Dengue is a disease endemic in Latin American countries, like Venezuela, and has become one of the most important public health problems. We report five complete coding sequences of dengue virus serotype 2 (DENV-2) isolated from DENV-infected patients in Venezuela. Phylogenetic analysis placed the isolates within the American/Asian genotype. Dengue virus (DENV) infection continues to be one of the most prevalent arboviral diseases in tropical and subtropical regions, with an estimated burden of 390 million cases/year worldwide . Infecti1–We report five complete coding sequences of dengue virus serotype 2 (DENV-2) isolated from DENV-infected patients in Venezuela in 2015, of whom two patients had dengue with warning signs. The study was approved by the ethics review committee of the Biomedical Research Institute, Carabobo University [Aval Bioetico numbers CBIIB(UC)-014 and CBIIB-(UC)-2013-1], Maracay, Venezuela, and the Ethics, Bioethics and Biodiversity Committee of the National Foundation for Science, Technology and Innovation, Caracas, Venezuela. For sequencing, RNA isolation was performed with the QIAamp viral RNA isolation kit . Libraries were prepared with the TruSeq V2 RNA , which includes a cDNA synthesis step. Sequencing was performed on a MiSeq instrument with the MiSeq reagent kit version 2 (Illumina) that generated 150-bp paired-end reads. The sequences were assembled and analyzed using the CLC Genomics Workbench version 10.1.1 software (Qiagen). Genome annotation was performed using the plugin MetaGeneMark version 1.4.The complete open reading frame (ORF) of the DENV-2 polyprotein was obtained in all samples with a coverage of greater than 177-fold. The lengths of the genomes sequenced were 10,694 nucleotides (nt), 10,619 nt, 10,712 nt, 10,711 nt, and 10,704 nt, with the lengths of the 5ʹ/3ʹ untranslated regions being 87/431 nt, 87/356 nt, 84/452 nt, 86/449 nt, and 77/451 nt, respectively. These are slightly shorter than the 96/451 nt reported for the DENV-2 reference genome (GenBank accession number NC_001474). The phylogenetic analysis based on the complete ORF using the maximum likelihood method revealed that the isolates belong to the American/Asian genotype. Strains clustered in two different subpopulations, sharing a common ancestor within the Venezuelan clade.in situ recombination has happened before in Venezuela; consequently, such potential changes should be monitored to reveal evolutionary trends , MH069496 (DENV-2/VE/IDAMS/921096), MH069497 (DENV-2/VE/IDAMS/921095), MH069498 (DENV-2/VE/IDAMS/910121), and MH069499 (DENV-2/VE/IDAMS/910105).The complete coding sequences of the five DENV-2 strains described here have been deposited in GenBank under the accession numbers


We found that the nprl2 mutation decreased Drosophila lifespan. Furthermore, the nprl2 mutant had a distended crop, with food accumulation at an early age. Interestingly, the inappropriate food distribution and digestion along with decreased crop contraction in nprl2 mutant can be rescued by decreasing TORC1 activity. In addition, nprl2-mutant flies exhibited age-related phenotypes in the midgut, including short gut length, a high rate of intestinal stem cell proliferation, and metabolic dysfunction, which could be rescued by inhibiting TORC1 activity. Our findings showed that the gastrointestinal tract aging process is accelerated in nprl2-mutant flies, owing to high TORC1 activity, which suggested that TORC1 promotes digestive tract senescence.Aging and age-related diseases occur in almost all organisms. Recently, it was discovered that the inhibition of target of rapamycin complex 1 (TORC1), a conserved complex that mediates nutrient status and cell metabolism, can extend an individual’s lifespan and inhibit age-related diseases in many model organisms. However, the mechanism whereby TORC1 affects aging remains elusive. Here, we use a loss-of-function mutation in  Aging involves the progressive loss of physiological functions in all organs and tissues and therefore, increases vulnerability to death . Age-relTarget of rapamycin (TOR) signaling is strongly implicated in the aging processes of diverse organisms, including yeast, worms, flies, and mammals . TOR is Drosophila, Rag GTPase is comprised of a heterodimer of RagA and RagC [Drosophila and mammals, depleting any of the GATOR1 or TSC complex components results in a dramatic increase in TORC1 activity and cell size [Drosophila, and mice [In and RagC , 7. Whenand RagC . Two comand RagC , 11. TSCand RagC . The GATand RagC , 14. In ell size , 14. Thuand mice –17. Althand mice , 4.One system that is affected by aging in humans is the digestive system or the gastrointestinal (GI) tract. The GI tract functions as a digestive system to absorb energy and nutrients from the environment and as a barrier epithelium to protect the animal from the exterior environment. Disruptions in this epithelial barrier negatively affect animal health and significantly decrease lifespan . The humDrosophila digestive tract is divided into three discrete domains: foregut, midgut, and hindgut [Drosophila intestine as a model system to study intestinal tissue homeostasis, immune responses, and stress signaling [The  hindgut , 24. The hindgut , 26. Fun hindgut , 28. Recignaling , 24. Damignaling . The agiignaling . Howevernprl2-mutant flies are phenocopied in aged wild-type flies. Moreover, we observed that the nprl2-mutant midgut displays age-related defects in gut morphology, ISC populations, metabolism, and immune responses. Our findings highlight the importance of TORC1 activity in aging and age-related GI tract disease and suggest that the Drosophila crop can be a useful model system to investigate the mechanisms of gastric emptying dysfunction in elderly individuals.Here, we used mutations in the GATOR1 complex component, Nprl2, to explore how TORC1 activity promotes age-related physiological defects in the GI tract. Importantly, we found that crop emptying and GI tract motility defects in nprl2, which have been previously shown to increase TORC1 activity, might affect Drosophila longevity [nprl2 null mutants, 1nprl2, with the lifespan of the control stock, yw, under standard culture condition. As shown in 1nprl2 flies (34 d) was markedly shorter than that of wild-type flies (64 d). Thus, mutations in nprl2 resulted in significantly decreased Drosophila lifespan.Recently, the TOR pathway has emerged as an important modulator of aging . Geneticongevity . To testDrosophilanprl2 mutants, which appear to have accelerated aging, exhibit age-related gastric dysfunction. The Drosophila crop, a foregut organ that is functionally comparable to the mammalian stomach, is primarily a reservoir for food and nutrients [nplr2-mutant flies often developed a swollen crop as they increased in age. To better define the extent of the distended crop phenotype, we scored the crops for size and placed them in one of four categories, with 1 being the smallest crop and 4 being the largest , which was nearly a five-fold increase relative to control adults. With the age increased, approximately 70% of nprl2-mutant adults displayed large crop at 25 d. As the crops of nprl2 mutants were small at 3 d  and tracked the time taken to expel the dye. As shown in nprl2-mutant flies still contained blue dye in their digestive tracts after 60 hours. Thus, crop (gastric) emptying was much slower in nprl2-mutant flies  method [nprl2-mutant flies. Next, we counted the number of feces to determine the differences in the rate of food digestion between nprl2-mutant and control flies. Interestingly, nprl2-mutant flies generated markedly less feces than control flies  method . As showol flies . The redol flies . Thus, tnt flies , which snprl2-mutant flies were related to hyperactivated TORC1. Thus, we placed one copy of the Tor mutation, A948VTor, into nprl2 mutants, which might decrease TORC1 activity in nprl2-mutant flies. We determined the TORC1 activity through detecting the phosphorylation of the downstream effector 4E-BP. The phosphorylated 4E-BP level was significantly increased in nprl2 mutant, which could be rescued by mutant one copy of Tor to A948VTor . SDS causes gut damage to flies and can be lethal [1nprl2 flies (24 hours) was much shorter than the median lifespan of wild-type flies  and Beadex, were shown to have enlarged crops and reduced fecal deposition at a very early stage and the crop is almost empty. This suggests that the Drd and Beadex genes are essential for the development of specific tissue required to maintain crop function [Drosophila C virus infection can also induce a distended crop, possibly by affecting crop muscle function [nprl2, which encodes an inhibitor of TORC1, also resulted in an enlarged crop. Interestingly, the distended crops in nprl2 mutants were accompanied by food accumulation in the crops and this phenotype emerged a few weeks after eclosion, which suggested that the crop defect was due to premature aging, rather than improper development. By using a blue dye, we further showed that mutation of nprl2 affected the rate of food movement and food distribution in the digestive tract. Furthermore, decreased TORC1 activity through genetic manipulation, mutant one copy of Tor to A948VTor, or pharmacy treatment, using TORC1 specific inhibitor rapamycin, rescued the crop phenotypes of distend size, food accumulation and contraction rate in nprl2 mutant, which suggests that hyper TORC1 activity promotes age-related GI dysfunction.Recently, two mutants, function , 48. Morfunction . Here, wnprl2-mutant flies had shorter lifespans and age-related GI dysfunction. In addition, our previous works showed that nprl2-mutant flies display some defects such as female fecundity, energy storage and locomotion [nprl2-mutant flies may serve as an important tool to address questions regarding the mechanism of TORC1 activity and aging.Although the phenomenon that decreasing TORC1 activity increase lifespan in many organisms, the mechanism remains still unclear. One important question is whether activated TORC1 can promote aging. Nprl2 mediates amino acid levels and acts as an important TORC1 inhibitor. Our results showed that comotion , 34, 50.nprl2 mutant displayed an increase in ISC proliferation and a decrease in the number of differentiated EE cells, which was consistent with a previous report on mutations in the TORC1 inhibitor, TSC1 [nprl2-mutant midguts, which can also be seen in aged flies [nprl2-mutant flies. Recently, a theory, proposed by Mikhail Blagosklonny, suggests that the hyperfunction of TORC1 and other nutrient-sensitive signaling is the cause of aging [C. elegans, a model system that also develop age-related pathology including digestive dysfunction and tumor formation [nprl2 mutant flies might be contributed by the hyperfunction of TORC1.TORC1 activity plays important roles in ISC maintenance. Mutation of the TORC1 inhibitor, TSC2 results in defects in ISC division and differentiation, which impairs tissue homeostasis and increases the susceptible of flies to tissue damage . On the or, TSC1 . Furthered flies . Recentled flies . Notablyed flies , which mof aging , 54, whiormation , 56. Hernprl2-mutant flies decreased lifespan and promoted the occurrence of age-related crop enlargement, digestive dysfunction, and midgut senescence. Our findings suggested that Nprl2 is essential for the maintenance of GI function during aging, by suppressing TORC1 activity.In conclusion, hyperactivated TORC1 in yw, 1nprl2, and UASp-3×FLAG-3×HA-Nprl2, have been described previously [A948V/CyOy, w; Tor was kindly provided by Thomas P. Neufeld [tsy w; esg-Gal4, UAS-GFP, tub-Gal80 (TB00137) were obtained from Tsinghua Fly Center . Flies were cultured at 60% humidity in a temperature-controlled incubator at 25 °C, with a 12 h on/off light cycle, unless otherwise noted. Only male flies were used in this study. The standard food consisted of 1% agar, 3% brewer’s yeast, 1.9% sucrose, 3.8% dextrose, and 9.1% cornmeal . The dyed medium was prepared using standard medium with FD&C Blue Dye No. 1  added at a concentration of 0.5% wt/vol. For rapamycin treatment, rapamycin  was dissolved in methanol and mixed into the media when preparing food vials, to a final concentration at 200 μM. For SDS treatment, SDS was dissolved in 5% sucrose to a final concentration at 0.6%. For ISC experiments using tsesg-Gal4, UAS-GFP, tub-Gal80, fly crosses were performed at 18°C and newly enclosed flies of the appropriate genotypes were kept at 18°C and then transferred to 29°C for 3 days before dissection.The stocks eviously . y, w; T Neufeld . y w; esDrosophila flies were homogenized in RIPA buffer containing complete protease inhibitors and phosphatase inhibitors . Western blotting was performed as described previously [Male eviously . AntibodDrosophila gut was dissected in phosphate-buffered saline (PBS) media, fixed for 20 min in 4% formaldehyde, and then washed twice and sealed with nail oil. Images were captured using a compound microscope. Gut length and crop area were quantified using Image J software.Enclosed male flies were collected and cultured on standard medium until the day of analysis. The Flies were allowed to consume dyed food for 72 hours prior to quantification. After 72 hours, the dye was clearly observed in the digestive tract. The flies with dye in the gut were fed standard food and the ratio of flies with observed dye in the digestive tract was determined at the indicated time points.For immediate food distribution, flies were starved in complete starvation medium (PBS with 1% agarose) for 10 hours and then fed dyed food for 30 min prior to quantification. To determine stable-state food distribution, flies were fed dyed food for 72 hours prior to quantification. The fly gut was dissected, and the crop was carefully separated from the entire gut. Collected tissues were homogenized in 100 μL of PBS. The absorbance values at 630 nm were measured using a spectrophotometer. The dye concentration in crops and guts was calculated by comparison with a standard curve.The crop motility assay was performed as previously described . The malThe CAFE assay was modified from previously described . BrieflyThe defecation rate detection was modified from previously described . Flies wDrosophila guts were dissected in PBS, fixed in 4% formaldehyde for 20 min, and then washed three times with PBS. The guts were incubated with Nile red for 30 min, washed twice, and mounted with ProLong ® Gold Antifade Reagent containing DAPI .The EdU labeling assay was modified from previously described with a Click-iT EdU Alexa Fluor 594 Imaging Kit  . Male flImmunofluorescence staining was performed as previously described . The mouSupplementary Figures


Neuropilin-1 and Neuropilin-2 form a small family of plasma membrane spanning receptors originally identified by the binding of semaphorin and vascular endothelial growth factor. Having no cytosolic protein kinase domain, they function predominantly as co-receptors of other receptors for various ligands. As such, they critically modulate the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. This review highlights the diverse neuropilin ligands and interacting partners on endothelial cells, which are relevant in the context of the tumor vasculature and the tumor microenvironment. In addition to tumor cells, the latter contains cancer-associated fibroblasts, immune cells, and endothelial cells. Based on the prevalent neuropilin-mediated interactions, the suitability of various neuropilin-targeted substances for influencing tumor angiogenesis as a possible building block of a tumor therapy is discussed. Cancer, one of the most common causes of death, appears in many ways, and is individually different in almost every patient. Although the causes of cancer are complex and elusive, it actually always arises in only three ways, i.e., by accumulation of genetic defects, by carcinogenic environmental factors, or by innate hereditary defects. Essentially, three key processes can be affected: (i) cell fate, (ii) cell survival, and (iii) genome maintenance. In their regulation, twelve signaling pathways are involved using approximately 140 genes . ViabiliNRPs are involved in a wide variety of signaling pathways and have pleiotropic effects on axon guidance and remyelination, immune response, angiogenesis, cell survival, migration and invasion ,8,9,10. NRPs are evolutionarily conserved cell surface proteins that are expressed by all vertebrates and are widely distributed in adult tissues ,16. TheyBoth NRP proteins can be glycosylated to varying degrees in different cell types ,30,31. MTwo NRP molecules non-covalently associate into a homodimer, presumably via their MAM domain and via motifs within their α-helical transmembrane domain ,36. In phttps://www.uniprot.org/uniprot/O14786, accessed on: 23 January 2019).NRP1 and NRP2 are differently distributed in tissues. First identified on neuronal cells of the central nervous system, especially in the developing embryo, NRP1 was also found to be expressed in blood vessels of different tissues, especially in arterial vessels. In the earliest stages of development, NRP1 and NRP2 are differentially expressed in arteries and veins, respectively . Later iNRP2, on the other hand, is expressed in neural crest-derived cells, such as hepatocytes and epithelial cells of proximal and distal renal tubules . In the reg) and basophilic granulocytes express NRP1, dendritic cells, macrophages and microglial cells express both NRP1 and NRP2, . Th. Th95]. Among the ligands of NRP1-containing signal complexes, VEGF-A is the best-understood binding partner of NRP1 and forms the molecular basis for the development of pharmaceutical compounds that affect VEGF-A binding to the NRP1/VEGFR2 complex ,101. Suc165 and VEGF-A189, but not the smaller variants with 145, 121, or 120 amino acids in length [145 in addition to VEGF-A165, but not VEGF-A121 [165 is of particular importance as it binds to a binding pocket within the NRP1-b1 domain formed by the side chains of residues Y297, Y353, D320, S346 [VEGF-A, encoded in a gene locus on chromosome 6p21.1, occurs in six different splice variants . All VEGn length ,107,108.EGF-A121 . Two VEGEGF-A121 , which cEGF-A121 ,111. FouEGF-A121 ,102,112.20, S346 ,101. Sev20, S346 .165) was first identified to be a ligand for NRP1 [121, dimeric VEGF-A165, binds with its C-termini to the NRP1 tandem domain b1/b2 and concurrently to the ectodomain of the VEGF receptor-2 (VEGFR2) [165/VEGFR2/NRP1 signaling complex [121 can also bind directly to NRP1, it is incapable to induce the formation of a NRP1/VEGFR2 holoreceptor [The 165 amino acid long variant of the vascular endothelial growth factor (VEGF-Afor NRP1 . Unlike (VEGFR2) ,106,107. complex . This co complex . Althougreceptor .145 and VEGF-C. Upon binding its ligand, NRP2 forms a ternary complex with VEGF-C and VEGFR3 on lymphatic ECs and is involved in lymphangiogenesis [The repertoire of NRP2 ligands within the VEGF family comprises VEGF-Aogenesis ,116.Recently, other soluble ligands and other receptor partners of NRP1 have been reported, demonstrating an amazing versatility of NRP1 in this respect. Interestingly, by binding to several growth factors other than VEGF-A, NRP1 becomes a coreceptor for the respective growth factor receptor, thus promoting the formation of a ternary signal complex. In such a way, the following growth factors and receptors may interact with NRP1: Placenta growth factor (PlGF), which is a member of the VEGF protein family and its receptor PlGFR ,118, hepWith the exception of the receptor-type serine kinases TGF-βRI and TGF-βRII that activate SMAD2 and SMAD3, all NRP1-associated growth factor receptors are receptor-type tyrosine kinases that autophosphorylate upon agonistic stimulation and recruit adapter proteins to trigger an intracellular signaling cascade . As partFurthermore, several members of the semaphorin family also bind to NRPs ,129. As The seven class 3 semaphorins, SEMA3A-G, are secreted members of a large family of guidance factors that regulate processes, such as developmental and tumor angiogenesis, by signaling through receptors composed of NRP1 or NRP2 and plexinA or plexinD, respectively . ECs expSemaphorin SEMA3A binds to the a1/a2 tandem domain of NRP1 and causes collapse and retraction of the nerve growth cone . The intNRP2 has been originally described as receptor for SEMA3F (at that time referred to as sema IV) that mediates its repulsive effect on growing neurons and is able to form heterooligomers with NRP1 . In compIntegrins have also been reported to interact with NRPs ,141,142.Another coreceptor of NRP1 is the L1 cell adhesion molecule (L1CAM) which belongs to the immunoglobulin superfamily (IgSF), mediates intercellular contacts between neurons, and interacts with NRPs in both cis- and trans-cellular manner ,158. The165 and PlGF-2, to the b1b2 domain of NRP1 when its chain length is at least 20–24 monosaccharides [165 and PlGF-2 interaction with NRP1 on ECs [Heparin and heparan sulfate have been described as binding partners of neuropilins . Heparincharides . By phys1 on ECs . As NRP11 on ECs . Heparan1 on ECs ,164,165.Calloselasma rhodostoma). Rhodocetin αβ (RCαβ) has been identified as the first non-enzymatic component of a snake venom that recognizes NRP1 on ECs [NRP1 is also a target of soluble toxins, such as the αβ subunit of rhodocetin, a venom component of the Malayan pit viper  vasculature is still unclear, as NRP1 may bind several FGFs  but doesThe interaction of NRP1 with PDGF-A and -B might be relevant for EMT ,189. StiNRP1 and NRP2 can also interact as coreceptors for HGF with the hepatocyte growth factor receptor/scatter factor receptor MET on ECs ,197. HGFOften, oncogenes switch signal pathways in such a way that they become essential regulators of proliferation and survival of tumor cells, termed oncogene addiction . DownregNRP1 can function as a coreceptor for TGFβ by forming holoreceptors with TGFβ-receptors I, II, and III ,201. By NRP1 has a negatively charged cleft in its b1 domain which allows binding of various ligands, such as TGF-β ,125. NRPThe binding and signaling of VEGF and related growth factors seem to be antagonized by class 3 semaphorins . AdditioAlthough NRP1 is the specific SEMA3A ligand, NRP1, with its short intracellular domain, cannot transduce SEMA3A signals on its own, but relies on complex formation with plexinA receptors . By bindBy binding to NRP1 and plexinD1 in ECs, SEMA3C induces internalization of VE-cadherin and shutdown of VEGF-induced signaling via AKT, FAK, as well as p38MAPK, which then causes disassembly of EC junctions and focal adhesions and related cytoskeletal rearrangement . Thus, SSEMA3F overexpressing melanoma cells form poorly vascularized tumors because Nrp2 inhibits tumor development and metastasis by a strong antiangiogenic cascade ,212. In Recruitment of pericytes to nascent vessels is essential for development, stabilization and maturation of the vasculature ,216,217,NRPs interact with specific integrins and activate them to bind to ECM molecules ,156,222.In a similar way, NRP2-mediated VEGF signaling in breast cancer and prostate carcinoma cells is subject to modulation by integrin α6β1 ,227. NRPGalectins are β-galactoside-binding proteins with mostly angiostimulatory activity, possibly by modulating receptor endocytosis ,231. In Binding of the snake venom component, rhodocetin αβ (RCαβ) to NRP1 induces the formation of a ternary complex with MET on endothelial and tumor cell membranes . This leUpon binding of VEGF, NRP1 triggers RAS activation and consecutively phosphorylation of ERK1/2 and AKT . NotablyBinding of GIPC1 to the cytoplasmic SEA (Ser-Glu-Ala) motif of NRP stimulates internalization of integrin α5β1 in Rab5-positive early endosomes . MoreoveMembrane trafficking also plays a key role in regulating signaling pathways . EndocytNRP1 and NRP2 can form docking sites for the endocytic adapter protein GIPC1  ,239,240.NRPs can activate signaling pathways that protect cancer cells from cytotoxic drugs and apoptosis. NRP1 and NRP2 are major positive regulators of the Hedgehog (HH) signaling pathway, which is relevant to angiogenesis and wound healing. It promotes EMT, cancer stem cell (CSC) survival, and tumor growth ,244,245.In medulloblastoma, tumor derived Sonic hedgehog (SHH) induces PlGF production in the cerebellar stroma, which promotes tumor cell survival through NRP1 independent of VEGFR1 . The majIn contrast, NRP1 expression is induced by Wnt/β-catenin signaling in mammary stem cells and in mouse mammary tumor virus (MMTV)-Wnt1 tumor xenografts . In breaNRP2 has been implicated in metastatic progression, although it usually occurs in carcinomas just in small amounts . Presuma165 and inhibiting its interaction with membrane-bound receptors [165 to VEGFR2 expressing ECs promoting angiogenesis [The binding of VEGF and related growth factors to NRPs and corresponding signaling seem to be antagonized by class 3 semaphorins . Additioeceptors . Accordieceptors . Artificogenesis .A soluble splice variant of NRP2, s9NRP2, scavenges VEGF-C and inhibits VEGF-C/NRP2 signaling in prostate cancer. This, suggests that s9NRP2 may be used therapeutically in the treatment of tumors that strongly depend on the VEGF-C/NRP2 axis for survival .Expression of NRP1 is induced by growth factors via the RAS/MAPK signal pathway ,250,258.NRP1 is instrumental in microRNA-based intercellular communication, because it efficiently binds Argonaute2 (AGO2) and AGO2/miR complexes without any involvement of its VEGF binding site, and it promotes their internalization into cells, whereupon they can perform their function and promote, for example, proliferation, migration and angiogenic tube formation . MicroRNThe transcription factor SOX10 induces among others the expression of miR338-p, which in gastric cancer and oral squamous carcinoma, inhibits the expression of NRP1 via phosphorylation of ERK1/2, MAPK, and AKT ,267. WhiSimilar to miR-338 in melanoma cells, in medulloblastoma cells, miR148a  and in nIn glioblastoma multiforme, miR-124-3p acts as a suppressor of NRP1, which promotes tumor cell proliferation and migration as well as tumor angiogenesis via PI3K/AKT/NFκB signaling . In contNRP expression levels correlate with tumor growth, invasiveness, angiogenesis, and poor prognosis . Cancer Furthermore, Nrp1 promotes EMT via TGF-β, HH, and HGF/Met signaling , therebyTumor cells have been in the scientific and therapeutic focus for long . Doubtleregs (CD4+CD25+Foxp3+ T-cells). In the tumor microenvironment, TAMs express SEMA4D on their surface, which forms a cell-cell contact with NRP1 on Tregs and activates them and other cells, such as ECs [regs characteristically express NRPs, via which they crosstalk with the different cells of the innate and adaptive immunity. Thus, they orchestrate immunosuppressive processes [reg cells express abundant NRP1, and the increased expression of NRP in Tregs correlates with poor prognosis, presumably because of its immunosuppressive properties against other CD4+-Thelper cells, CD8+ cytotoxic T-cells and natural killer cells [Dendritic cells and other antigen presenting cells, which surveil the tumor tissue, express NRPs ,57. Evenh as ECs ,300,301.h as ECs ,92,184. rocesses . Conspicer cells ,170. Bluer cells .Another cell type within solid tumors are ingrowing ECs. Their diverse interactions with each other and with tumor cells involving NRPs are highlighted in the following sections. In tumors, NRP1 is involved in evading the control by the immune system and also in angiogenesis and formation of tumor-characteristic vasculogenic mimicry (VM) vessels that significantly contribute to resistance to antiangiogenic therapy .NRPs are involved in the (patho) physiological regulation of (lymph) angiogenesis . InitialThe multifaceted tumor vasculature can arise in various ways : EssentiA variety of factors, especially the tumor microenvironment, determines the type of vascularization in the tumor mass ,325. ConNRP1 is essential for VGFA-triggered and angiogenesis-inducing signaling in ECs and in tumor cells . In partIn developmental angiogenesis of mice, plexinD1 mediates signals from Sema3E, –A, and –F . NRP/SEMSEMA3C is released by pericytes and SMCs and counteracts VEGF-triggered angiogenic signaling in neighboring ECs and, thus, attacks immature vessel sprouts in pathological angiogenesis, rather than quiescent ECs in established vessels ,332. ThiWhile SEMA3C inhibits tumor lymphangiogenesis and metastasis, its furin-cleaved form p65-SEMA3C has tumor-promoting properties in NRP2-expressing cancer cells, which was found at least in vitro, however, not yet in vivo ,336,337.An important aim in tumor treatment is a normalization of the tumor vasculature, rather than inhibition of angiogenesis, so that hypoxia is reduced by improved perfusion and drugs can reach tumor cells better ,340. SEMIn tumors, NRP enhances angiogenesis probably by blocking receptor endoytosis via trans-cellular interactions and thereby stabilizing receptor signaling . In thisIndependent of VEGFR1, PlGF promotes invasion and VM in melanoma via NRP1 . Unlike In cancer cells, signaling cascades are often activated by the constitutive activation of an oncogene, upon which they become dependent (‘oncogene addiction’), and on which a therapy inhibiting this signaling can be based . UnfortuSince NRPs can associate with diverse receptors into holoreceptors, they are promising targets for tumor therapies, and development of highly specific and highly potent NRP1 inhibitors is of outstanding interest ,350. In NRP1 is generally considered as a tumor-promoting coreceptor, but the situation is not entirely clear . In coloThe use of soluble sNRP as a decoy receptor has been investigated in animal models in tumor therapy. sNRP1 caused extensive hemorrhage, damaged vessels, and apoptotic tumor cells in tumors of rat prostate carcinoma cells and inhibited tumor angiogenesis and growth in murine granulocytic sarcoma (chloroma) ,358. LikBlocking NRP1 with antibodies selectively inhibits angiogenesis and in combination with anti-VEGF therapy, it further reduces tumor growth, suggesting that NRP1 antibodies may render tumor vessels more responsive to anti-VEGF therapy .NRP1 is overexpressed by angiogenic ECs of the tumor vasculature and in diverse tumor cells ,360,361.A monoclonal antibody against NRP1 inhibits EC migration and tumorigenesis in mouse tumor models ,131. TarIn renal cancer, angiogenesis inhibition by blocking NRP1/VEGFR2-mediated signaling with bevacizumab in combination with the RTK inhibitor sunitinib had toxic hematologic and vascular side effects and caused hypertension .A phase I trial with a human monoclonal IgG1 antibody (MNRP1685A) targeting NRP1’s VEGF-binding domain was promising, but another phase Ib trial with concomitant inhibition of NRP1 and VEGF in combination with chemotherapy showed an unexpectedly high proteinuria and toxicity ,365. ThuDCLPRC), DG1 (CRRPRMLTC) and DG2 (CRSRRIRLC) have suppressive effects on angiogenesis, tumorigenesis, and invasion, respectively, in preclinical mouse and primate models of human retinal diseases [The first NRP-specific small molecule peptide inhibitor is EG00229 (HY-10799) . Similardiseases  and in adiseases ,371. Thediseases .reg cell activation with good stability in vivo [Distinguishing between the two NRP isoforms, the small molecule inhibitor EG01377 selectively binds to the arginine-binding pocket of NRP1 without binding to NRP2, and it functionally inhibits VEGF-A-induced angiogenesis, cell migration, melanoma cell invasiveness, and T in vivo .165 with NRP1 [Based on the inhibitory peptide KPPR, branched pentapeptides in which the K side chain has been extended by an additional homoarginine have been developed, which are up to 30 times more active than ATWLPPR with respect to their inhibition of the interaction of VEGF-Aith NRP1 ,368,374.Targeting the transmembrane domain of NRP1, a peptide corresponding to the NRP1 transmembrane domain, termed pTM-NRP1, shows antiangiogenic activity in a xenograft mouse model of glioma and inhibits breast cancer growth and metastasis ,376.The NRP1-binding motif has been harnessed to generated peptides that are directed to and penetrate into the tumor. Such NRP1-specific tumor targeting peptides effectively enhance the uptake of various therapeutic agents . These pDual targeting compounds, such as the peptide RGD-ATWLPPR, encompassing ligands of NRP1 and integrin αvβ3 that are both overexpressed in the tumor microenvironment accumulate with higher specificity in the tumor and, unlike uncoupled NRP1 and integrin ligands, reduce turnover and/or surface internalization of NRP1 .A phase 1 clinical trial of the integrin-binding, tumor tissue-homing peptide CEND-1  in combination with nanoparticle albumin bound (nab)-paclitaxel and gemcitabine in metastatic exocrine pancreatic cancer has started this year LD22-4 is an N-terminal fragment of FGF2 that binds with its CendR motif-corresponding C-terminus (KDPKR) to NRP1 and inhibits in vitro migration of ECs, tumor cells, and fibroblasts, and in vivo, it suppresses tumor angiogenesis and growth in animal models of breast, prostate, and lung carcinoma without being cytotoxic, inducing apoptosis, or affecting cell proliferation rates ,386,387.An immunoglobulin FC-fused tumor tissue penetrating peptide, Fc-TPP11 (HTPGNSKPTRTPRR), binds to the VEGF-binding site of NRP1 with 1000-fold higher affinity as compared to NRP2 . After bThe disulfide-bridged TT1 (CKRGARSTC) and its linear analogon LinTT1 (AKRGARSTA) home to breast cancer in mouse models and enhance the antitumor potency of coadministered therapeutics ,390. AftDocetaxel-loaded nanoparticles that dually target NRP1 and CD44 with the tLyP-1 peptide (CGNKRTR) and hyaluronic acid, respectively, home in on metastatic tumor cells and metastasis-supporting neovasculature and suppress tumor cell invasion and inhibit lung metastasis in three mouse models of triple-negative breast cancer .Diagnostically, lipid microbubbles with conjugated NRP1-targeted peptides CRPPR and ATWLPPR specifically bind to NRP1-expressing cells and allow ultrasound imaging of angiogenic tumors .Theranostically, polysiloxane nanoparticles combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer and the NRP1-targeting motif KDKPPR can be employed to destroy tumor neovessels in glioblastoma, because they accumulate in the tumor vessel wall and show no cytotoxicity unless after exposure to light during photodynamic therapy .CendR peptides also bind to NRP2 and are subsequently internalized . A truncThe risk that tumor-penetrating peptides might also promote the spread of metastases by inducing increased vascular permeability at the tumor site, in addition to improved drug permeation, appears to be limited, as they not only not increased metastasis but also reduced them ,395,396.In addition to reducing intratumoral hypoxia and elevated oncotic pressure and improving the accessibility with chemotherapeutic agents by normalization of the tumor vasculature, as a further possible approach, a tumor-specific vessel disruption in combination with chemotherapeutic agents is also conceivable for some cancers. In xenograft fibrosarcoma and epidermoid carcinoma mouse models, intravenously injected NRP1-specific rhodocetin-αβ selectively destroys the vasculature only in tumors and results in hemorrhage, but not in other tissues .As in normal arteries, NRP1 occurs on the basolateral side of TEC of intrinsically leaky tumor vessels . MoreoveBinding to the b1/b2 domain of NRP1, rhodocetin-αβ triggers NRP1/MET signaling only in cells that expose NRP1 to the bloodstream ,167. TheMacrovipera lebetina inhibits tumor angiogenesis by reducing the expression of NRP1 and VEGF in a quail embryonic chorio-allantoic membrane system as well as in a human colon adenocarcinoma xenograft mouse model [Lebein, a snake venom disintegrin from se model .NRPs, as coreceptors of important RTKs, integrins, and other receptors, are of paramount importance for formation and functioning of the tumor vasculature. In this context, NRPs modulate cellular responses by capturing ligands, regulating growth factor expression, endocytosis and recycling, and by signaling independently. The complex interplay of different cell types within the tumor microenvironment causes dysregulated angiogenic signaling resulting in pathological tumor angiogenesis. The highly irregular shape and comparatively poor functionality of the tumor vasculature complicates treatment with drugs administered via the bloodstream. To promote tumor therapy with cytostatic drugs, vessel normalization is sought. NRPs represent a potential therapeutic target due to their multifaceted roles and the fact that they are highly expressed on tumor ECs and tumor cells. As NRP also plays a key role in the uptake of nutrients by cells, NRP appears to be particularly suitable for introducing drugs into both TECs and tumor cells.


Endometriosis is a disease of theories, but none has succeeded to explain the whole picture. Most widely available drugs for endometriosis aim to relieve symptoms and improve fertility. Unfortunately, many short and long-term side-effects are associated with the treatments. To overcome this problem, researchers have developed many novel therapeutic agents, including non-invasive technique. We aim to provide new insights on pathogenesis model and novel non-surgical treatments for endometriosis, including drugs already available in the market and also drugs which are still under research. Seven novel treatment modalities are recognized, namely dienogest, aromatase inhibitor (AI), gonadotrophine-releasing hormone (GnRH) antagonist, anti tumor necrosing factor (TNF)-α, selective estrogen receptor modulator (SERM), selective progesterone receptor modulator (SPRM), and high-intensity focused ultrasound (HIFU). Dienogest, AI, and GnRH antagonists are effective novel treatments with good tolerance and safety. SERM and SPRM show inconsistent results, while anti-TNF-α is still in the animal experimental stage. HIFU is a potential futuristic treatment. However, it is still a long way until this technology is truly applicable. Endometriosis causes a wide spectrum of symptoms and inflicts heavy socio-economic burden to patients. Endometriosis occurs in about 2-10% of women of reproductive age . The ecoClinical diagnosis of endometriosis is often difficult due to the wide spectrum of symptoms which most are non-spesific. Visual observation through laparoscopy and hystopathological sampling are the gold-standards ,6. The mThe efficacies of conventional therapies are good, but when given for a longer period, some aspects should be considered: 1) significant potential side-effects, especially for reproductive-aged women as the result of hipoestrogenic environment; 2) high relapse rate despite optimal medical therapy, and 3) costly treatments ,10. AlonAmong existing theories on endometriosis pathogenesis, Sampson’s retrograde menstruation theory is the most popular, because it is scientifically proven, easy to understand and widely acceptable. The theory is supported by laparoscopic findings from women on perimenstrual period, of which menstrual blood components were found in peritoneal cavity on 90% of patients ,13,14,15In endometriosis, various biomolecular changes are involved in the development of lesions, including: impaired immune system response, increased cytokines and pro-inflammatory mediators, increased angiogenic activity, excessive estrogen production, and progesterone resistance. Ectopic tissues may avoid normal apoptotic and phagocytosis mechanisms, presumably due to decreased expression of metalloproteinases, CD36 and increased production of dissolved intercellular adhesion molecule-1 .        Increased inflammatory activity is also present in endometriosis, through the overproduction of Interleukin (IL)-1, IL-6, IL-8, monocyte chemo-attractant protein-1, RANTES, tumor necrosis factor (TNF)-α and TNF-β. These mediators will further stimulate the prostaglandins production and triggers the release of vascular endothelial growth factor that serves as pro-angiogenic agent .The most important factor in the pathophysiology of endometriosis is the estrogen hormonal dysregulation and progesterone resistance. Hypomethylation of the CpG cluster changes the balance of estrogen receptors, from alpha subtypes (ERα) dominance into beta subtypes (ERβ) dominance. In endometrial tissue, ERβ binds to the promoter of ERα, suppressing the production of ERα, thereby reducing the formation of progesterone (PR) receptor, resulting in resistance to progesterone. ERβ regulates cell cycle progression, and contributes to the proliferation of endometriotic cells ,21. ProsAn understanding of the biomolecular processes in endometriosis has now brought about the possibility of potential new therapies. These new therapies aim specific pathophysiologic mechanisms that have not been targeted by conventional methods. Although promising, some has not been fully tested in humans, and some are still in the early phase of clinical trials ,25. Dienogest (DNG) is an oral progestin that has been recognized as single-drug therapy for endometriosis in Europe, Japan, Australia and Singapore ,27. DNG DNG 2 mg/day has been shown to significantly inhibit the expression of genes and proteins associated with aromatase and cyclooxygenase (COX)-2, as well as prostaglandin E2 (PGE2) production ,31. DNG The administration of aromatase inhibitors (AI) in endometriosis patients may directly decrease aromatase activity in endometriotic tissue and estrogen level, thereby suppressing COX-2 activity, decreasing PGE2 level, and breaking the positive feedback loop ,36,37,38AIs when combined with progestogen, COC, or GnRH agonist significantly decrease endometriotic pain intensity, thereby improving patient’s quality of life. AI is superior in preventing postoperative recurrence when compared to GnRH or Danazol, within 6 months period ,41. AI iGnRH antagonists act by competitively block GnRH receptor. When compared to GnRH agonist, this class of drugs shows no-flare period, faster therapeutic onset, and unchanged pituitary sensitivity to GnRH after discontinuation of therapy ,46,47,48The most common side effects are hot flush, nausea and headache. With long-term use up to 6 months, these side effects are increased by 10%. Approximately 25% of patients become amenorrhea after 8 weeks of therapy with a dose of 150 mg per day, but this number decreases to 7.6% after 24 weeks . ElagoliIn vitro studies have shown that regression of lesion size, as well as decreased expression of inflammatory cytokines after anti-TNF-α administration  or soluble TNF-α receptors  ,52,53. Istration ,59,60,61stration ,62.The selective estrogen receptor modulator (SERMs) are agents that have the effect of estrogen antagonists on the target organ, and the agonistic effects on bones and blood vessels ,64. Therin vivo (Selective progesterone receptor modulator (SPRMs) are PR ligands with specific clinical effects: agonists, antagonist, or agonist-antagonist combination on progesterone target tissues in vivo . The idein vivo ,70,71. Hin vivo . In animExperimental study on primates by giving asoprisnil and asoprisnil ecamate, resulted in amenorrhoea, endometrial proliferative suppression, and endometrial atrophy . In phasHigh-intensity focused ultrasound (HIFU) is a new technique that utilizes local heating phenomenon. This technique was first introduced by Zhang and Wang  in 1940 Abnormal tissue ablation with USgHIFU in the case of adenomyosis provides good safety and effectiveness as well as significant improvement of clinical symptoms . HIFU haEndometriosis is a gynecologic disorder highly associated with chronic pelvic pain and infertility. Dienogest, AI, and GnRH antagonists have been proven effective as endometriosis therapy in many clnical studies, with good tolerance and safety. Studies on SERM and SPRM are mostly still in phase I and II clinical trials, that show inconsistent results. Anti-TNF-a is still studied in the animal model. HIFU is a potential futuristic treatment. However, it is still a long way until this technology is truly applicable.


Recent advances in the diagnostic and metagenomic investigations of the feline enteric environment have allowed the identification of several novel viruses that have been associated with gastroenteritis in cats. In the last few years, noroviruses, kobuviruses, and novel parvoviruses have been repetitively detected in diarrheic cats as alone or in mixed infections with other pathogens, raising a number of questions, with particular regards to their pathogenic attitude and clinical impact. In the present article, the current available literature on novel potential feline enteric viruses is reviewed, providing a meaningful update on the etiology, epidemiologic, pathogenetic, clinical, and diagnostic aspects of the infections caused by these pathogens. Feline viral gastroenteritis is considered a common worldwide disease, especially in cats younger than one year of age living in high-density cat environments, such as catteries and shelters. The feline panleukopenia virus (FPV) ,2,3, felWithin the last decade, there has been a resurgence of interest for viral gastroenteritis that was sparked by the identification of novel viruses associated with diarrhea either alone or in mixed infections, occasionally resulting in more severe clinical signs. For many years, viral detection was restricted to a few specialized laboratories with EM equipment, and the etiology of a large portion of viral gastroenteric cases remained unknown. However, the introduction of diagnostic molecular tools mainly based on the use of broadly reactive primers, genus- or family-specific, targeting highly conserved genomic regions, increased the viral detection rate significantly, revealing that additional viruses, may be involved in the feline enteritis disease ,13,14,15Information on the epidemiology and genetic heterogeneity of these newly described viruses are still limited, and it is unclear whether these viruses may play a role as enteric pathogens of cats and to which extent they impact on feline health. The aim of this review is to provide an update on novel enteric viruses that have most recently been identified in association with enteritis signs in cats, focusing the main attention on feline norovirus, feline kobuvirus, and novel feline parvoviruses.Noroviruses (NoVs) are a major cause of epidemic gastroenteritis in children and adults. They cause nearly half of all gastroenteritis cases and > 90% of nonbacterial gastroenteritis epidemics worldwide . NoVs haNorovirus in the family Caliciviridae [NoVs are small non-enveloped viruses of 27–40 nm in diameter, belonging to the genus iviridae . The icosahedral capsid surrounds a 7.5–7.7 kb positive-sense single-stranded RNA genome covalently linked to Viral Protein g (VPg) at the 5′ end and polyadenylated at the 3′ end. NoV genome is organized into three Open Reading Frames (ORFs) . ORF1 isThe viral capsid is composed of 180 copies (90 dimers) of VP1, which consists of a shell (S) domain and two protruding (P1 and P2) domains . The P2 Based on the full-length VP1 amino acid sequence, NoVs are classified into at least eight genogroups (GI to GVIII) and >40 genotypes . Only GIFelidae to NoV infections was documented in 2006, in a four-week old lion (Panthera leo) died of severe hemorrhagic enteritis at the zoo of Pistoia, Italy [The first evidence on the possible susceptibility of a member of the family a, Italy . Upon sea, Italy , the novShortly, after this first identification, additional evidence was found on the circulation of NoVs among carnivores. In 2007, a novel NoV strain (dog/Bari/170/07/ITA) was detected in a stool sample collected from a 60-day-old mixed-breed pup with diarrhea and vomiting, hospitalized at the Faculty of Veterinary Medicine of Bari  . In the Since then, by implementing the diagnostic algorithms of gastroenteritis cases with NoV-specific molecular assays, canine NoVs have been repeatedly detected ,45,46,47Direct evidence on the circulation of NoVs in cats was obtained in 2012 in New York State , in whicMore recently, two novel feline NoVs have been identified in Japan  and ItalAlong with the accumulation of point mutations, recombination is a powerful mechanism strongly influencing the evolution and epidemiology of human NoVs . RecombiThe identification of the GIV.2 NoV strain lion/Pistoia-387/06/IT in the lion cub  also repExcept for one study in which GIV.2 NoV like-sequences were identified by the metagenomic approach analyzing pooled fecal samples from clinically healthy cats , to dateThe broadly reactive primers p289–p290  targetinExcept for the murine NoV , a reproSerological studies of NoVs necessarily rely on the expression of synthetic antigens, and the baculovirus system appears to be particularly adequate since the baculovirus-expressed full-length VP1 of NoVs is assembled into virus-like particles (VLPs) morphologically and antigenically indistinguishable from wild-type NoVs . ELISAs Kobuvirus, within the family Picornaviridae [Feline kobuvirus (FeKoV) is a newly discovered virus, belonging to the genus aviridae .Kobuvirus, was first discovered in 1989 as the cause of oyster-associated nonbacterial gastroenteritis in humans in Aichi Prefecture, Japan [Human Aichi virus (AiV) strain A846/88, the prototype strain of the genus e, Japan . Since te, Japan ,70,71,72Recently, novel kobuviruses genetically closely related to human AiVs have been identified in domestic and wild carnivores including dogs ,74, catsKobuvirus currently includes six officially recognized species: Aichivirus A (formerly Aichi virus), Aichivirus B (formerly Bovine kobuvirus), Aichivirus C (porcine kobuvirus), Aichivirus D (kagovirus 1), Aichivirus E (rabbit kobuvirus) and Aichivirus F (bat kobuvirus 1). Feline kobuvirus (FeKoV) is classified within the species Aichivirus A, along with the human AiV, canine kobuvirus (CaKoV), murine kobuvirus (MuKoV), Kathmandu sewage kobuvirus, and roller kobuvirus  of 646–717 nucleotides (nt), one single open reading frame (ORF) of 7308–7467 nt and 3′ UTR of 241–244 nt. The unique ORF encodes a single large polyprotein of 2436–2437 amino acids (aa) that undergoes protease processing to yield a leader protein (L), three structural capsid proteins  and seven non-structural proteins (NSPs) . The FeKoV genome, like that of other kobuviruses, could be divided into three functional regions: the P1 encoding the structural proteins VP0, VP1 and VP3, P2 encoding the NSPs 2A-2C and P3 encoding the NSPs 3A–3D ,89. PutaGenetic distance calculations based on the analysis of the full-length polyprotein of the six FeKoV strains to date available on GenBank database revealed a high identity to each other (92.9%–93.4% nt and 96.8%–98.5% aa), that is consistent with the genetic similarities found for human AiVs and CaKoVs. More recently, further information on the genetic diversity of these group of kobuviruses has been obtained by Niu et al.  in a molThe first evidence on the susceptibility of cats to kobuvirus infections was obtained in the UK  by a serAlthough, the relationship between infections and gastroenteritis in cats is far from elucidated and still requires further epidemiological and experimental studies, the frequency of detection of FeKoV has been found to differ significantly between symptomatic and asymptomatic cats ,86. In aIn most cases, FeKoV-infected cats were also co-infected by other viral pathogens like FPV, FeCoV, feline NoV, and feline bocavirus (FBoV) ,86,87. FKobuviruses [Conventional RT-PCR is the primary assay for the detection of FeKoVs. The first identification of FeKoVs RNA was obtained by using the primers set 10f and 10r  targetinuviruses . These buviruses ,77,80,85Replication in vitro of kobuviruses has been demonstrated only for AiV strain A846/88  and boviAn ELISA and an immunofluorescence assay have been set up using the human AiV strain A846/88-infected cells as antigen. These assays were successfully used to assess the exposure of domestic carnivores to kobuviruses . HoweverParvoviridae) are non-enveloped icosahedral viruses of 22–25 nm with a positive-sense single-stranded (ss) DNA genome of 4.5–5.5 kb with complex hairpin structures at the 5′ and 3′ ends, and it encodes three or fpur proteins; non-structural (NS) 1, nucleoprotein (NP) 1, and viral protein (VP) 1 and VP2 [Parvoviridae family is divided into two subfamilies, Parvovirinae and Densovirinae [Parvovirinae subfamily is enclosed the genera Bocaparvovirus and Protoparvovirus, whose members have been identified in a wide range of mammalian species, including domestic carnivores.Parvoviruses , causes feline panleukopenia, the oldest known viral disease of cats [Feline parvovirus (FPV), currently classified within the species  of cats ,3 and ce of cats . Infecti of cats .Bocaparvovirus and Protoparvovirus have been identified in cats [More recently, novel parvoviruses belonging to the genera  in cats ,14,16,17 in cats ,14, bloo in cats .Bocaparvoviruses (BoVs) have been detected in a number of mammalian species including humans and non-human primates ,99,100, Bocaparvovirus is classified into twenty-three officially recognized species, with at least five species detected in domestic carnivores and designated Carnivore bocaparvovirus 1–5; a sixth species (Carnivore bocaparvovirus 6) includes bocaparvoviruses detected in minks [Carnivore bocaparvovirus 3 [Carnivore bocaparvovirus 4 [Carnivore bocaparvovirus 5 [Common structural features of the bocaparvoviruses include the approximately 5.5 kb ssDNA genome and two main ORFs coding the NS1 (ORF1) and the capsid protein precursor VP1/VP2 (ORF2). A third additional ORF (ORF3), located between ORF1 and ORF2, encodes the NP1 , a highlin minks . Feline ovirus 3 , Carnivoovirus 4 , and Carovirus 5 .Carnivore bocaparvovirus 3. Subsequently, by metagenomics analysis of feces collected from a healthy cat in Portugal [Carnivore bocaparvovirus 4 [Carnivore bocaparvovirus 5, has been detected in California [FBoV DNA was firstly identified by assessing fecal, nasal, urine, kidney, and blood samples collected from stray cats during a large molecular survey conducted in Hong Kong . The neaPortugal , an addiPortugal . Accordiovirus 4 . The thilifornia  by next-lifornia , both FBlifornia ,115,116.lifornia ,121,122.lifornia . In dogslifornia . Furtherlifornia ,125.The pathogenicity of FBoV has not been experimentally assessed and it is not clear if this virus may play a role in enteric or extra-enteric diseases in cats. FBoV DNA was first identified in multiple tissues, specifically in 26 (7.2%) of 363 fecal samples, six (1.6%) of 364 urine samples, three (0.8%) of 361 blood samples, one (0.3%) of 364 nasal samples, and one (2.0%) of 51 kidney samples collected from a total of 364 stray cats in Hong Kong, suggesting a wide tissue tropism . After tCarnivore protoparvovirus 1 (genus Protoparvovirus) includes genetically and antigenically related viruses such as FPV, canine parvovirus (CPV-2), mink enteritis virus (MEV), and raccoon parvovirus (RaPV) [The species s (RaPV) , causingFPV, isolated for the first time in 1965 , has beeCarnivore protoparvovirus 1, while the closest relatives to CBuV were protoparvoviruses identified (47.2%–51.4% aa identity in NS1) in human and non-human primates, commonly termed as bufaviruses (BuVs) [Protoparvovirus, for which the name “Carnivore protoparvovirus 2” has been proposed [In 2017, a novel protoparvovirus distantly related to FPV and CPV-2 was identified in cats, by assessing respiratory specimens collected from cats with or without signs of upper respiratory tract disease (URTD) and stool samples from diarrheic animals . Sequencs (BuVs) ,143,144.s (BuVs) , the newproposed . More reproposed , which tproposed .Whether these protoparvoviruses are associated with any disease in cats is not yet known. Until now, BuVs have been identified in humans and recently in wild carnivores (wolves and foxes), exclusively in the enteric tract ,146, butAstroviridae, are small, non-enveloped, spherical viruses of approximately 28–30 nm in diameter, with a positive single-stranded RNA genome of 6.8–7.3 kb in length organized in three ORFs  and a poly A tail at the 3′ end [Mamastrovirus  and Avastrovirus (AstVs of avians) [Mamastrovirus 2, formerly known as feline astrovirus. Sequence analysis of the complete ORF2 gene revealed that the feline strains clustered within two distinct groups that have been recently proposed as different genotypes (group 1 and 2) [In addition to noroviruses, kobuviruses, and parvoviruses, other viruses such as astroviruses, rotaviruses, novel circular replication-associated protein-encoding single-stranded (CRESS) DNA viruses , and Lyoe 3′ end . AstVs h avians) . AstVs a avians) . Feline  avians) ,153. In 1 and 2) . Further1 and 2)  have occ1 and 2) ,156.To date, FAstVs have been detected in stool samples of cats in UK , AustralRotavirus, family Reoviridae) are characterized by a 70–75 nm non-enveloped multi-layered virion with 11-segmented double-stranded RNA encoding six structural proteins  and other five or six NSPs  [Felis catus shared a monophyletic root, being most closely related to the two currently known rotavirus species I strain detected in the feces of two sheltered dogs in Hungary [Rotaviruses (RVs) have been recognized as a major cause of human acute gastroenteritis since 1973 . They prP5/NSP6) . To dateP5/NSP6) , and a tP5/NSP6) . Among tP5/NSP6) . The genP5/NSP6) . CurrentP5/NSP6) ,167. FirP5/NSP6) . SubsequP5/NSP6) , while oP5/NSP6) . Early eP5/NSP6)  to 100% P5/NSP6) , while EP5/NSP6)  either iP5/NSP6) . SequencP5/NSP6) . More reP5/NSP6) . Phyloge Hungary . Overall Hungary . HoweverCircoviridae family [Circoviridae, but into a distinct clade to that of circovirus and cyclovirus. Further investigations aimed to acquire information on the genetic features, epidemiology and pathogenesis of these viruses are needed. Polyomaviruses (PyVs), belonging to the Polyomaviridae family, are non-enveloped double-stranded DNA viruses with a circular genome of approximately 5.0 kb in length. PyVs DNA have been occasionally identified in fish, birds, rodents, and primates [Alphapolyomavirus detected in saliva and skin samples of human origin [Mayor CRESS DNA viruses detected in animals are represented by circoviruses and cycloviruses belonging to the e family . The suse family ,178. In e family ,180. By e family ,17. By me family , viral Dprimates ,182. Intprimates . Upon sen origin  and provIn the last few years, a number of viruses have been found in association with enteric disease in cats. Many of them have been discovered serendipitously, using advanced molecular techniques to screen feline stool samples. The epidemiology of these newly discovered viruses is still largely unexplored, but several pieces of evidence suggest their possible role as primary causative pathogens or synergistic agents in feline gastrointestinal disease. In order to obtain a complete picture, each novel enteric virus should be included in the panel of pathogens for routine testing of cases of feline enteritis. Furthermore, large structured epidemiological studies and experimental infections might help to clarify any possible association with enteric diseases.Interestingly, some of the viruses considered in this review have also been identified in the canine fecal virome, suggesting the possibility of inter-species circulation between the two carnivore species. Cats and dogs may harbor NoVs of the same genogroups and genotypes, GIV.2 and GVI.2 ,26,48. BThe global distribution of cats and their close contacts with humans represents an additional reason to better understand the composition of their enteric virome . Histori


Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 − log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4–24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant  Staphylococcus aureus represents a widely disseminated and invasive pathogen causing clinically important and life-threatening infectious diseases [S. aureus colonizes 30% or more of all humans, primarily the vestibules of the nasal cavity, the risk for subsequent infections originating from the patients’ own flora is significantly increased [S. aureus (MRSA) drastically aggravates the treatment by limiting the therapeutic options and necessitates expansive preventive measures, therefore resulting in additional morbidity, mortality, and costs [S. aureus infections, particularly those due to MRSA, are frequently based on the topical application of the antibiotic agent mupirocin [diseases ,2. Sincencreased ,5,6,7. Tnd costs ,8,9. Resnd costs ,11,12,13upirocin ,15,16, bupirocin ,18,19. Pupirocin ,22,23,24Beyond these classical resistance mechanisms, resistance based on microbial biofilm lifestyle or the formation of the small-colony variant (SCV) phenotype may render microbial isolates functionally resistant even if they are susceptible to antibiotics. Moreover, the administration of antibiotics, in particular long-term exposure, may select for the generation of SCVs ,26. The The ability of SCVs to persist within eukaryotic cells and their changed physiology is also of particular therapeutic significance, as the activity of many antibiotic compounds may be attenuated. In particular, β-lactams, which in general represent the most effective anti-staphylococcal agents, are not bioavailable in the intracellular milieu . MoreoveS. aureus infections due to classical and functional resistance mechanisms necessitate alternative prophylactic and therapeutic options. Bacteriophage-derived endolysins exhibit the ability to disrupt bacterial cell walls in a highly specific manner [Pseudomonas aeruginosa [Streptococcus spp. [Staphylococcus spp. [Obviously, the limited treatment options of c manner . Recombic manner . In the c manner . Only inruginosa , Streptocus spp. ,53,54,55cus spp. ,60,61,62cus spp. ,64,65. S. aureus [S. aureus, this CHAP domain cleaves between d-alanine at the termini of the tetrapeptide and glycine of the pentaglycine crossbridge, and is thereby responsible for the lytic mode of action [Staphylococcus aureus (MSSA) and MRSA isolates [The recombinant endolysin HY-133  used in this study represents a stability-optimized derivative of endolysin PRF-119 ,60. The . aureus ,62. Its . aureus . Within f action . HY-133 isolates ,60,61,62S. aureus strain pairs, each comprising WT and stable SCV phenotypes. Since the pulsed-field gel electrophoresis (PFGE) fragment patterns of the WT-SCV pairs were indistinguishable or varied in only one band, all strain pairs were considered clonally identical according to published guidelines [We previously demonstrated the very fast and high bactericidal in vitro activity of the recombinant endolysin HY-133 against several clinical MSSA and (LA-)MRSA isolates ,60,61,62idelines  . hemB mutant SCV IIb13. MIC50/90 and MBC50/90 for HY-133 tested against the 12 clinical strain pairs were similar for the different phenotypes or growth phases. The same applied for oxacillin which, however, exhibited slightly higher MIC50/90 and MBC50/90 values for clinical strains than HY-133  when measured from cultures in stationary growth phase. Measurements from logarithmic growth cultures revealed the same MIC and MBC values against 6850 (WT) and IIb13 (SCV), being 0.25 mg/L for both strains and reflecting values detected during the stationary phase. However, HY-133 MIC and MBC values measured against the gentamicin-selected SCV JB1 were increased (1 mg/L) in this growth phase. For oxacillin, MIC and MBC were substantially lower against the 6850-derived SCVs JB1 and IIb13 than against the corresponding WT and the clinical strains, irrespective of the growth phase .n = 2/48) of cases with an MBC:MIC ratio of 2. In either case, the SCV phenotype tested from stationary growth cultures was affected. However, when strains were treated with oxacillin, 20.8% (n = 10/48) showed a higher median MBC than MIC with 40% (n = 4/10) being WTs and 60% (n = 6/10) being SCVs, respectively, and an MBC:MIC ratio of 2 in each case.Furthermore, for HY-133, the median MBCs were higher than the median MICs in 4.2%  strain ATCC 29213 showed oxacillin values in the range as required by the Clinical & Laboratory Standards Institute (CLSI) guidelines . There iTime-kill curves were performed for the two representative clinical strain pairs OM299 and 4652 as well as for the strains of triplet 6850. Time-kill curves revealed distinct growth characteristics of the two phenotypes with clinical and 6850-derived SCVs exhibiting a clear growth retardation compared to the WTs, as described in previous studies ,43,70. Fp ≤ 0.001 for all concentrations, except for 0.25 mg/L HY-133 against strain 6850), already after 1 h of incubation. At this time point, a ≥3 − log10 decrease in CFU/mL (99.9% killing) was shown for all strains irrespective of the phenotype when applying a concentration of 4 mg/L (p ≤ 0.001). A concentration of 1 mg/L of HY-133 was bactericidal for most of the strains within 1 h except for the clinical WT strain OM299-1 as well as for SCVs JB1 and IIb13 , borderline oxacillin-resistant isolates, as well as isolates that were found to be resistant to ceftaroline/ceftobiprole [S. aureus isolates as mupirocin or daptomycin, with an even faster mode of killing [In recent years, several in vitro and in vivo studies revealed the high potential of endolysins for the treatment of several bacterial infections . HY-133 obiprole ,60,61,62S. aureus clonal lineages possessing polyribitol phosphate wall teichoic acid (WTA) [S. aureus PS 187, which belongs to a distant S. aureus lineage  often isolated from dogs [In contrast to the vast majority of id (WTA) , the unurom dogs , was lesrom dogs . Also SCrom dogs ,46,47,73rom dogs ,43,74. AKayvirus bacteriophage with broad S. aureus host range, was shown to exhibit growth phase-dependent activity [To consider the divergent growth characteristics of SCVs , the detactivity . The kilactivity . The obsmenD SCV mutant, hyper-susceptibility to β-lactams in a macrophage cell model has been shown [Whereas MIC and MBC values for oxacillin did not show significant differences to MIC and MBC values of HY-133, time-kill studies revealed that the onset of bactericidal effect of oxacillin (4 mg/L) was not as fast as that detected for HY-133 within 1 h and was reached only after 4 to 24 h. For strain pair OM299, including an SCV exhibiting a considerable growth retardation compared to its WT, the detected killing effect of the SCV by oxacillin was clearly delayed compared to its WT. For β-lactams, tolerance is a known phenomenon in slow-growing or dormant bacteria ,35, whicen shown . For HY-S. aureus, thus inducing cellular lysis. This form of cell death occurs independently of cellular processes. In contrast, most conventional antibiotics operate by inhibiting essential cellular functions [This study highlights one of the most relevant advantages of HY-133, i.e., its particular mode of action. The modular design of HY-133 is responsible for the species-specific cleavage of the peptidoglycan of unctions , e.g., iunctions . Furtherunctions ,39,40,79For HY-133, the killing kinetics revealed an in vitro regrowth phenomenon for all strains tested appearing after prolonged incubation. This regrowth occurred even after an initial killing of 99.9% of the bacerial population. Its extent depended on both the concentration of HY-133 and the strain used. A similar in vitro regrowth phenomenon was previously shown for clinical MSSA and MRSA isolates . FurtherS. aureus cells as well as for bacterial cells with impaired growth. S. aureus SCVs possess the capacity to enter and persist in host cells [Streptococcus pyogenes without damaging the host cell [The present study demonstrates that HY-133 caused very rapid cell death independent of the rate of bacterial growth, the consistency of the cell wall, and the distinct growth phase. Although mutants resistant to the bacteriocin lysostaphin have been created ,81, selest cells ,79,87,88ost cell .S. aureus WT and SCV isogenic strain combinations, irrespective of their exhibited phenotype or genetic background. Due to its highly bactericidal activity and its high specificity, HY-133 is a promising candidate for future anti-staphylococcal therapy, decolonization, and prevention, particularly for difficult-to-treat phenotypes such as SCVs.In summary, HY-133 was shown to be highly bactericidal in the logarithmic as well as in the stationary growth phases of all tested clinical S. aureus WT isolates and their corresponding stable SCVs were obtained from patients of the University Hospital Münster (UKM), Münster, Germany  after approximately 48 h of incubation, decreased pigmentation, and reduced hemolytic activity [Twelve clinical many see  [39,89,9lsewhere ,92,93. Iactivity . The pheS. aureus skin abscess [Furthermore, the highly cytotoxic MSSA 6850—originally isolated from a patient with a complicated  abscess —and the Strains were cultivated on Columbia blood agar at 37 °C for 24–48 h. Unless otherwise stated, liquid cultures were grown aerobically overnight in 10 mL cation-adjusted Mueller Hinton II broth  in 100-mL glass baffled flasks at 37 °C and 160 rpm.SmaI macrorestriction analyses of total bacterial DNA and resolving the digests with the use of PFGE, as previously described [Insofar as this has not already been done in previous studies ,89,90, tescribed . Strainsescribed .5 CFU/mL. Furthermore, to test the activity of HY-133 and oxacillin under logarithmic growth conditions, colonies from overnight Columbia blood agar cultures were used to prepare 10 mL tryptic soy broth  cultures with a starting OD578 of 0.05. After 3 h of incubation at 37 °C and 160 rpm, starting inocula of 5 × 105 CFU/mL were prepared. Microplates were incubated at 37 °C for 18–20 h for HY-133 and 24 h for oxacillin.The MIC and MBC values of HY-133  and oxacillin  were determined in sterile 96-well microplates  using standard CLSI broth microdilution methodology for staphylococci ,97. CAMHS. aureus ATCC 29213 was used as a QC strain. For the evaluation of the MBC, 10 µL of culture medium from each microwell displaying the MIC and at least one concentration above were inoculated onto tryptic soy agar  plates. After incubation at 37 °C overnight, CFUs were counted. The MBC was defined as the concentration of antimicrobial substance generating <500 CFU/mL  . MICs an5 CFU/mL, the adjusted cultures were diluted in either CAMHB for HY-133 or CAMHB with 2% NaCl for oxacillin and supplemented with the appropriate amount of antimicrobial substance. MICs of the strains used for time-kill studies differed slightly depending on the growth phase tested (see 10 of the numbers of CFU/mL) versus the time were plotted in graphs for each analyzed strain and antimicrobial (mean ± standard deviation).In addition to MIC and MBC determination, the killing kinetics of HY-133 and oxacillin were evaluated by means of time-kill curves for two representative strain pairs (OM299 and 4652). Moreover, the killing kinetics of HY-133 were determined for triplet 6850. Time-kill curves were performed in triplicate with the macrodilution method in accordance with CLSI guideline M26-A . Brieflysted see . For a dp value of ≤ 0.001 was considered significant.Statistical analysis and graphs were made using GraphPad PRISM software version 5.0 . Changes in the number of CFU/mL after 1 h of incubation analyzed by time-kill curves were compared by one-way analysis of variance (ANOVA) with Dunnett’s test. A


Mitogen-activated protein kinases (MAPKs) are key regulators of signal transduction and cell responses. Abnormalities in MAPKs are associated with multiple diseases. Dual-specificity phosphatases (DUSPs) dephosphorylate many key signaling molecules, including MAPKs, leading to the regulation of duration, magnitude, or spatiotemporal profiles of MAPK activities. Hence, DUSPs need to be properly controlled. Protein post-translational modifications, such as ubiquitination, phosphorylation, methylation, and acetylation, play important roles in the regulation of protein stability and activity. Ubiquitination is critical for controlling protein degradation, activation, and interaction. For DUSPs, ubiquitination induces degradation of eight DUSPs, namely, DUSP1, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, and DUSP16. In addition, protein stability of DUSP2 and DUSP10 is enhanced by phosphorylation. Methylation-induced ubiquitination of DUSP14 stimulates its phosphatase activity. In this review, we summarize the knowledge of the regulation of DUSP stability and ubiquitination through post-translational modifications. Mitogen-activated protein kinases (MAPKs) are the important components of cell signaling pathways. MAPKs regulate physiological and pathological responses to various extracellular stimuli and environmental stresses ,4,5,6,7.The DUSP family phosphatases dephosphorylate both threonine/serine and tyrosine residues of their substrates. All DUSPs have a common phosphatase domain, which contains conserved Asp, Cys, and Arg residues forming the catalytic site. A subfamily of DUSPs contains the MAP kinase-binding (MKB) motif or the kinase-interacting motif (KIM) that interacts with the common docking domain of MAPKs to mediate the enzyme–substrate interaction ,15. DUSPDUSPs do not always require phosphatase activity to regulate the function of substrates. For example, DUSPs can control functions of MAPKs by sequestering them in the cytoplasm or nucleus ,17,18. BDUSPs are critical for the regulation of MAPK activity and are thus subject to complex regulation. Gene expression and phosphatase activity of DUSPs are regulated by gene transcription, protein modification, or protein stability. This review will focus on the regulation of DUSP protein stability and ubiquitination by post-translational modifications. Ubiquitination regulates many biological functions such as cell proliferation, cell apoptosis, and immune responses . UbiquitDUSP1 (MKP1) is a nuclear phosphatase . DUSP1 bDUSP4 (MKP2) preferentially inhibits ERK and JNK . In seneDUSP5 displays phosphatase activity toward ERK; DUSP5 overexpression results in both inactivation and nuclear translocation of ERK . DUSP5 iDUSP6 (MKP3) preferentially inhibits ERK ,42. ReduDUSP7, an ERK phosphatase, is ubiquitinated under hypoxic stress . HypoxiaDUSP8 (M3/6) preferentially inactivates JNK and maybe p38 ,49,50. TDUSP9 (MKP4), an ERK phosphatase, is associated with maintenance of the stemness of embryonic stem cells (ESCs) . The lonDUSP16 is also regulated by ubiquitin-mediated proteasomal degradation, and their ubiquitinations are regulated by phosphorylation (see below). In addition to Lys48-linked ubiquitination, one DUSP (DUSP14) is regulated by Lys63-linked ubiquitination see .DUSP1 stability is differentially regulated by sustained or transient activation of ERK. Sustained activation of ERK phosphorylates DUSP1 on Ser296 and Ser323 residues . The SerDUSP1 stabilization results in inhibition of JNK and p38 activation, as well as subsequent inhibition of TNF-α and IL-6 production . GlucocoDUSP2 stability is induced by the atypical MAP kinase ERK4 . Wild-tyDUSP4 is rapidly induced after ERK activation . ERK intDUSP6 can also be a substrate of ERK. Upon serum stimulation, ERK phosphorylates DUSP6 on Ser159 and Ser197 residues in fibroblast cells . The ERKDUSP6 is ubiquitinated and degraded by proteasome in the early phase of platelet-derived growth factor-B chains (PDGF-BB) stimulation; the process requires MEK-induced phosphorylation of DUSP6 on Ser174 residue [DUSP10 is phosphorylated by mTORC2 on Ser224 and Ser230 residues upon insulin stimulation, leading to stabilization of DUSP10 and subsequent inactivation of p38 in glioblastoma cells .DUSP16 preferentially inactivates JNK  and maybDUSP1 and DUSP4 proteins can be oxidized under oxidative stress. Oxidation of catalytic cysteine within the active site of DUSPs inactivates DUSP phosphatase activities and triggers their proteasomal degradation ,79. Supe2+)-induced oxidative stress, DUSP4 is oxidized by glutathione disulfide (GSSG), leading to DUSP4 degradation and cell apoptosis [2+)-induced apoptosis [DUSP4 is redox sensitive. Under cadmium ion  is a MAP kinase phosphatase that inactivates JNK, ERK, and p38 in vitro . DUSP14 f DUSP14 ,87. Methf DUSP14 ,86,87 (FFH) differentiation [DUSPs are involved in immune cell homeostasis, inflammatory responses, metabolic regulation, and cancer development/progression ,88. For ntiation . DUSP6 kntiation . Moreoventiation ,92. DUSPntiation . DUSP3 dntiation ; consistntiation . DUSP5 intiation ,97. In antiation ; consistntiation . DUSP22 ntiation ,101. TheThe DUSP family phosphatases are key regulators of MAPK activity . BecauseThe protein stability of DUSP1, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, and DUSP16 are regulated by ubiquitin-mediated proteasomal degradation. Protein stability of DUSP2 and DUSP10 is increased by ERK and mTORC2, respectively ,75; howe2+-induced oxidative stress triggers DUSP4 oxidation by glutathione disulfide (GSSG), resulting in protein degradation of DUSP4 [In addition to phosphorylation, oxidation of DUSPs also induces protein degradation. Asbestos or TNFα stimulation generates ROS that oxidizes DUSP1 and induces DUSP1 proteasomal degradation ,82. Cd2+of DUSP4 . Ubiquitination not only controls protein stability but also induces protein activity. The ubiquitin E3 ligase TRAF2-mediated Lys63-linked ubiquitination of DUSP14 enhances its phosphatase activity. It would be interesting to study whether Lys63-linked ubiquitination also regulates the activity of other DUSPs.To our knowledge, eight DUSPs  are known to be degraded by the proteasome, and it is important to understand whether other members of the DUSP family are also regulated by ubiquitin-mediated proteasomal degradation. Lastly, it would be useful to study whether other modifications, such as sumoylation, neddylation, acetylation, and glycosylation also control DUSP ubiquitination and protein stability.


BRAF (primarily p. V600E) and RAS oncogenes. Ultrasound (US) examination provides significant diagnostic data in the management of thyroid nodules, as many sonographic features of thyroid lesions are correlated with the potential risk of thyroid carcinoma. The aim of the study was to analyze the current literature in regard to the potential associations between genetic landscape and sonographic features of PTC. Based on the current literature, sonographic features of PTCs correlate with their molecular drivers, particularly between tumors harboring V600EBRAF versus activating RAS mutations, although many of these findings appear to be dependent on the tumor variant. Suspicious US findings, such as hypoechogenicity, spiculated/microlobulated margins, non-parallel orientation/taller-than-wide shape, and the presence of microcalcifications, are typical for PTC positive for V600EBRAF mutations. On the contrary, tumors with RAS mutations are most frequently hypo- or isoechoic and ovoid-to-round in shape, with smooth margins and without calcifications. There are also some US features typical for PTCs harboring other mutations, including K601EBRAF, RET/PTC rearrangements, PAX8-PPARγ, CTNNB1, and APC. However, further research is necessary, as some rare PTC variants still cannot be reliably analyzed due to the scarce published data.Papillary thyroid carcinoma (PTC), the most common thyroid cancer, is predominantly driven by mutations in  BRAF (predominantly p. V600E) and activating RAS mutations. Less frequent drivers include other non-V600E BRAF mutations, APC and CTNNB1 mutations, as well as NTRK, ALK, and BRAF fusions [Papillary thyroid carcinoma (PTC) is the most common thyroid cancer, accounting for over 90% of all thyroid malignancies. Several mutations comprise the underlying molecular etiology of PTC, including Ultrasound (US) examination is a precise diagnostic tool in the management of thyroid nodules as many sonographic features of thyroid lesions provide information about the potential risk of thyroid carcinoma. Moreover, sheer wave sonoelastography was demonstrated to provide significant additional data facilitating the assessment of the risk of malignancy . Many USIt seems reasonable to believe that the presence of a specific mutation leading to PTC may affect the ultrasound image of the lesion. Recently, we demonstrated that genetic drivers can significantly affect the US pattern of thyroid lesions in subacute thyroiditis . It can The aim of this review is to answer the question whether—on the basis of the most recent literature—there is a relationship between the genetic drivers of PTCs and their ultrasound image. A study selection flowchart is available in the The most recent improvements in thyroid nodule classifications began in March 2015, when the Endocrine Pathology Society reclassified encapsulated follicular variant of PTC (EFV PTC) or clearly delimited non-invasive follicular variants of papillary thyroid carcinoma under the proposed nomenclature “non-invasive follicular thyroid neoplasm with papillary-like nuclear features” (NIFTP) ,11. ThisThe new WHO classification schema also introduced two additional diagnoses under this new category: A follicular tumor of uncertain malignant potential (FT-UMP) and a well-differentiated tumor of uncertain malignant potential (WDT-UMP), both of which were borderline between follicular adenoma and carcinoma or follicular adenoma and a follicular variant of papillary carcinoma. In these new entities, nuclear features of PTC as well as lack of capsular or vascular invasion are not so clearly expressed as in NIFTP .Importantly, a new classification of PTC variants was provided, with the following variants being included: (1) Conventional/classic variant; (2) papillary microcarcinoma; (3) encapsulated PTC—applicable to fully encapsulated classic PTC; (4) follicular variant (FV)—invasive PTC with an exclusively or almost exclusively follicular pattern, and no developed papillae; (5) diffuse sclerosing variant; (6) tall cell variant; (7) columnar cell variant; (8) cribriform-morular variant; (9) hobnail variant—new entity; (10) PTC with fibromatosis/fascitis-like; 11) solid/trabecular variant; 12) oncocytic variant; (13) spindle cell variant and giant cell carcinoma; (14) clear cell variant; (15) Warthin-like variant; and (16) combined papillary and medullary carcinoma [ solid/tr oncocytiIn recent years, several classifications of thyroid nodules based on the presence of US high-risk features were introduced ,5,6,7,8.ATA classification distinguishes five groups of US risk—benign, very low suspicion, low suspicion, intermediate suspicion, and high suspicion . The lasBRAF in PTC, N-K-H-RAS in PTC and/or follicular thyroid carcinoma (FTC), RET/PTC in PTC, and PAX8/PPARγ in FTC [RET/PTC1, RET/PTC3, and RET/PTC4 are formed as a result of intrachromosomal rearrangements (paracentric inversions within the long arm of chromosome 10). Several other RET/PTC rearrangements result from inter-chromosomal translocations [Initiating mutations or gene fusions in the thyroid carcinoma include: γ in FTC . RET/PTCocations .BRAF in 40% to 45%, RET/PTC in 6% to 30%, telomerase reverse transcriptase (TERT) promoter in up to 23.5%, RAS in 10% to 20%, and TRK in <5%; FTC: RAS in 40% to 50%, PAX8-PPARγ in 10% to 35%, PIK3CA in <10%, and PTEN in <10%; poorly differentiated carcinoma: RAS in 25% to 30%; CTNNB1 in 10% to 20%, TP53 in 20% to 30%, and BRAF in 10% to 15%; familial forms of medullary carcinoma (MTC): RET in >95%; and sporadic MTC: RET in 40% to 50% [The prevalence of mutations according to the thyroid tumor type was demonstrated as follows: PTC: % to 50% ,18,19.PIK3CA, p53, CTNNB1, and TERT promoter mutations [Some of the mutations can serve as molecular markers of the aggressiveness of differentiated thyroid carcinoma (DTC). Such markers include the presence of >1 driver mutation, and utations ,20.BRAF mutations have been reported in classic, Hürthle, tall cell, columnar cell, hobnail, Warthin, and diffuse sclerosing variants. V600EBRAF is a common mutation characteristic for PTC displaying a papillary or mixed follicular-papillary growth pattern [V600EBRAF mutation. Lee et al. [V600EBRAF mutation was associated with the following US features: Solid composition, marked hypoechogenicity, irregular margin, taller-than-wide shape, and the presence of microcalcifications. Similarly, Khadra et al. [V600EBRAF positivity. Unfortunately, they did not analyze the tumor shape. Additionally, they observed that V600EBRAF-positive PTCs were associated with a higher rate of lymph node metastasis and the rate of extrathyroidal extension [V600EBRAF mutation and extrathyroidal invasion, as well as US features of the higher ATA risk category of PTC [V600EBRAF mutation was associated with hypoechogenicity, microcalcifications, and a nodule diameter <1 cm. Kabaker et al. [V600EBRAF positivity was associated with such suspicious US findings as a taller-than-wide shape, ill-defined margins, hypoechogenicity, micro/macrocalcifications, and an absent halo. On the contrary, V600EBRAF mutations were not associated with non-cystic composition. The authors stated that BRAF mutation can be predicted with a positive predictive value of 82% when at least three of the above-mentioned suspicious US features are present [ pattern . Wang et pattern  demonstre et al.  found tha et al.  observedxtension . Very rey of PTC . In the y of PTC , BRAFV60r et al.  observed present . A talle present . Those o present ,25,26,27V600EBRAF mutation is the most frequent (90%) genetic driver of this variant [The classic variant is the most common PTC variant (32–49%), and the  variant ,29,30. A variant . V600EBRAF was detected in tumors displaying a papillary or a mixed growth pattern but not in those with a follicular pattern [In the Hürthle cell variant,  pattern . In US,  pattern .V600EBRAF mutations, is reported as 4% to 17% of PTCs [V600EBRAF mutation, which include a solid structure, hypoechogenicity, with or without microcalcifications, a spiculated/microlobulated margin, and non-parallel orientation with frequent nodal metastass [The incidence of tall cell variant, which is also frequently (over 92%) associated with  of PTCs . Ultrasoetastass ,31.V600EBRAF mutation occurs in one-third of these PTCs [V600EBRAF mutation is frequent in these more aggressive tumors while the encapsulated ones are often V600EBRAF negative [V600EBRAF is an adverse prognostic factor in the columnar cell variant due to its association with unfavorable clinicopathological characteristics. Older patient age, male gender, and distant metastasis were highly prevalent features for these BRAF-positive tumors [V600EBRAF mutation in 58% of cases. [The very rare columnar cell variant also presents typical malignant features, and ese PTCs . Encapsuese PTCs ,32. Intef cases. . In US, f cases. ,33. TumoV600EBRAF mutations are less frequent than in the classic variant (65% vs. 84%) [BRAF mutation is still the most common genetic driver. The most common US features of WV PTCs are a solid composition, hypoechogenicity, and taller-than-wide shape [V600EBRAF mutations may differ depending on the presence of Hashimoto’s thyroiditis. Microcalcifications were observed as typical for V600EBRAF-positive nodules without Hashimoto’s thyroiditis while a solid nodule structure was more common in V600EBRAF-positive nodules with Hashimoto’s thyroiditis [The Warthin-like variant (WV) is associated with Hashimoto’s thyroiditis in 93% to 100% of all cases . Typicalvs. 84%) , but BRAde shape ,35. The roiditis .BRAF mutation, with a US pattern typical for these mutations [Papillary thyroid microcarcinoma (PTMC) constitutes approximately 30% of all PTC cases, and it is also typically associated with utations ,30.V600EBRAF mutations are rare (24%) in the diffuse sclerosing variant (DSV) [nt (DSV) , but theV600EBRAF mutations frequently demonstrate typical US high-risk features, including hypoechogenicity, microcalcifications, irregular and/or ill-defined margin, taller-than-wide shape, and extrathyroidal invasion. On the basis of the presented literature review, PTCs harboring V600EBRAF mutation is presented in A sonographic pattern typical for PTCs bearing the V600EBRAF mutations and sonographic findings in PTC are widely described, although there are also a few reports in which such associations were not observed [V600EBRAF-positive and –negative tumors without any assessment of the PTC variants. The authors claimed not to observe an association with the US pattern, although correlations between the presence of V600EBRAF mutations and features, such as a larger tumor size, extrathyroidal extension, and lymph node metastases, were described [The correlations between the presence of observed ,38. The escribed .K601EBRAF mutations are present in most cases of the infiltrative follicular variant (FV) [ant (FV) . In US, ant (FV) ,39. The RET/PTC rearrangements are the second most common genetic alteration in PTC, mainly being a genetic driver of the classic variant. More rarely, it can be associated with other variants of PTC, especially with the diffuse sclerosing variant (DSV) [RET/PTC1 and RET/PTC3. The prevalence of RET/PTC rearrangements in PTC is 6% to 30%, and they occur more commonly after radiation exposure and/or in children [RET/PTC3 rearrangement [RET/PTC rearrangement is the most common genetic feature in DSV [RET/PTC rearrangements seems to be different in RET/PTC1- and RET/PTC3-positive cancers. Typical diffuse involvement with scattered microcalcifications without any visible nodule is much more frequent in RET/PTC3-driven tumors. The coexistence of DSV and Hashimoto’s thyroiditis is characteristic for RET/PTC1 mutations. In these last cases, microcalcifications are less frequent than in RET/PTC3 tumors [V600EBRAF mutations are rare (24%) in DSVs [RET/PTC1 fusions and are significantly different from those with RET/PTC3 rearrangement. In US, V600EBRAF-positive DSVs are more similar to other PTCs that harbor V600EBRAF than a typical DSV pattern, as the nodules are usually clearly visible and diffuse microcalcifications occur in only 33% of cases [RET/PTC3 rearrangement are presented in nt (DSV) ,40. The children ,19. Datachildren ,19,25,37e in DSV ,19. The e in DSV . Despitee in DSV . This vaRAS mutations are typical for follicular tumors (including adenomas and carcinomas), but they also occur in the follicular variant (FV) of PTC. The follicular variant is the second most common type of PTC after the classic variant [RAS mutation (30–59%), including N-RAS, H-RAS, and, less commonly, K-RAS mutations [V600EBRAF mutation, detected by molecular assays or immunohistochemistry, and lack of V600EBRAF-like mutations or other high-risk mutations  were included into the 2018 revised diagnostic criteria for NIFTP as secondary criteria [V600EBRAF in NIFTP and demonstrated that, initially, the tumors were probably inappropriately classified as NIFTP, but in a further assessment, the presence of papillae and/or even capsular invasion was observed. K601EBRAF mutation was found in a subset of NIFTP cases (4%) [RAS mutations, tumors with other genetic alterations, such as PPARγ and THADA mutations, were also described [ variant ,40. Nucl variant . NIFTP iutations ,42,43,44criteria . Rosariocriteria  analyzedses (4%) ,48, but escribed . NIFTP tescribed ,50,51. Rescribed  demonstrescribed . Similar TERT, TP were incRAS mutation or PAX8-PPARγ and it constitutes 4% of all PTCs. Infiltrative FV accounts for 6% of all PTCs and is mainly caused by BRAF mutation, usually K601EBRAF, or, less often, RAS mutation [BRAF mutations are therefore typical for the more aggressive infiltrative FV PTC while RAS mutations are often found in the less aggressive invasive EFV or the above-described NIFTP. US features of FV can suggest the tumor behavior and molecular landscape, as it can have typical PTC-like US patterns with hypoechoic lesions with irregular blurred margins and, sometimes, microcalcifications or rather follicular neoplasm-like patterns, without these markers of potential malignancy. Infiltrative FV is frequently a hypoechoic lesion, with spiculated/microlobulated margins. Microcalcifications can be present in about 20% of the cases. Nodules are often ovoid to round in shape, with a non-parallel orientation and mixed vascularization. The presence of microcalcifications is less common in the FV than in the classic variant of PTC (41.2% vs. 17.6%) [RAS mutations were presented by Rossi et al. [RAS mutations were also larger than nodules with V600EBRAF mutations (>1 cm) [N-RAS mutation. US examination is a very good prognostic tool in examining follicular-patterned lesions with papillary nuclear features as the infiltrative FV PTC may be differentiated from the benign NIFTP on the basis of the US pattern. However, US-based discrimination between invasive EFVs and NIFTPs is rather impossible in clinical practice [RAS mutations is usually associated with less aggressive forms of PTC and a more benign US image , and infiltrative FV, which is not encapsulated . Invasivmutation ,21,28,40. 17.6%) ,39. Inva. 17.6%) ,39. Simii et al. , who dem (>1 cm) . Interes (>1 cm)  observedpractice ,55. The US image . RAS mutations. In such cases, the US findings are significantly different from those of NIFTP or invasive encapsulated FV of PTC. The differences in the sonographic pattern of thyroid follicular adenoma and carcinoma are well described, despite the fact that both of them harbor RAS mutations [RAS mutations often have more benign US patterns than those with BRAF mutations, the presence of RAS mutations cannot be determined by any particular sonographic findings. In RAS-positive highly aggressive thyroid carcinomas, the US pattern is commonly very suspicious, definitely belonging to the ATA high-risk group [However, follicular adenomas and carcinomas , and eveutations . Thus, ask group .TERT promoter mutation is reported as 21%, 12%, and 9% in the classic, hobnail, and cribriform-morular variant (CMV) of PTC, respectively [TERT promoter mutation is strongly associated with older age, less-differentiated PTC, and a high risk of recurrence. TERT promoter mutations were suggested to be useful surrogate markers for high-risk PTC [TERT promoter mutations. Tumors are typically multifocal, microlobulated, and hypoechoic with microcalcifications, and multiple lymph node metastases are frequently present [TERT promoter-mutated PTC in patients older than 50 years. The frequency of the ectively ,33,59. Trisk PTC . The US  present  recentlyDICER1 mutation in a solid/trabecular variant [VK600-1EBRAF mutation was also described in this variant of PTC [Several different gene mutations, rarely occurring in PTCs, were described in uncommon variants, such as the  variant . The sol variant . Extratht of PTC .CTNNB1, APC [TERT promoter mutations [K-RAS mutation [RET/PTC rearrangement, [PIK3CA [Several mutations were described in the exceptionally uncommon cribriform-morular variant (CMV)—a variant of PTC that is usually associated with autosomal dominant inherited familial adenomatous polyposis (FAP). Sporadic isolated cases are very rare ,64. TumoNB1, APC ,66,67. TNB1, APC ,67,68,69utations . Severalmutation , RET/PTCngement, , and PIK [PIK3CA . UnfortuAssociations between the genetic landscape and US features of PTC may be observed only for the most common mutations, due to the lack of published data in the cases of rare genetic alterations. Thus, we summarized the reports that provide the largest number of cases and the most detailed description of both US features and molecular findings. The observed correlations between molecular and sonographic aspects of PTC are presented in V600EBRAF versus activating RAS mutations, although many of these findings appear to be dependent on the tumor variant. Suspicious US findings, such as hypoechogenicity, spiculated/microlobulated margins, non-parallel orientation, taller-than-wide shape, and the presence of microcalcifications, are typical for PTC harboring V600EBRAF mutations. On the contrary, tumors with RAS mutations are most frequently hypo- or isoechoic, and ovoid to round in shape, with smooth margins and without calcifications. There are also some US features typical for PTCs harboring other mutations, including K601E, RET/PTCBRAF rearrangement, PAX8-PPARγ, CTNNB1, and APC. However, further research is necessary, as some rare PTC variants still cannot be reliably analyzed due to the scarce literature, and some authors question even the widely described correlations between V600EBRAF and the typical suspicious US pattern.On the basis of the current literature data, sonographic features of PTCs correlate with their molecular drivers, particularly between tumors harboring