study cohort except for one had multiple relevant comorbidities, consistent with the previous reports , 12, 16.All COVID-19 study patients showed strong systemic inflammation, documented by high plasma levels of acute-phase proteins , and interleukin 6. Systemic immune activation affects the CNS, resulting in so-called sickness behavior including lethargy, malaise, and fatigue . As micr+ T lymphocytes in some COVID-19 brains, this was similarly encountered in controls with sepsis or systemic inflammation. Likewise, in none of the brains of our study cohort, intraparenchymal hemorrhages or acute/subacute ischemic infarcts were found. Furthermore, we did not detect microthrombi or fibrinoid necrosis in intracerebral or leptomeningeal blood vessels, indicating that the pathomechanism of disseminated microthrombotic pulmonary vessel occlusions [We did not find any evidence for a COVID-19-related meningitis/encephalitis with increased lymphocytic infiltration of the brain or the leptomeninges. Although we detected sparse perivascular and leptomeningeal infiltrates of CD3clusions  is not gclusions , 9, perhclusions , 10 and/clusions  in severclusions , 25.With regard to the widely accepted concept that CNS inflammation can contribute to the progression of neurodegenerative diseases , it is wAnosmia is a frequent early neurological sign of infection with SARS-CoV-2 . In addiACE2 expression also by olfactory bulb (and brainstem) neurons, as well as by few astrocytes . In particular, as the expression of ACE2 was not upregulated in COVID-19 brains when compared to controls, it remains unclear whether its upregulation plays a role in the context of COVID-19. These findings may indicate that ACE2 expression below levels detectable by immunohistochemistry is sufficient for SARS-CoV-2 entry into target cells.As angiotensin-converting enzyme 2 (ACE2) has been identified as an entry receptor for SARS-CoV-2 , we inveOur findings represent endpoints of particularly severe disease, with single time point qRT-PCR measurements precluding conclusions about a potentially dynamic brain viral load during the course of disease. It is possible that reversible brain alterations such as reactive inflammatory processes that had potentially occurred earlier during the clinical course, went unnoticed. These limitations notwithstanding, based on our observations, it seems unlikely that irreversible changes such as acute demyelination or ischemic lesions are part of the usual spectrum of COVID-19 with brain involvement. Instead, several histological brain abnormalities previously postulated to occur in association with COVID-19 can likewise be seen in SARS-CoV-2 negative, critically ill patients. Our observations suggest that specific SARS-CoV-2 induced neuropathological abnormalities are absent in the majority of COVID-19 patients.Supplementary file1 (PDF 36557 kb)Below is the link to the electronic supplementary material.


The need for proven disease-specific treatments for the novel pandemic coronavirus SARS-CoV-2 necessitates a worldwide search for therapeutic options. Since the SARS-CoV-2 virus shares extensive homology with SARS-CoV and MERS-CoV, effective therapies for SARS-CoV and MERS-CoV may also have therapeutic potential for the current COVID-19 outbreak. To identify therapeutics that might be repositioned for treatment of the SARS-CoV-2 disease COVID-19, we strategically reviewed the literature to identify existing therapeutics with evidence of efficacy for the treatment of the three coronaviruses that cause severe respiratory illness . Mechanistic and in vitro analyses suggest multiple promising therapeutic options with potential for repurposing to treat patients with COVID-19. Therapeutics with particularly high potential efficacy for repurposing include camostat mesylate, remdesivir, favipiravir, tocilizumab, baricitinib, convalescent plasma, and humanized monoclonal antibodies. Camostat mesylate has shown therapeutic potential, likely by preventing viral entry into epithelial cells. In early research, the targeted antivirals remdesivir and favipiravir appear to benefit patients by decreasing viral replication; clinical trials suggest that remdesivir speeds recovery from COVID-19. Tocilizumab and baricitinib appear to improve mortality by preventing a severe cytokine storm. Convalescent plasma and humanized monoclonal antibodies offer passive immunity and decreased recovery time. This review highlights potential therapeutic options that may be repurposed to treat COVID-19 and suggests opportunities for further research. Coronaviridae family [Coronaviridae family is a cohort of viruses with single-stranded, positive-sense RNA genomes that typically cause both respiratory and enteric diseases [The new pandemic coronavirus disease 2019 (COVID-19) is caused by the novel Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which belongs to the diseases .betacoronavirus genus [The three coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2 all belong to the us genus . Sequencus genus , whereasus genus . While tEach of these three coronaviruses causes significant morbidity and mortality in humans. In 2003, a SARS-CoV outbreak started in China and caused 8,098 confirmed cases worldwide with 775 fatalities . The SARin vitro, or in vivo antiviral activity against SARS-CoV or MERS-CoV to strategically identify therapeutic options that can be used or repurposed for the treatment of SARS-CoV-2.Given the lack of vaccines or specific treatments for the novel coronavirus SARS-CoV-2, it is crucial to identify therapeutic options to both limit the replication of this virus and prevent further spread. Based on the extensive homology shared by SARS, MERS, and COVID-19, we hypothesize that drugs with evidence of benefit in treatment of SARS or MERS are likely to benefit patients with COVID-19. This review summarizes drugs and therapies with either theoretical, PubMed, CINAHL, EMBase, and Google Scholar were searched to identify articles that supported therapeutic benefit for a pharmacological substance in SARS-CoV or MERS-CoV that could be repositioned for treatment of SARS-CoV-2 infections. Four of the authors conducted independent searches to evaluate various search strategies. These combined strategies were shared and optimized with the Prisma Health System staff medical librarians. The aggregate search strategy includes articles limited to those available prior to June 15, 2020. Search criteria included the keywords coronavirus, severe acute respiratory syndrome, Middle East respiratory syndrome, SARS, MERS, SARS-CoV, MERS-CoV, SARS-CoV-2, COVID-19 in conjunction with the terms treatment or therapeutic as well as the following Medical Subject Headings: antiviral agents/therapeutic use, coronavirus infections/drug therapy, coronavirus infections/therapy, drug repositioning, Middle East respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus/drug effects, SARS virus, SARS virus/drug effects, severe acute respiratory syndrome/drug therapy, and treatment outcome. The initial search strategy identified a total of 353 articles. Next, the names of the individual medications reviewed in this article were also used in conjunction with the above search criteria to identify an additional 99 articles, and CENTRAL was searched for relevant trials. Finally, bibliographic references of the pertinent articles were also searched for relevant literature not identified in the original search strategy. Each article was reviewed by a minimum of two authors to assess suitability for inclusion in this review. The authors reviewed the literature for inclusion based on critical evaluation of the relevance and methods of candidate papers. Articles were accepted for inclusion based on a modified Delphi method.The RNA-dependent RNA polymerase (RdRp) enzymes replicate and transcribe the viral genome of RNA viruses, including coronaviruses. Since these essential enzymes are highly conserved across RNA viruses, they are attractive targets for anti-viral therapies. The prodrugs remdesivir and favipiravir inhibit RdRp and are now under evaluation for treatment of COVID-19. Other RdRp inhibitors, such as sofosbuvir, have not been evaluated for activity againstcoronaviruses .Remdesivir is a promising investigational nucleoside analog  with potp < 0.001) [Expanded Access or Compassionate Use programs have provided remdesivir to patients with confirmed, severe SARS-CoV-2 infections in the United States, Europe, and Japan . Althoug< 0.001) . Even th< 0.001) , these eFavipiravir is an RdRp inhibitor that blocks the replication of multiple RNA viruses by terminating elongation at the incorporation site as a nucleoside analogue. Like remdesivir, it functions as an inhibitory RdRp substrate in its phosphorylated form, favipiravir-triphosphate, and inhibits the SARS-CoV-2 RdRp . It is cNeuraminidase inhibitors, such as oseltamivir, are used to treat Influenza A and Influenza B ,19. The Protease enzymes, which contribute to multiple protein processing, signaling, and degradative cellular pathways by cleaving peptide bonds, are important drug targets across a range of diseases ,23. ThesLopinavir is a human immunodeficiency virus (HIV) protease inhibitor that also inhibits SARS-CoV viral replication. Ritonavir, another HIV protease inhibitor, increases lopinavir concentrations by inhibiting the cytochrome P450 3A4-mediated metabolism of lopinavir . CombinaTransmembrane Protease, Serine 2 (TMPRSS2) is a serine protease that is required for SARS-CoV-2 entry into host cells; TMPRSS2 cleaves the viral spike (S) protein to prime it for binding to the host cell angiotensin-converting enzyme 2 (ACE2) . The S pDisulfiram inhibits hepatic aldehyde dehydrogenase and is approved by the U.S. Food and Drug Administration (USFDA) for alcohol aversion therapy . In vitrThe angiotensin-converting enzyme 2, or ACE2, belongs to the angiotensin-converting enzyme (ACE) family of dipeptidyl carboxydipeptidases. ACE2 inactivates angiotensin II and generates angiotensin 1–7, a heptapeptide with potent vasodilator function via activation of the Mas receptor. ACE2 performs a vital role in the cardiovascular and immune systems but is used as a functional receptor by SARS-CoV-2 to infect human cells. SARS-CoV and SARS-CoV-2 endocytosis depends on viral spike protein binding to ACE2 , which iN-(2-Aminoethyl)-1-aziridineethanamine (NAAE), which inhibits SARS cell fusion in vitro, as a potential therapy for SARS [Although prior structural analyses identified the ACE2 inhibitor for SARS , this cofor SARS .Kinase enzymes transfer phosphate groups from ATP to recipient proteins in a process called phosphorylation. This process transfers physiological signals, often via cascades of sequential reactions. Since cancer often deregulates signaling pathways, protein kinases are key targets for oncological drug development. Similarly, since many viruses hijack host signaling pathways, kinase inhibitors identified in other contexts may be repositioned for use as broad-spectrum or targeted antiviral therapy .Imatinib, also known as Gleevec, is an oral tyrosine kinase inhibitor with demonstrated activity against Ebola and poxviruses . Imatini. Since baricitinib decreases viral entry and blunts cytokine storm [Baricitinib is a Janus kinase (JAK) inhibitor used to treat rheumatoid arthritis . Recent ne storm , it is aRibavirin is a purine nucleoside analog with antiviral activity that is currently approved for treatment of chronic hepatitis C . As an ain vitro, it does not appear to inhibit SARS-CoV [Ribavirin inhibits MERS-CoV viral replication when administered as combination therapy with other antivirals . In patiSARS-CoV . FurtherSARS-CoV . New eviSARS-CoV . In a reSARS-CoV . UnfortuSARS-CoV , hypocalSARS-CoV ,51; it iConvalescent plasma containing SARS-CoV-2 antibodies is being administered to severely ill patients with COVID-19 as an adjunct immunotherapy based on the hypothesis that the plasma contains neutralizing antibodies for SARS-CoV-2. Previous work showed that antibodies to the S protein were present for more than 7 months in the serum of SARS patients . In a seHuman monoclonal antibodies also have robust potential for supporting passive immunity, whether via use as pre-exposure prophylaxis or for treatment of severe cases .The humanized antibodies mAb 4C2h and mAb 5H10 are murine antibodies that neutralize the spike protein receptor-binding domain of the MERS and SARS viruses, respectively. In mice, treatment with the mAb 4C2h antibodies significantly decreases MERS-CoV viral titers . The mAbTocilizumab and sarilumab are humanized monoclonal antibodies specific for the interleukin 6 (IL-6) receptor; both are USFDA approved for the treatment of rheumatoid arthritis . Multiplin vitro. Early results of interferon-beta-1b as combination therapy have been encouraging, and trials to evaluate the use of type 1 interferons as adjunctive therapy for SARS-CoV-2 are ongoing [Interferons are cytokines that activate the host immune system and thus broadly combat viral infections. Empirically, several interferons have in vitro antiviral activity ,65. Inte ongoing ,68.Cyclosporine A is a calcineurin inhibitor used as an immunosuppressant and as an antiviral treatment for hepatitis C  and MERSMycophenolic acid (MPA) is also an immunosuppressant that may have antiviral activity . AlthougCorticosteroids have been investigated extensively as adjunctive viral therapy due to their immunomodulatory effect. Since corticosteroids suppress host inflammatory responses, they offer theoretical benefit against excessive inflammasomal responses. However, this mechanism also suppresses natural defense mechanisms. Thus, corticosteroids may cause more harm and even increase mortality when used as adjuncts to antiviral therapies. When corticosteroids were used as adjunct therapies for treatment of SARS and MERS ,75, a plSurprisingly, recently released, preliminary, non-peer-reviewed data from the Randomised Evaluation of COVID-19 therapy (RECOVERY) trial described reduced mortality in severely ill patients with COVID-19 who were treated with dexamethasone for 10 days . AlthougSeveral glycopeptide antibiotics have been studied for treatment of coronaviruses. Teicoplanin is a glycopeptide which is used to treat complicated infections associated with commonly susceptible organisms including aerobic and anaerobic gram-positive bacteria. Teicoplanin is marketed in the United Kingdom but is not available in the United States . TeicoplNitazoxanide (NTZ) was originally developed to treat protozoal infections. By inhibiting the pyruvate ferredoxin oxidoreductase enzyme-dependent electron transfer, NTZ uncouples the oxidative phosphorylation pathways of energy metabolism . Howeverp < 0.01), without significant adverse events [Umifenovir (Arbidol) is an anti-influenza drug used in Russia and China with in vitro activity against SARS-CoV. It is thought to inhibit viral fusion, but its mechanism remains unclear . While te events ,86.Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) that inhibits the cyclooxygenase (COX) enzymes; this inhibition decreases inflammation by lowering COX-mediated prostaglandin production. However, the use of NSAIDS has been controversial in the setting of COVID-19. NSAIDS improve outcomes in some viral respiratory infections, but they may mask symptoms and delay further treatment. Hypothetically, NSAIDS may increase viral uptake by increasing ACE2 receptor expression. Prior studies showed that indomethacin inhibits SARS-CoV RNA synthesis and blocks SARS-CoV replication in both Vero and human lung cell cultures . SimilarThe anti-malarial agent chloroquine and its analog, hydroxychloroquine, exert antiviral effects by multi-factorial mechanisms that include increasing intracellular vacuole pH, manipulating pathways involved in protein degradation, and modifying ACE2 receptor glycosylation ,90. In vChlorpromazine hydrochloride, a dopamine receptor antagonist used as an antipsychotic and antiemetic, also inhibits the clathrin-mediated endocytosis pathway that SARS-CoV uses to enter cells . In vitrToremifene citrate is an estrogen receptor antagonist used to treat breast cancer. Its potential for antiviral repurposing has led to the discovery that toremifene destabilizes the Ebola virus membrane glycoprotein; this drug is thought to inhibit viral fusion and prevent viral replication ,99. ToreLoperamide is an opioid receptor agonist that is commonly used as an anti-diarrheal agent because it slows gut motility. It activates the mu opioid receptor, primarily within the gastrointestinal tract. Loperamide inhibits SARS-CoV and MERS-CoV replication in cell culture, though its anti-viral mechanism of action is not understood . Howeverbetacoronavirus genus [The novel pandemic COVID-19 is an emergent threat to global health . COVID-1us genus , and SARus genus . This reus genus . The tarus genus , it did us genus . ConvaleSuggestions for further research include the repurposing and development of drugs that are thought to inhibit specific viral targets. The discovery of the structure of SARS-CoV-2 has provided essential information for drug targets and clinical trials ,30,103. While an inspiring global research effort seeks to develop a vaccine against SARS-CoV-2, there are multiple existing therapies that are likely to benefit patients with COVID-19. This review summarizes currently available evidence for medications that are likely to be effective for treatment of SARS-CoV-2 based on past studies in the context of the highly homologous SARS-CoV and MERS-CoV. Extensive further research will be needed to identify the therapeutics that best treat patients with COVID-19.


Advancements in cell-free synthetic biology are enabling innovations in sustainable biomanufacturing, that may ultimately shift the global manufacturing paradigm toward localized and ecologically harmonized production processes. Cell-free synthetic biology strategies have been developed for the bioproduction of fine chemicals, biofuels and biological materials. Cell-free workflows typically utilize combinations of purified enzymes, cell extracts for biotransformation or cell-free protein synthesis reactions, to assemble and characterize biosynthetic pathways. Importantly, cell-free reactions can combine the advantages of chemical engineering with metabolic engineering, through the direct addition of co-factors, substrates and chemicals –including those that are cytotoxic. Cell-free synthetic biology is also amenable to automatable design cycles through which an array of biological materials and their underpinning biosynthetic pathways can be tested and optimized in parallel. Whilst challenges still remain, recent convergences between the materials sciences and these advancements in cell-free synthetic biology enable new frontiers for materials research. We live in a material world . There aThe field of synthetic biology has emerged over the last twenty years into a highly dynamic community of interdisciplinary researchers and societal stakeholders, that are working toward the responsible development and implementation of cutting-edge biotechnologies . SynthetHowever, biological systems are highly complex and initial attempts at engineering biological systems to fulfill specific application goals, are often only partially successful. To help overcome these challenges, synthetic biology employs the concept of the design-cycle, through which biotechnologies are iteratively designed, built and tested . Learninin vitro biotechnologies. Broadly, the term cell-free synthetic biology refers to different methods and technologies for engineering or using biological processes outside of a cell. For example, cell-free protein synthesis reactions enable the production of proteins within biochemical reactions. Thus, cell-free reactions typically make use of isolated cellular components  and/or cell extracts, rather than live whole-cells. In the context of this review four commonly used cell-free reaction formats will be discussed (Cell-free synthetic biology is a broad term that encompasses many different iscussed . We descEscherichia coli using purified histidine (His)-tagged proteins , by sonication or osmotically  approaches can reconstitute entire biosynthetic pathways using either cell extracts from diverse species, engineered cells and/or cell-free synthesized recombinant enzymes  Figure . Also, cde novo bioproduction of biological materials, including biopolymers or their monomers, cellulosic materials and nanoparticles (R)-3-hydroxybutyrate-CoA (32.87 ± 6.58 μM)  biosynthetic pathways from phaCAB operons that also enhanced in vivo PHAs production -3-h6.58 μM) , silver 6.58 μM)  and silk6.58 μM) . Poor ce6.58 μM) . Becauseoduction . Furtheroduction . Thus, hin vitro gold or silver nanoparticle production studies reported desirable nanoparticle characteristics  and/or easier purification protocols within cell-free bioproduction reactions than whole-cell production methods  PHAs production has been commercially manufactured at industrial scales over the last several decades. Unfortunately, the commercial impact of PHA-based bioplastics has been historically prohibited by their higher production costs than oil-derived plastics  than comparative, standard liquid-format cell-free reactions  or disease biomarkers . Several studies have described comparability between DNA regulatory elements and genetic circuits tested in vitro (cell-free) and in vivo (whole-cells) across several model   (in vivo (whole-cell) applications. Cell-free workflows are also amenable to automation. Indeed, several studies have successfully utilized acoustic liquid handling robots to rapidly setup large-scale, low-volume (≤10 μl) prototyping cell-free reactions in a 384 well plate format . These data sets also enable accompanying quantitative modeling that can potentially predict unknown model parameters, such as transcription factor binding affinities or cell-free energy utilization (in vivo (whole-cell) contexts. Also, cell extract processing breaks down cellular organelles and other intracellular compartments, thus perturbing native biomolecule localization. Yet, despite these potential limitations complex genetic circuits , that are functional in vivo, have been prototyped using cell-free forward-engineering design cycles   and non-riegens)  organisme format . Microfle format , droplete format  or multie format  strategilization . These dlization  for use lization . High-thn cycles . This ren cycles . It is tn cycles .de novo biological components . These A panoply of recent examples, including those discussed in this review, are indicative that biological materials are the next frontier for cell-free synthetic biology – but challenges remain. A deeper understanding of the compositions and biochemical activities of processed cell extracts and cell-free reactions are needed  to mitigIn summary, cell-free synthetic biology is a powerful, highly customizable and promising biotechnology that is beginning to have a positive impact on the industrialization of sustainable materials production. Importantly, synthetic biology companies continue to champion interdisciplinary collaborations with designers and materials scientists as part of the development process. We envision that a continuation of these trends will result in a new frontier for sustainable cell-free materials production and the growing bioeconomy.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.