PTCH1 or gain-of-function mutations in SMO, which occur in the majority of basal cell carcinoma (BCC), SHH-subtype medulloblastoma and rhabdomyosarcoma. Besides the classical canonical ligand-PTCH1-SMO route, mounting evidence points toward additional, non-canonical ways of GLI activation in cancer. By non-canonical we refer to all those mechanisms of activation of the GLI transcription factors occurring independently of SMO. Often, in a given cancer type canonical and non-canonical activation of HH-GLI signaling co-exist, and in some cancer types, more than one mechanism of non-canonical activation may occur. Tumors harboring non-canonical HH-GLI signaling are less sensitive to SMO inhibition, posing a threat for therapeutic efficacy of these antagonists. Here we will review the most recent findings on the involvement of alternative signaling pathways in inducing GLI activity in cancer and stem cells. We will also discuss the rationale of targeting these oncogenic pathways in combination with HH-GLI inhibitors as a promising anti-cancer therapies.The Hedgehog-GLI (HH-GLI) pathway is a highly conserved signaling that plays a critical role in controlling cell specification, cell–cell interaction and tissue patterning during embryonic development. Canonical activation of HH-GLI signaling occurs through binding of HH ligands to the twelve-pass transmembrane receptor Patched 1 (PTCH1), which derepresses the seven-pass transmembrane G protein-coupled receptor Smoothened (SMO). Thus, active SMO initiates a complex intracellular cascade that leads to the activation of the three GLI transcription factors, the final effectors of the HH-GLI pathway. Aberrant activation of this signaling has been implicated in a wide variety of tumors, such as those of the brain, skin, breast, gastrointestinal, lung, pancreas, prostate and ovary. In several of these cases, activation of HH-GLI signaling is mediated by overproduction of HH ligands , loss-of-function mutations in  Hedgehog-GLI (HH-GLI) signaling is a conserved pathway that plays critical roles during embryonic development, cellular proliferation, differentiation and stem cell maintenance . The mamR) that repress transcription of HH target genes (C) and the other on N-terminus (degron DN), preventing inappropriate signaling activity  into the nucleus, where they activate transcription of HH target genes, including factors involved in cell proliferation, survival, self-renewal and invasiveness. Among targets there is also GLI1 itself , SHH-subtype medulloblastoma, rhabdomyosarcoma); (ii) autocrine/juxtacrine ligand-dependent activation, in which tumor cells increase HH ligand expression and respond to the same HH stimulation in a cell-autonomous manner ; (iii) paracrine ligand-dependent activation, where HH ligands secreted by tumor cells turn on HH signaling in the surrounding stroma, which, in turn, stimulates growth and survival of the tumor and vice versa   that occur independently of SMO. The existence of these non-canonical mechanisms appears relevant to allow the development of novel therapeutic approaches to eradicate tumors dependent on HH-GLI signaling.2H2-Kruppel type ZNF motifs constituting the specific DNA binding domain. ZNF4 and ZNF5 bind specifically to a 9 base pair DNA consensus sequence (9-mer) 5′-GACCACCCA-3′ within the GLI-target gene promoters (GLI proteins are members of the Gli-Kruppel family of zinc-finger (ZNF) containing transcription factors (TFs), with five Cromoters , whereasromoters . A nucleromoters   , which into GLIR . Abnormagression .GLI1 gene was first identified by Vogelstein and colleagues as a putative oncogene amplified in glioblastoma . Further, it does not show a preferential expression in cancer tissues  respond to HH ligand stimulation, but whether and how they interact with the other components of the pathway has not yet been determined.The human blastoma , and itsblastoma . In the blastoma . The N-t tissues . Conversno acids . This istastasis . SimilarIn addition to the canonical activation of GLI by the HH-PTCH-SMO route, which is typical of normal cells and ciliated tumors, growing evidence points to a SMO-independent stimulation of GLI activity in cancer. These non-canonical mechanisms are involved both in transcriptional activation of GLI genes and in post-translational modifications of GLI proteins  and leads to the hyperactivation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2), which promote tumor cell growth . SeveralThe first hint of a positive regulation of HH pathway by the RAS-RAF-MEK-ERK signaling came from a study showing that constitutively active MEK1 increases GLI1 activity, which is totally abrogated by inhibition of MEK1/2. Authors identified a region containing the first 130 amino acids of GLI1 responsible for sensing the status of ERK1/2 signaling. Indeed, its deletion yields to an active GLI1 protein showing decreased response to MEK1 signaling . A subseQ61K) and HRAS (HRASV 12G) are able to enhance transcriptional activity and nuclear localization of GLI1. The cytoplasmic retention of GLI1 by SUFU is prevented by both oncogenes. Since inhibition of MEK1/2 abolishes the effect of oncogenic RAS on GLI1, it is likely that the MEK1/2-ERK1/2 module acts as the main effector of RAS   cooperates with a dominant active form of Gli2 that lacks the N-terminal repressive domain to initiate PDAC tumorigenesis in vivo (in vitro (The HH-GLI pathway is important for the initiation and progression of pancreatic adenocarcinoma (PDAC), which is also characterized by high incidence of activating f cases) . KRAS cath UO126 . Interac in vivo . Anotherin vitro .In gastric cancer, KRAS positively modulates tumor proliferation increasing GLI1 transcriptional activity and expression of HH target genes. EGF-induced stimulation of GLI is insensitive to SMO inhibition and is blocked by MEK1 inhibition . Similarin vitro and in vivo (In lung adenocarcinoma (LAC) cells and in their CSC compartment, GLI1 is active despite SMO is often epigenetically silenced. Consistently, genetic silencing or pharmacological inhibition of GLI1, but not of SMO, reduces LAC tumor growth and stemness  in vivo . Mechani in vivo .In multiple myelomas, the MEK1/RSK  cascade sustains non-canonical HH pathway activation through the stabilization of GLI2. RSK2 promotes GLI2 nuclear localization and impairs its degradation through inhibitory phosphorylation of GSK3β on Ser9 . In thesVarious upstream growth factor receptor tyrosine kinases (RTK) can have an impact on GLI activity independent of SMO, through the activation of the RAS-RAF-MEK1/2-ERK1/2 signaling. In human normal keratinocytes, co-expression of EGF and GLI factors enhances the activation of the HH-GLI pathway and promotes oncogenic transformation activating the MEK1/2-ERK1/2 pathway . IntegraSurprisingly, recent studies reported that the apical members of the MAPK cascade, the mitogen activated kinases MEKK1 and MEKK2/3, exert a negative action on GLI1. Indeed, MEKK1 overexpression strongly decreases transcriptional activity of GLI1, causing the inhibition of HH-GLI signaling and reduction of cell growth and viability of medulloblastoma cells. MEKK1 directly phosphorylates multiple residues in the GLI1 C-terminal region, promoting the association with the 14-3-3 cytoplasmic protein and reducing GLI1 transcriptional activity . The reaThe PI3K-AKT-mTOR signaling contributes to a variety of cellular processes, such as nutrient uptake, proliferation and survival in both physiological and pathological conditions, including cancer . The firPI3K signaling has been also reported to increase GLI1 activity via members of the ribosomal S6 kinase family (S6K/p70-S6K), which are the downstream effectors of the PI3K-AKT-mTOR axis. For instance, in EAC cells, mTOR activation by TNFα is responsible for S6K1 phosphorylation and activation. Activated S6K1 directly phosphorylates GLI1 at Serine residue at position 84, thus preventing its association with SUFU . Anotherin vitro proliferation and clonogenicity, and in vivo tumor growth in squamous cell lung tumors  reduces GLI2 mRNA levels and cell proliferation (GLI2 promoter (Transforming growth factor β (TGF-β) signaling exerts important functions during embryonic development and homeostasis of adult tissues. During carcinogenesis, TGF-β signaling can act both as tumor suppressor or oncogene depending on tumor type and stage . An incrferation . A furthpromoter . In addipromoter . GLI1 inpromoter .Several mouse models have shown that TGF-β signaling is also critical for Hh-mediated carcinogenesis. For instance, in a model of pancreatic cancer, SMO-independent Gli1 activation promotes transformation and requires both TGF-β and KRAS signaling . Similarin vitro and in vivo. Consistently, GLI2 silencing reduces invasion of extracellular matrix in vitro and bone metastasis in mice . Interestingly, GLI2 inhibition sensitizes cells to cisplatin treatment and reduces tumor proliferation, and both CD105 and GLI2 are proposed as promising targets to overcome resistance . In coloThe protein kinase C (PKC) is a serine/threonine kinase consisting of three members: calcium-dependent conventional PKC , calcium-independent novel PKC , and calcium-independent atypical PKC . The role of PKCα and PKCδ in controlling GLI activity is controversial. Constitutive activation of PKCα reduces the transcriptional activity of GLI1, whereas that of PKCδ enhances GLI1 transcriptional activity in HEK-293T cells independent of MAPK signaling . In contOn the other hand, aPKCι/λ activates GLI1 downstream of SMO, through phosphorylation of two residues (Ser243 and Thr304) in the zinc finger DNA binding domain of GLI1, leading to increased DNA binding and transcriptional activity . FurtherDual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are serine, threonine and tyrosine kinases containing a DYRK-homology box. Among the five DYRK members, DYRK1A and DYRK1B play a dual role in GLI regulation, whereas DYRK2 is mainly an inhibitor. Overexpression studies indicated that DYRK1A can act as a positive regulator of GLI1, promoting its nuclear translocation  through DYRK1B can act as activator or repressor. It inhibits GLI2 function and promotes the formation of the GLI3 repressor form . Convers5′ Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a serine/threonine kinase that supervises cellular energy status in response to the nutrient supply and environmental conditions; it is activated by increased AMP/ATP ratio and controls different energetic processes . Activated AMPK phosphorylates GLI1 at Serine/Threonine residues , decreasing both transcriptional activity and protein stability . Howeverin vitro and in vivo  fusion oncogene, that characterizes Ewing Sarcoma Family Tumors (ESFT), has been shown to induce  protein . Indeed, protein  and thisr growth .In addition, the SRY-related high mobility group (HMG) box transcription factor SOX9 has been reported to regulate GLI1 by suppressing its association with β-TrCP and subsequent ubiquitination . Indeed,Han and colleagues showed that the Forkhead box C1 (FOXC1) stimulates SMO-independent HH signaling in basal-like breast cancer (BLBC) by binding GLI2 . This inGLI1. Recently, the beta subunit of the IKK complex (IKKβ), which is induced in response to TNFα, has been reported to increase GLI1 protein levels and transcriptional activity in diffuse large B-cell lymphoma  has been shown to cooperate with HH signaling to increase tumor formation in SHH-dependent medulloblastoma . Similarignaling . Interesignaling . These signaling . WIP1 phignaling .Non-canonical activation of the HH pathway can also be mediated by the interaction of the androgen receptor (AR) with the GLI transcription factors. A recent report showed that transcriptionally active AR increases GLI transcriptional activity in prostate cancer cells. AR binds to the GLI3 protein processing domain at the C-terminus, impairing GLI3 ubiquitination and degradation . AR prevIn addition to the various oncogenic signals that positively influence GLI activity, increasing evidence suggests that tumor suppressive signals play also a crucial role in the control of GLI function and activity.TP53 gene  in a proline-rich motif in the C-terminal tail of GLI1, and recruits it into a complex with Numb. This results in the ubiquitination and proteasome-dependent degradation of GLI1  are responsible for post-transcriptional regulation of gene expression via binding to 3′-UTRs of target mRNAs, resulting in their degradation or inhibition of translation. miRNAs are involved in several biological processes, such as proliferation, differentiation, development, and metabolism, and have been implicated in tumorigenesis by targeting developmental pathways involved in tumor formation . With reand GLI3 . miR-326and GLI3 . Indeed,and GLI3 . Downregand GLI3 .Beside genetic modifications and other oncogenic inputs, alterations of the epigenetic machinery appear critical for tumor development. Recently, several lines of evidence pointed to an epigenetic control of HH signaling that occurs downstream of SMO and SUFU.GLI1 and GLI2 promoters  family of chromatin adaptors bind acetyl-lisine residues on open chromatin and facilitate gene transcription at super-enhancers through their interaction with the positive transcription elongation factor b (P-TEFb) and RNA polymerase II (PolII) . Recentlromoters . Of note in vivo . BET pro in vivo . However in vivo .KCTD11, which is an adaptor subunit of the Cullin-3 (Cul3)-based ubiquitin ligase complex, that targets HDAC1 for ubiquitination and subsequent proteasome-degradation, in order to ensure proper development and prevent tumorigenesis  and histone deacetylases (HDACs) catalyze, respectively, the addition or the removal of an acetyl group on lysine residues within the N-terminal histone tails as part of gene regulation . Severaligenesis . Subversigenesis . In suppigenesis . Recentlocessing . Indeed,ocessing , althougIn addition to HDAC1/2, the class II protein HDAC6 is found upregulated in medulloblastoma tumors, and its inhibition shows tumor suppressive effects in preclinical models of Shh-medulloblastoma. Remarkably, HDAC6 effects seem to be independent of GLI acetylation, and to occur most likely through the transcriptional control of GLI2 expression and stabilization of GLI3 protein . In lineHistone acetyltransferases have been also implicated in the regulation of non-canonical HH signaling. First, it has been shown that the HAT co-activator p300 functions as a crucial transcriptional checkpoint during morphogen-dependent development . Indeed,GLI1 promoter and recruits PRMT5 to repress GLI1 expression, at least partially through the PRMT5-catalyzed histone arginine methylation on histone H4 (H4R3m2). Thus, mutations in the MEN1 gene result in activation of GLI1 through increased binding of transcriptionally active GLI1 at its promoter, leading to the development of neuroendocrine tumors (Members of the protein arginine methyltransferase (PRMT) family regulate several cellular processes, including gene transcription, DNA repair, mRNA splicing and signal transduction. Aberrant activity of PRMT has been reported in several tumors . PRMT1 ce tumors  , such as vismodegib (GDC-0449), sonidegib (NPV-LDE-225), saridegib (IPI-926), BMS-833923, glasdegib (PF-04449913) and taladegib (LY2940680) . These SHowever, acquisition of resistance due to specific missense mutations at SMO level  represents the major challenge to the success of therapies, as well documented in BCC . This liin vitro cytotoxicity , will be needed to test the role of each aberrantly activated signaling pathway and provide personalized therapeutic options for individual patients. In addition, it will be equally important to establish sensitive and validated biomarkers of HH-GLI pathway activation, such as reliable antibodies against human GLI1 and GLI2 and their phosphorylated forms. For treatment involving SMO inhibitors , a better biomarker for SMO activation should be developed, such as phospho-specific antibody to monitor SMO phosphorylation (In general, canonical activation of HH-GLI signaling occurs in cancers with mutations in reported . In the rylation . The avaIn summary, both canonical HH signaling and the confluence of multiple SMO-independent oncogenic pathways can regulate the activity of the GLI transcription factors. Thus, a more effective therapeutic approach to fight cancers harboring non-canonical HH pathway activation would be to inhibit the GLI rather than SMO. Often in a given cancer type, canonical and non-canonical HH pathway activation co-exist. In some cancer types, more than one mechanism of non-canonical HH activation occurs simultaneously. Therefore, combined targeted therapy will be more effective than single treatment in blocking GLI-dependent tumor growth and progression.SP and BS conceptualized the content, and reviewed and edited the manuscript. All authors wrote and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In particular, the ORF-II proteins, HTLV-1 p30II and HTLV-2 p28II, suppress Tax-dependent transactivation from the viral promoter—whereas p30II also inhibits PU.1-mediated inflammatory-signaling, differentially augments the expression of p53-regulated metabolic/pro-survival genes, and induces lymphoproliferation which could promote mitotic proviral replication. The ubiquitinated form of the HTLV-1 p13II protein localizes to nuclear speckles and interferes with recruitment of the p300 coactivator by the viral transactivator Tax. Further, the antisense-encoded HTLV-1 HBZ and HTLV-2 APH-2 proteins and mRNAs negatively regulate Tax-dependent proviral gene expression and activate inflammatory signaling associated with enhanced T-cell lymphoproliferation. This review will summarize our current understanding of the pX latency-maintenance factors of HTLV-1 and HTLV-2 and discuss how these products may contribute to the differences in pathogenicity between the human PTLVs.Of the members of the primate T cell lymphotropic virus (PTLV) family, only the human T-cell leukemia virus type-1 (HTLV-1) causes disease in humans—as the etiological agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other auto-inflammatory disorders. Despite having significant genomic organizational and structural similarities, the closely related human T-cell lymphotropic virus type-2 (HTLV-2) is considered apathogenic and has been linked with benign lymphoproliferation and mild neurological symptoms in certain infected patients. The silencing of proviral gene expression and maintenance of latency are central for the establishment of persistent infections in vivo. The conserved  The primate T-cell lymphotropic virus (PTLV) family consists of the simian T-cell lymphotropic virus types 1–5 (STLV types 1–5) and human T-cell lymphotropic virus types 1–4 (HTLV types 1–4), which includes the human T-cell leukemia virus type-1 (HTLV-1) and the related human T-cell lymphotropic virus type-2  . The HTLpX region  protein, HBZ, is encoded by the antisense  strand of the integrated proviral DNA and the transcriptional initiation of hbz occurs from the 3′ LTR . The antisense strand of HTLV-2 also encodes a nonconventional bZIP protein, APH-2, which is a synologue of HTLV-1 HBZ. These genes are expressed through alternative mRNA-splicing, and many , and HBZ-SP2 (spliced-2), that negatively regulate proviral gene expression and modulate host lymphoproliferative signaling [hbz-sp1 mRNA which codes for the most abundant isoform (HBZ-SP1) present in ATLL cells  spliced to an acceptor site at position 1767 on the antisense RNA strand [Hbz is the only viral gene detectable at every stage of infection in chronically-infected cell-lines and PBMCs derived from HTLV-1+ asymptomatic carriers, HAM/TSP, and ATLL patients. Despite the fact that the 5′ LTR is frequently inactivated in ATLL clinical isolates as a result of DNA hypermethylation or proviral deletions, the 3′ LTR and hbz mRNA and protein expression are usually intact, alluding to their pivotal roles in viral pathogenesis [26–34 peptide epitope, effectively selected against HTLV-1-infected CD4+ T-cells which expressed a HLA-A*0201 major histocompatibility class I molecule that binds to HBZ-SP1 with high-affinity. These findings suggest a CD8+ cell-mediated immune-response could select for HTLV-1 proviral clones with reduced, steady-state levels of the antisense hbz products and intermittent tax expression in vivo.The antisense strand of HTLV-1 encodes unspliced and alternatively-spliced transcripts, ignaling –33. ThesA strand . Hbz is ogenesis . The HBZogenesis  demonstrTax-responsive elements (TREs) of the HTLV-1 promoter [RelA subunit [hbz expression. The HBZ protein suppresses AP-1-dependent transcription through interactions with the bZIP factors, c-Jun and Jun B [human telomerase reverse transcriptase (hTERT) gene promoter [RelA subunit which inhibits p65RelA DNA-binding and NF-κB transactivation [The antisense bZIP protein of HTLV-1, HBZ, antagonizes Tax-dependent viral gene expression through its nuclear interactions with the transcriptional coactivators p300/CBP and chromatin-remodeling components. The HBZ protein localizes in nuclear speckles and contains three nuclear localization signals, with NLS1 (aa 87–92) and NLS2 (aa 116–120) located within the two upstream basic domains and NLS3 aa 137–163) in the DNA-binding region , while also activating the noncanonical Wnt5a signaling pathway which could promote ATLL cell proliferation . In 2013siveness .hbz is dispensable for the infection and immortalization of primary T-cells by HTLV-1 in vitro, it is required for viral persistence and the maintenance of a high proviral titer in vivo [Gag) and viral persistence in vivo as measured by quantitative RT-PCR is dependent upon hbz gene expression. A study of the kinetics of viral gene expression demonstrated the levels of tax/rex, gag/pol, and env mRNAs decreased and inversely correlated with higher levels of the hbz transcripts in infected rabbits [hbz mRNAs are compartmentalized and sequestered in the nuclei of cultured HTLV-1-infected T-cell clones established from ATLL and HAM/TSP patients. Valeri et al. [hbz is required for viral persistence in rabbits and Rhesus macaques inoculated with lethally-irradiated 729 B-cell-lines expressing the wild-type HTLV-1 provirus or mutants ablated for hbz expression (or other ORF-I and ORF-II products), with genetic reversion to the wild-type sequence observed in 3 out of 4 seropositive macaques inoculated with the HBZ-knockout mutant. The hbz mRNA and HBZ protein differentially promote T-cell activation, lymphoproliferation and cell survival [hbz mRNA protects against programmed cell-death and induces the expression of cell-cycle regulatory and anti-apoptotic genes  in transduced primary murine T-cells. Both the HBZ protein and mRNA were able to induce T-cell lymphoproliferation and aberrant S-phase entry [hbz expression blocked the in vitro proliferation of HTLV-1-transformed T-cell-lines and ATLL cells [hbz, exhibit reduced cell proliferation, tumorigenesis, and secondary tissue infiltration in engrafted NOD/scidγchain−/− animals. The hbz mRNA posttranscriptionally increased the expression of oncogenic microRNAs, miR17 and miR21, in CD4+ T-cell clones established from HTLV-1-infected HAM/TSP patients [hbz-transgenic mice resulted in the formation of skin and lung lesions associated with systemic inflammation and lymphocyte infiltration [reg cells which resulted in increased inflammation [reg cells. Esser et al. [granzyme B promoter-hbz transgenic mice developed CD45+ mixed-cellular tumors, with enlargement of the spleen, elevated white blood cell counts, and osteolytic bone metastases associated with the increased expression of inflammatory cytokines and factors involved in hypercalcemia, including RANKL, PTHrP and DKK1.Although  in vivo . By inoc in vivo  demonstr rabbits . Rende e rabbits  have repi et al.  have fursurvival , 34. These entry . Small-iLL cells , 34. ArnLL cells  also dempatients . Importaltration , 63. Manammation . In 2011ammation  demonstrr et al.  have furGag) by the pH6neo HTLV-2 clone, suggesting APH-2 antagonizes Tax-2 functions and promotes viral latency in vivo. The repression of Tax-2-dependent transactivation and binding to CREB were dependent upon the C-terminal modulatory sequence, LXXLL, of APH-2 [aph-2 mRNA is constitutively expressed in chronically-infected cell-lines and PBMCs derived from HTLV-2-infected carriers [aph-2 mRNA levels coincided with the proviral loads in HTLV-2-infected patients, neither the aph-2 mRNA nor APH-2 protein were able to induce lymphoproliferation in vitro [aph-2 transcripts are sequestered in the nuclei of HTLV-2-infected cells, similar to the subcellular compartmentalization of hbz mRNAs. In 2012, Yin et al. [RelA-dependent transcriptional activation; however, by contrast, APH-2 does not augment TGF-β signaling [survivin gene associated with the cellular anti-apoptotic response [survivin promoter transactivation by the NFAR NF110a [The antisense protein of HTLV-2, or APH-2, is a functional synologue of the HTLV-1 HBZ factor and is generated through alternative mRNA-splicing which uses a donor site at position 8544 and a splice acceptor site at position 7173 on the antisense strand of the pH6neo molecular clone of HTLV-2 . APH-2 iof APH-2 . Unlike of APH-2 . The aphcarriers . Althougin vitro . Bender in vitro  have repn et al.  demonstrignaling . The HBZignaling . While bignaling  have furresponse . The siRR NF110a . These rpX regions of HTLV-1 and HTLV-2 encode the ORF-II products: p30II and p28II, respectively, through alternative mRNA-splicing, which negatively regulate the Tax-dependent expression of viral antigens [II and p28II differ significantly in their capacity to modulate host signaling pathways and cooperate with other viral and cellular oncoproteins and, therefore, these factors are likely to have divergent roles in pathogenesis [The ORF-II proteins which suppress the expression of proviral antigens represent an understudied area in the HTLV field, yet—in the light of recent evidence, there is reason to believe these factors may have key ancillary functions that could provide clues into the different pathogenic properties of the human PTLVs. The conserved antigens –20, 75 aantigens , 24, 26.antigens . Howeverogenesis , 76–86. II protein  is comprised of 241 aa residues, contains arginine and serine/threonine-rich regions, and shares aa sequence similarities with the Oct-1/Pit-1/POU-family of homeodomain transcription factors  also corresponds with the reading frame for the p13II protein sequence  within its N-terminus shares 78% sequence homology with residues 193–241 of HTLV-1 p30II [II has been reported to contain two intrinsically disordered sequences, spanning aa residues 75–155 and 197–241 [II protein is predicted to contain at least five alpha-helices which may contribute to its unique biological functions and molecular interactions  with exon 2 (nts 4641–4831) spliced to an acceptor site at position 6478 of the downstream pX sequence that is also used for the bicistronic pX-tax/rex mRNA [pX mRNAs, including pX-orfII-p30II, have been detected by RT-PCR in cultured HTLV-1-infected T-cell-lines and primary uncultured ATLL clinical isolates, as well as in cells from asymptomatic HTLV-1-infected carriers [pX-orfII mRNA is expressed in chronically-infected T-cell-lines; and Cereseto et al. [pX-orfII mRNA in HTLV-1-transformed T-cell-lines, PBMCs from HTLV-1-infected carriers, and cells isolated from HAM/TSP patients using a non-PCR-based ribonuclease-protection assay. The HTLV-2 alternatively-spliced pX-orfII-p28II mRNA, which is generated by splicing exon 1 (nts 316–449) to an acceptor site at position 6944 of the pX sequence, has been detected in the chronically-infected MoT cell-line by RT-PCR analysis [II and p13II peptides from HTLV-1-infected carriers, HAM/TSP and ATLL patients, suggesting these proteins are chronically expressed and could contribute to the establishment of persistent infections in vivo as well as viral pathogenesis.The HTLV-1 p30-1 p30II , 93. How 197–241 , the p30rex mRNA , 95. Thecarriers , 90, 96.analysis  have isoII and p28II proteins can be divided into: (a) the establishment of early-stage latency and viral persistence in vivo, and (b) their interactions with host proliferative-signaling pathways and cooperation with other viral  and cellular factors to drive mitotic proviral replication. The HTLV-1 p30II protein contains three putative nuclear localization signals (NLS1/2/3) as well as a nucleolar retention sequence  of transduced human PBMCs selected on blasticidin and cultured in the presence of recombinant interleukin-2 (IL-2), although these transiently-amplified clones were observed to undergo a second crisis at around 7 months and it is presumed that other viral and/or cellular factors are necessary for T-cell immortalization [II-defective HTLV-2 mutant provirus, derived from the pH6neo molecular clone, exhibited viral infectivity and immortalized T-cells in vitro similar to the wild-type virus, but failed to promote proviral replication and T-cell survival in vivo in a rabbit model of HTLV-2 pathogenesis [pX-tax/rex mRNA were inversely correlated with the expression of the other pX-orfII, pX-orfI, and hbz-sp1 transcripts [pX-orfII mRNA in vitro [pX-tax/rex and pX-orfII-p28II transcripts were detected at comparable levels in the chronically-infected cell lines, MoT and BJAB-Gu, and in PBMCs isolated from 2 out of 3 HTLV-2-infected patients. The HTLV-1 p30II protein has been shown to promote aberrant S-phase entry and lymphoproliferation, and induces the expression of T-cell activation and pro-survival genes [II enhanced the survival of transduced 293T and Jurkat T-cells, associated with p30II-interactions with the ataxia telangiectasia mutated (ATM) and REGγ proteins. A follow-up study by Doueiri et al. [II protein interacts with the nuclear 20S proteasome activator REGγ. This study combined biochemical affinity purification with mass spectrometry analysis and identified several unique binding partners and three common interacting factors  that associate with the HTLV-1 p30II and HTLV-2 p28II proteins [The functional roles of the ORF-II p30ribution , 98. Usiribution  demonstrin vitro , 100, tho et al.  have demlization . By contogenesis . A studynscripts . A similin vitro . By contin vitro  investigal genes –86, 101.al genes  demonstri et al.  demonstrproteins .II and HTLV-2 p28II proteins negatively regulate proviral gene expression and function as latency-maintenance factors which could help HTLV-infected cells evade host immune-surveillance pathways for the establishment of persistent infections in vivo. In 2000, Zhang et al. [II fusion constructs and luciferase reporter assays and demonstrated that p30II contains a functional transcriptional activation domain . The p30II protein binds to the KIX domain of the transcriptional coactivators, p300/CBP, and competes against the viral transactivator Tax for the recruitment of p300/CBP to CREB/21-bp-repeat TRE complexes on the HTLV-1 promoter and represses the expression of viral antigens [II was dependent upon a single lysine residue at position K106 within the p30II protein  in transfected cells. The inhibitory effect of p30II upon PU.1-dependent transcriptional activation was countered through overexpression of the p300 coactivator. p30II also inhibited the pro-inflammatory cytokines, MCP-1, TNF-α, and IL-8, and increased the release of the anti-inflammatory factor IL-10 following the stimulation of TLR4 in THP-1 monocytic cells with lipopolysaccharide, suggesting p30II might interfere with adaptive immunological signaling in the early stages of viral pathogenesis [II inhibits the expression of interferon-responsive genes by interfering with the PU.1-dependent expression of TLR4 in THP-1 monocytes and dendritic cells. The inhibition of interferon-responsive signaling and innate immunity by p30II could contribute to the early-stage establishment of infection and viral persistence in vivo. Valeri et al. [II is required for the productive infection of human dendritic cells by HTLV-1, and further demonstrated that Rhesus macaques inoculated with an irradiated 729 B-cell-line, containing an ACH.p30-knockout mutant ablated for p30II expression, either failed to seroconvert or exhibited genetic reversion to the wild-type ACH sequence.The HTLV-1 p30g et al.  used Galain Fig. a, and thantigens . The traein Fig. a, and reogenesis . These fogenesis  which dei et al.  have alsII posttranscriptionally inhibits the nuclear export of the doubly-spliced bicistronic pX-tax/rex mRNA and negatively regulates HTLV-1 gene expression. For these studies, 293T cells were cotransfected with an HTLV-1 molecular clone, p-BST, and an expression construct for p30II and the inhibition of viral gene expression was demonstrated by Anti-p19Gag ELISAs and the nuclear accumulation of pX-tax/rex transcripts was detected with RT-PCR. This study further demonstrated that p30II was associated with the splice-junction of the pX-tax/rex mRNA using biotinylated RNA-precipitation experiments, and that lentiviral p30II negatively regulated proviral gene expression in transduced HTLV-1-transformed T-cell-lines  [II is dependent upon its interactions with large ribosomal subunit L18a within cell nucleoli. Younis et al. [II and the synologue p28II posttranscriptionally inhibit proviral gene expression from molecular clones of HTLV-1 or HTLV-2. The HTLV-2 p28II (AU1-tagged) protein was immunoprecipitated bound to pX-tax/rex mRNA complexes in vivo; and p28II resulted in increased nuclear sequestration of the pX-tax/rex mRNA as detected by RT-PCR [II and p28II are recruited to their respective RNA targets through co-transcriptional interactions with the retroviral transactivator protein Tax on the promoters of the HTLV-1 and HTLV-2 proviruses [II and p28II interact with Tax-1 or Tax-2 and are recruited and travel with RNA Polymerase II-containing transcriptional elongation complexes until they reach their nascent RNA targets. Using confocal microscopy and biochemical glutathione-S-transferase (GST)-pull-downs, Baydoun et al. [II interacts with the retroviral splicing regulator, Rex, and sequesters Rex/CRM1 complexes in nucleoli in cotransfected COS and 293T cells. However, this somewhat contradicts findings by Sinha-Datta et al. [II interact with Rex complexed with the Rex-responsive RNA element (RxRE) of tax/rex mRNA transcripts, but this interaction did not interfere with the shuttling of Rex/CRM1/mRNA complexes out of the nucleus. The regulation of HTLV-1 gene expression and latency by Tax, HBZ, and p30II  is orchestrated by coordinate and dynamic molecular interactions at the transcriptional and posttranscriptional levels. Choudhary and Ratner [hbz-sp1 anti-sense mRNA indirectly increases expression of the Tax transactivator from a molecular clone of HTLV-1 by reducing the expression of the pX-orfII-p30II mRNA, which lends additional complexity to the control of proviral gene expression by pX products.In 2004, Nicot et al.  reportedHUT-102) . GhorbelHUT-102)  have shos et al.  subsequey RT-PCR . Interesn et al.  have shoa et al.  which ded Ratner  have alsII protein of HTLV-1 helps to accomplish this by activating cellular oncogenic and pro-survival pathways through its molecular interactions with the TIP60 acetyltransferase—a transcriptional cofactor for both c-Myc and p53. In 2005, Awasthi et al. [II cooperates with the c-Myc oncoprotein, induces aberrant lymphoproliferation in Molt-4 T-cells, and enhances the oncogenic potential of c-Myc in cellular transformation/foci-formation assays using human fibroblasts. Mechanistically, aa residues 99–154 of p30II were shown to interact with the MYST-family acetyltransferase TIP60, using biochemical GST-pull-downs and co-immunoprecipitation experiments  was primarily responsible for acetylation of the c-Myc oncoprotein [p53 tumor suppressor is a downstream target of c-Myc; and acute and lymphoma-stage ATLL clinical isolates frequently contain c-Myc overexpression and elevated levels of wild-type p53 [p53 gene is rarely mutated in HTLV-1-infected ATLL patient samples [II protein induces p53 and inhibits TIP60-mediated K120-acetylation of p53, and transcriptionally activates the expression of p53-regulated pro-survival genes. Most notably, lentiviral p30II induced the expression and mitochondrial targeting of the TP53-induced glycolysis and apoptosis regulator  recruitpromoter . p30II telements , 85. Usiof TIP60 . At leasIP60 [II , 84, 101type p53 –108. Int samples –112, whi samples –115. In  samples  and Hutcr (TIGAR )—a fructr  from an Asian monkey species, Macaca arctoides, which lacked the methionine initiation codons for both p12I and p13II and serologically more closely resembled HTLV-2. The mitochondrial targeting signal (MTS) of p13II spans the amino-proximal residues 20–35  [II remain to be completely defined, Andresen et al. [II protein is mono-ubiquitinated on a non-lysine residue and localizes in nuclear speckles in the presence of the viral transactivator Tax and, consequently, interferes with recruitment of the p300 coactivator to Tax-containing complexes and represses transcriptional activation from the HTLV-1 5′ LTR. These findings suggest that ubiquitinated-p13II may help to promote viral latency for the establishment of persistent infections in vivo. The unmodified p13II protein targets the inner membrane of mitochondria, induces membrane depolarization and mitochondrial swelling, opens the apoptogenic permeability transition pore, and results in an increased flux of K+ and Ca2+ ions and the production of ROS [II  polymerase (PARP)-cleavage, and also enhanced the levels of nuclear phospho-CREB in response to Ca2+-stimulation in histamine-treated cells [II proteins localize to mitochondria and interact with farnesyl pyrophosphate synthetase (FPPS)—a cofactor involved in targeting oncogenic Ras to the plasma membrane, suggesting there may be some functional overlap between these factors. Further, a mutant BLV proviral clone, defective for G4 production, was impaired in its pathogenic potential and failed to induce leukemia or lymphosarcomas in infected sheep [II inhibited the growth of Jurkat T-cells at high culture densities, and sensitized these cells to apoptosis induced by either Fas Ligand or ceramide-treatment. The ability of p13II to promote cellular apoptosis was countered by treating p13II-expressing Jurkat cells with a farnesyl transferase-inhibitor which prevents the posttranslational modification of the Ras protein and interferes with its membrane localization [II was associated with the activation of resting primary T-cells which was countered by ROS scavengers, whereas p13II sensitized transformed Jurkat T-cells to apoptosis under conditions of glucose-deprivation. It is thus intriguing to speculate that the ORF-II products, p13II and p30II, could act coordinately in HTLV-1-infected cells to promote carcinogenesis—with p30II suppressing ROS-dependent apoptosis by p13II through the p53-regulated induction of the antioxidant effector, TIGAR [II synologue and dissimilar functions of HTLV-1 p30II and HTLV-2 p28II, in terms of their ability to activate cellular pro-survival signals, may, at least in part, account for the different pathogenic properties of these related PTLVs.The HTLV-1 p13155 Fig. a, b [25,ion 6875 . Interesx et al.  identifi–35 Fig. b which an et al.  have shon of ROS , 127–129n of ROS  have sho[II Fig. b interaci et al.  demonstred cells . By comped cells  have demed sheep  demonstrlization . Silic-Br, TIGAR . The absIt remains an enigmatic mystery why the HTLV-1 is the only member of the PTLV family that is pathogenic in humans. Both the HTLV-1 and HTLV-2 can infect and immortalize primary T-cells cultured in vitro. Despite its similar genomic organization and structural relatedness, the HTLV-2 is not causally linked with any specific disease, although it has been associated with non-malignant lymphoproliferation and mild neurological symptoms in some infected patients , 10, 14.pX-encoded latency-maintenance factors of HTLV-1 and HTLV-2 may provide clues into the differences in pathogenicity of these PTLVs. The antisense bZIP proteins, HBZ and APH-2, both repress Tax-dependent transactivation and gene expression from the viral 5′ LTR and inhibit NF-κB-signaling through interactions with the p65RelA subunit which prevents its binding to κB-responsive enhancer elements [hbz is required for HTLV-1 proviral persistence in vivo, Yin et al. [aph-2 (∆Aph-2) had higher antibody titers and proviral loads than animals infected with wild-type HTLV-2. These results are somewhat surprising and suggest that HBZ and APH-2 have different roles for the maintenance of viral persistence in vivo, and allude to the potential importance of the other pX latency factors: p30II, p28II, and p13II. While the HTLV-1 p30II and HTLV-2 p28II proteins are functionally similar in their ability to negatively regulate Tax-dependent transactivation from the proviral LTR [II, there is no evidence that p28II contains transcriptional activity. p30II interacts with the cellular acetyltransferases p300/CBP and TIP60 [II is required for viral persistence and the maintenance of a high proviral titer—presumably, through the suppression of viral antigens which could help HTLV-1-infected cells evade host immune-surveillance pathways [II could enhance the survival of infected T-cells by activating cellular pro-survival genes and antioxidant effectors, such as TIGAR, to prevent the accumulation of cytotoxic metabolic byproducts  and counter the oxidative-stress caused by the aberrant expression of viral (Tax and HBZ) and/or cellular oncoproteins [II protein has been shown to promote the activation of primary T-cells through the induction of low levels of ROS; however, higher levels of ROS, as can be present in oncogenically-transformed cells, induce apoptosis [II, p13II, and HBZ may act coordinately to promote enhanced lymphoproliferation and mitotic proviral replication, while preventing the buildup of excessive levels of damaging ROS. The absence of a p13II synologue, together with the disparate functions of APH-2 and p28II relative to their HTLV-1 pX counterparts, could provide a molecular explanation for the weakened pathogenic nature of HTLV-2.Similar to other transforming viruses that encode latency-maintenance factors, such as Epstein–Barr virus and the Kaposi’s sarcoma-associated herpesvirus, the HTLVs have developed several strategies to suppress the expression of viral antigens, while simultaneously driving mitotic proviral replication through the activation of cellular proliferative pathways. Indeed, the divergent and dissimilar functions of the elements , 67, 70.iral LTR –20, unlind TIP60 –86, intend TIP60 , and modnd TIP60 , 86, 101pathways , 24. Altproteins , 86. Thepoptosis . It is t


The model is particularly well suited for beyond visual line‐of‐sight operations, and is useful for UA operators for conducting risk assessment of planned operations as well as for regulators for determining appropriate operational requirements.Unmanned aircrafts (UA) usually fly below 500 ft to be segregated from manned aircraft. However, while general aviation (GA) usually do fly above 500 ft in areas where UA are allowed to operate, GA will at times also fly below 500 ft. Consequently, there is a distinct risk of near‐miss encounters as well as actual midair collisions (MACs). This work presents a model for determining this risk based on physical parameters of the aircraft and actual figures for the numbers of GA in a given airspace, as well as the probability of having GA below 500 ft. The aim is to achieve a prediction with a precision better than one order of magnitude relative to the true MAC rate value. The model is applied to Danish airspace and the MAC rate for unmitigated operations of UA is found to be approximately 10 As the number of UA increases in the airspace, the probability of midair collisions (MACs) between UA and other airspace users also increases. This work addresses the need for quantifying the risk that UA operating beyond the visual range of the operator pose to airspace users that may occasionally or often operate at very low altitude. This is done by providing a model that predicts the probability of MAC for such scenarios. This model can be used by operators, manufacturers, and authorities for planning flight operations, for conducting risk assessment, for development of guidance material, and for aviation regulation. This work was in part prompted by the Danish Transport, Housing, and Construction Authority (DTCHA) in an effort to determine risk of MAC for beyond visual line‐of‐sight (BVLOS) operations in Danish airspace.1.1As the number of UA in most national airspaces is growing rapidly, especially for amateur users, it has become evident through an increasing number of reports of near‐misses that collisions between UA and commercial aircraft are likely to occur, and the consequences could be catastrophic  operating in class G airspace below 500 ft where UA are indeed allowed to operate. As such, there is a distinct risk of MACs simply due to the fact that UA and GA (to some extent) share the airspace. Most GA operate solely according to visual flight rules (VFR) where the primary method for maintaining separation between airspace users is visual navigation. When operating BVLOS, UA are generally not able to comply with this method, and technologies that allow UA to reliably detect GA  are still both quite expensive and somewhat immature. For this reason, it is valuable to estimate in a quantitative way the actual probability of MAC. This estimation will allow authorities, aircraft manufactures, and operators to assess the risk, and plan and execute flights accordingly.1.2The vast majority of previous work in MAC modeling is associated with manned aviation, in particular with scenarios where aircraft are in controlled airspace following directions from either ATC (air traffic control) or following predetermined flight patterns. This is reviewed in Section 1.2.1Different models for determining the probability of aircraft actually colliding during flight have emerged over the years. Many date back to the 1960s and 1970s where the rise in air traffic over some parts of the globe made it necessary to thoroughly consider how to avoid MAC, and as part of this process, how to determine the likelihood of MAC. These models focus on aircraft following predefined air corridors, such as cross‐Atlantic flights. Some of the first to consider collision risk for such operations were Marks  and ReicA description of the development of collision risk models for commercial aviation from the 1960s to 1995 is given in Machol , which iAlthough there is plenty of literature on the risk and mitigation of MACs involving GA, there is less literature on the modeling of probabilities of MAC involving GA. The main focus in the GA‐related literature is on VFR and how that particular method of risk mitigation relates to collision avoidance. As an example, Koglbauer  describe1.2.2A data‐driven approach is presented in McFadyen and Martin , 2016b, 1.3The focus in this work is to develop a model that can predict the probability of a MAC between a UA and any form of GA in class G airspace with relatively good accuracy. The model is contingent on the GA operating “randomly,” that is not following specific flight routes or patterns, or that such patterns are unknown at the time of modeling.A quite thorough first principle approach similar to the one used in this work was derived in Endoh , albeit Unlike most existing models, the method proposed in this work is particularly well suited for BVLOS operations where the UA operator is not able to visually segregate the UA from any GA, and where no other effective means of detect and avoid are in place. Also unlike most other works, we do use the model to compute MAC rates for a specific airspace (mainland Denmark) to both demonstrate the use of the model and to show the estimated MAC rates for various types of GA.The aim of this work is to achieve an estimated MAC rate that can reliably be expected to be no more than one order of magnitude from the true MAC rate. This aim is partly based on the expected quality of the data and partly on the identified model errors that follow from the simplifying assumptions listed below. In addition, manned aviation safety typically operates with orders of magnitude classification of levels of safety and levels of hazard, lending credence to our aim.2The model presented in this work is developed for GA such as small fixed‐wing planes, such as the Cessna 172 and Piper Cub; for small rotorcraft, such as the Robinson 22; for gliders, ultralights, hang gliders, balloons, etc., that could occasionally operate below 500 ft, and thus appear in the airspace where light commercial UA almost always operate. The model is probabilistic and is set up to accommodate parameters that can reasonably be expected to be available from national aviation authorities, or other similar sources of data.The model structure and parameters are presented first in Section 2.1The model is probabilistic in nature and relies on a number of parameters, some deterministic and some probabilistic. It is solely for determining the probability of collision between a UA and a GA. It does not include collateral damage on the ground from falling debris and the like. The basic concept of the model is presented in Section 2.1.11.The UA and the GA fly independently.2.The UA fly below an upper altitude threshold.3.The probability of the GA being below this upper altitude threshold is known (or estimated).4.Flight directions for both aircraft are uniformly distributed from 0º to 360º.5.Geographical positions of both aircraft are uniformly distributed across a predefined area.6.Both aircraft fly only horizontally  when close to each other.7.Altitude of both aircraft is modeled probabilistically with predefined probability distributions.8.GA is mostly not below 500 ft in any area where UA can legally operate. See Section As a consequence of assumptions 4 and 5, the model is suited only for airspace where no external control is imposed to guide aircraft into certain patterns, typically class G airspace. Also, as a consequence of assumptions 6 and 7, collisions caused by sudden altitude change  are not modeled. However, since the vertical speed typically is somewhat less than the horizontal speed, and since the vertically traversed distance for a GA is significantly smaller than the horizontally traversed distance, this assumption has only negligible effect for all types of GA but parachutes and balloons in descent or ascent. This is detailed in Section The initial model setup is for a single UA and a single GA. Later, the model will be expanded to include multiple GA. The UA and the GA are represented by vertical cylinders approximating the physical extends of the two aircraft. The radius and height of the cylinders can either equal the width and height of the aircraft, or be safety zones for modeling near‐miss events. The model describes the probability that the “UA cylinder” will intersect the “GA cylinder.” Examples of cylinders are depicted in Fig. The model supports fairly general assumptions on the type of GA , as well as more specific assumptions where data are available for individual types of GA. The latter is the approach taken in Section 2.1.2pHC is the rate of “horizontal collision.” That is, the rate by which the two aircraft will be at the same two‐dimensional geographical location to allow a collision to occur. This rate is based on assigned circles around both aircraft , and the assumption that both aircraft have known horizontal speeds.The model for MAC is composed of four main probabilistic parts appearing in product form as:pVC is the conditional probability of “vertical collision” given GA is below threshold. That is, the probability that the two aircraft are at the same vertical location  and with zero vertical speed. This probability is based on conditional probability density functions for the altitude of both aircraft, as well as the height of both aircraft.The second term pbelow is the probability that the GA is below an altitude threshold, which in this work represents the maximum altitude at which UA would legally operate. How to determine an appropriate altitude is discussed later in Section The third term STM is the effect of strategic and tactical mitigations. The three previous factors assume complete randomness in the aircraft motions (subject to the assumptions described above), and thus do not capture any mitigating means that could be introduced to reduce collision probability. However, for some flight scenarios there will be some form of strategic mitigations  and/or tactical mitigation , and λSTM captures the effect of such mitigations. Examples of strategic mitigation are temporal and spatial constraints, and examples of tactical mitigation are detect and avoid sensors and transponders as well as visual observations by the GA operator. In this work, λSTM expresses how much the combined strategic and tactical mitigations will reduce the probability of collision. It is important to note that λSTM as implemented in this model is only valid for relatively modest mitigations; it does not represent well relatively effective means such as operations in controlled or closed airspace.The fourth and last term λpHC and pVC are the probabilities of the GA and UA being at the same physical location in an North‐East‐Down coordinate system, where pHC is the probability that the North–East coordinates sets for the GA and UA are equal (or sufficiently close for the cylinders to intersect) and pVC is the probability that the Down coordinates are equal. Since the motions (and thus locations) of the UA and GA are independent, so too pHC and pHC. The probability pbelow is solely a property of the GA, and expresses the likelihood of the GA being in the same air volume as the UA, making it independent from pHC and pVC. Finally, the last term λSTM may have minor dependency on both pHC and pVC if, for instance, the flight altitude for the UA is chosen such as to mitigate the risk of colliding with known, say, glider, operations nearby. However, for most mitigations, such as transponders and SAA, there is rather little dependence on the altitude and/or geographical position of the GA.These four products are largely independent; 2.1.3G. The number of GA available in this area is denoted as n. A GA has a given fraction of time airborne T . It flies at an average speed vGA, and the GA cylinder has a vertical extend hGA and horizontal radius of rGA. The UA similarly has values vUA, hUA, and rGA. The maximum altitude that we will consider is called zmax. This can be 500 ft, but it can also be lower depending on the regional or national limit for UA operations. We assume SI units for all parameters.Assume a given geographical area where the number and flight time of various GA are available, typically a nation. This area has size 2.2STM is briefly discussed in Section The following derivation is for a single UA flying in an airspace with a single GA. It is composed of a horizontal component and a vertical component. The horizontal component is presented in Section 2.2.1G′/G. The cylinders move over time as the GA and UA travel through the airspace, and the average time period the GA and UA cylinders intersect horizontally is:d is half the travel distance for the GA during safety zones intersection and v is the relative speed between the GA and the UA. See Fig. First, the average area covered by the cylinders  of one GA and one UA at any given time is:G′=πr GA +r UA 2T,r=r GA +r UA , and x is the distance between the center of the GA cylinder and the line that the center of the UA cylinder travels as perceived by the GA. We assume that x is uniformly distributed in the interval . The average value of d then becomes:v is the relative speed between the UA and GA, it is the norm of the difference of the two velocity vectors for the two aircraft:vGA and vUA are the norms, and θGA and θUA the angles  of vx and vGA, respectively. Assuming that both θGA and θUA are uniformly distributed on , the PDF for θ=θ UA −θ GA  is:cosθ in Equation to be the average time the cylinders are not intersecting horizontally. Then the probability of intersection can be expressed as:pHC in this formulation is hertz. Note that this can be simplified by the assumptions r GA ≫r UA  and v GA ≫v UA , resulting in:Now,2.2.2G as defined above is typically a state or nation because states and nations have well‐known areas, and databases for GA typically cover specifically states or nations. However, it means that a certain fraction of G inevitably are areas where UA cannot legally operate , and consequently Equation G∗⊂G, representing the UA legally accessible area of G, would not remedy the problem as the yearly operational time T covers all flight of the GA, including flight outside G∗. However, under the  assumption that the fraction G∗/G approximately equals T∗/T, where T∗ is the flight time spent in G∗, Equation G∗/G and T∗/T are close to 1.The geographical area 2.2.3Under the assumption that the UA cylinder and the GA cylinder are at the same geographical location, we will now determine the probability that the cylinders intersect in the vertical direction. The vertical location  of both aircraft is at the vertical middle of the respective cylinders. Thus, there is a collision if the difference in altitude between the two aircraft is less than the sum of the cylinder heights.f GA (z) and f UA (z) for the GA and UA, respectively. These are chosen so as to reflect how the aircraft actually behave in terms of altitude  the areas listed above as exceptions. The model is also based on the assumption that in most cases when GA is indeed below zmax in rural or urban areas, it is not a normal operational situation . Knowing approximately how often such abnormal operations occur allows for an estimate of how often GA is indeed below zmax. As described above, the model pbelow that expresses the likelihood of a GA being below zmax . Estimated values for Denmark are shown and discussed in Section In most countries, some geographical areas are designated as non‐UA areas . By definition, the complement to the set of these areas is the areas where UA can indeed fly. The model implementation has the assumption  before the model is practically useful. Our approach to this is described in Section To have a model that captures jointly all GA in the airspace , we want to determine the four right‐hand side factors in Equation pMAC from the model pMAC.A good approximation of the joint probability for a MAC is a weighted summation of The final model becomes:2.4pMAC. From an overall perspective, event rates and probabilities appearing in risk analysis will often be of small magnitude, whereas the accuracy by which they are given is logarithmic. That is, if results predict the correct order of magnitude, they will most likely be acceptable. This aligns with the aim of this work, as stated in Section The quantitative framework developed above for collision rate assessment is model based, and a number of simplifying assumptions were made for mathematical tractability. This of course introduces model errors, which manifest themselves into quantitative errors in the final result ti in Equation Ti from the ellipsoidal center of a vehicle with a speed vector  is found by:Vz=0 is given by:Vz2 to be at least 100 times smaller than Vx2+Vy2, we find Vz accounts for at most 16% of the result. In addition, assuming that Vz is only nonnegligible for a limited part of a normal flight, say 10% of the time, this further reduces the average error by that same factor. We get from Equation p HC  in Equation One significant simplification is the disregard of vertical flight speeds in the expression for PCI is less than 1, which directly affects the final result as a multiplicative error. However, PCI is still relatively close to 1, since most GA as well as UA will appear as a predominantly filled rectangle  in the flight direction. Thus, PCI and therefore the result is skewed much less than an order of magnitude  in available flight incident statistics. As described in Section pbelow in Table These types are provided by the DTCHA from its database of GA registered in Denmark, and the values for z max =100.Since the maximum altitude in Denmark for flights outside urban areas is 100 m, we set Three different types of UA are used; a generic smaller fixed‐wing aircraft, the DJI Matrice 600 multirotor, and the larger fixed‐wing UAV Factory Penguin C. The parameters for the aircraft are shown in Table 3.1.2The values from mitigation are shown in Table 3.1.3The GA distributions show how each GA type is distributed in altitude when below 100 m. This information is quite difficult to determine accurately, and in this work is based purely on considerations on how each type of GA could be expected to behave below 100 m. Three different distributions are used, a uniform between 0 m and 100 m, and two normal distributions, one with mean 100 m and one with mean 50 m. The used GA distributions are shown in Fig. Each GA type is assigned one of these distributions, as shown in Table Rotorcraft can occasionally conduct operations at lower altitude, but tend to stay some distance over the ground. The same goes for balloons, which tend to be some distance from the ground when below 100 m. Ultralights are powered and would typically also not fly low during most of the flight. All these GA are therefore assigned a normal distribution that puts them predominantly at the higher altitudes.Paragliders and hang gliders are unpowered and are expected to operate around 50 m during the ascent and landing. As such, they are assigned a normal distribution around 50 m.Do note that these distributions are speculative insofar as no research, to the best knowledge of the authors, has been done to actually determine such altitude distributions.3.1.41.z max =100).Uniform distribution from 0 to maximum altitude, exemplified with a generic aircraft conducting flight operations at varying altitudes, and modeled with uniform distribution U, that is, a normal distribution with mean 25 m and standard deviation 20 m.3.Near‐maximum altitude long endurance flight mission, exemplified with a Penguin C aircraft doing hub‐to‐hub parcel transport, and modeled with N.Both normal distributions are cropped to  and scaled to integrated to 1. The three distributions are shown on the right in Fig. The altitude profile for operations of UA is obviously varying with the type of mission, and can be anything from flying at very low altitudes for action photography over changing altitudes for inspection to long endurance operations at maximum altitude for surveying. We will use three different profiles here:3.2Using the parameters for Danish airspace shown in Table pVC along with the probability of each type being below zmax. The pVC numbers are shown in a separate plot since they are relatively high compared to pHC, and since they are probabilities (as opposed to rates).First, Fig. ρi set to 1 and ρi set according to Table −7 MAC per flight hour is also shown in each plot, as this is the order of magnitude that several sources report as being equivalent to the level of safety for GA , which may not necessarily be easy to obtain for a given airspace. However, this approach does provide some degree of certainty in the resulting probabilities in comparison to models based on stochastic assumptions only. The aim has been to achieve a precision of at least one order of magnitude relative to the true MAC rate.−6 collision per flight hour .The model is applied to Danish airspace and gives a generic MAC rate of approximately 10−7 MAC per flight hour given by FAA  the ability to provide a reasonable estimate of MAC for any airspace where only standard data held by the aviation authorities are available, and (2) the applicability to the most common type of airspace that UA operate in, now as well as in the foreseeable future.Although the model in Equation The assumption that GA is uniformly distributed in the relevant air volume is also a weakness of the model, insofar as UA pilots are aware of this and adapt their behavior accordingly . However, while a model that would include such geographically specific information would not be that complicated to implement, it would probably be rather difficult to obtain reliable data for such a model.4.2pbelow as well as λSTM, which at this point are estimations only.The primary effort in future work would be to address the separation of horizontal and vertical motions, that is, eliminating assumption 6 in Section


The aim of this study was to evaluate the reproductive performance of a new artificial insemination (AI) device specifically designed for gilts  by means of which the sperm is deposited deeply in the cervix . New AI techniques have arisen in recent decades in the porcine industry, such as post-cervical artificial insemination (PCAI), which involves depositing the sperm in the body of the uterus [through a catheter (outer tube)-cannula (inner tube)] rather than by CAI. Although the PCAI method has been successfully applied in farm conditions to reduce sperm doses without impairing the reproductive performance, this technique has limitations in gilts mainly because of the difficulty involved in introducing the inner cannula through the cranial part of the cervix. For this reason, the Dp-CAI method described herein may be considered as an alternative to CAI and PCAI methods in gilts.n = 1166) inseminated using 1.5 × 109 sperm/45 mL; 2) CAI (as a control group): gilts (n = 130) inseminated using 2.5 × 109 sperm/85 mL. The Dp-CAI method was successfully applied in 88.90% of the gilts, with no differences detected between gilts with 1 or 2 previous oestrus cycles, although the catheter could be introduced more deeply in 2 oestrus gilts (P < 0.05). As the length of the insemination device that could not be introduced increased (at the moment of insemination), so the success rate of the Dp-CAI device fell, as did the total number of piglets born. When the reproductive output in CAI and Dp-CAI was compared, none of the parameters analysed  showed significant differences.Gilts were divided in two experimental groups: 1) Dp-CAI: gilts  contains supplementary material, which is available to authorized users. Nowadays, more than 85% of swine insemination in developed countries is performed using assisted reproduction techniques , 2. The However, the use of PCAI has certain limitations that need to be considered. While the success rate of introducing the insemination device in multiparous sows is higher than 95% (75% at the first attempt) , 14, 15,The fact that PCAI cannot be performed consistently in young females could be due to the smaller dimension of the cervix, which would become the major barrier because the reproductive tract would not have undergone total physical development . With eaPrecisely, one of the critical points in the porcine breeding system is the high percentage (~ 50%) of replacement among multiparous sows on pig farms , 21, whi9 sperm/85 mL; Dp-CAI: 1.5 × 109 sperm/45 mL). In both studies, the number of previous oestrus cycles and the depth that the insemination device could be inserted (measured as the length of the device protruding from the gilt during AI) were related with the level of success and the reproductive performance attainable with the new device.For all the reasons mentioned above, the aim of this study was to test a new AI device, specifically designed for use in nulliparous females, taking into account their anatomical limitations and allowing the deposition of semen deep in the cervix. The study first evaluated the degree of success of the new device  in nulliparous females, and then the reproductive performance  obtained with the new AI method compared with CAI, but reducing the sperm number per insemination (CAI: 2.5 × 10The study was carried out under field conditions from March to July 2016 (Cherkizovo Group). The gilts used were F1 (Landrace Sire x Large White Dam) all sourced from the same farm. Boars (337 PIC and 1–4 years old) of proven fertility were used in this study. Gilts were fed ad libitum until insemination, while boars and inseminated gilts had a restricted feeding regime according to their nutritional requirements. Water was available ad libitum. Temperature levels were controlled automatically by a climate control system which maintained the temperature at 18–22 °C.9 and 1.5 × 109 spermatozoa, respectively. Semen doses were conserved at 16–18 °C and used for AI within 48 h after collection. Sperm doses from the same boar were divided in the same proportion among experimental groups to avoid any boar effect.The semen used for AI was delivered daily from a commercial boar stud belonging to the same company. Each boar was used to obtain semen every 5 d by a trained worker using gloves and a collection bag with filter . The semen ejaculate was analysed using a computer-assisted spermatozoa motility analyzer (CASA)  for spermatozoa motility, morphology and concentration. For the study, only boars with > 85% spermatozoa progressive motility and < 20% abnormal spermatozoa morphology were used. Ejaculates which met the minimum criteria were further diluted with commercial extender  and then pooled into groups of three before being stored. Semen doses were prepared in 85 and 45 mL bottles containing 2.5 × 10Exposure to boars to detect estrus was initiated at 25 weeks of age, allowing each gilt 10 min of nose to nose contact with a vasectomized mature boar while experienced workers applied back pressure. Standing reflex, vulva swelling, and redness were considered signs of estrus and were recorded. The criterion for insemination was a minimum of one oestrus cycle with any previous service. Each gilt received two AI sessions, the first on the day estrus was detected and the second, 24 h later. Females were weighed before insemination in order to ensure similar weights in all the experimental groups. The AI procedure was performed in individual crates.9 spermatozoa. As in CAI, the insemination dose was introduced slowly to reduce backflow because the deposition is still in the cervix but deeper. Moreover, during Dp-CAI the rear part of the cannula is attached to the back of the catheter  was performed using a new kit composed of a device which combined catheter and cannula Fig.   was performed with the same device used for Dp-CAI but without the inner cannula (using only the catheter). Gilts were inseminated with the boar present and using a non-spermicidal gel to introduce the catheter. An experienced worker applied back pressure while the semen flowed into the uterus. Each gilt received 1 dose per AI of 85 mL containing 2.5 × 10Gilts were checked for estrus 21 d after insemination by boar exposure but with no contact, while an experienced worker applied back pressure in search of a standing reflex. Any gilt which showed estrus signs was taken out and re-allocated to another pen. Pregnancy was confirmed 35 d after AI by transabdominal ultrasonography .n = 130; average weight: 142.35 ± 8.06 kg) and Dp-CAI . First, the application of the new Dp-CAI device for gilts  or 2 (n = 1045)]. Subsequently, reproductive performance was compared in the two experimental groups: CAI (2.5 × 109 sperm cells/85 mL) and Dp-CAI (1.5 × 109 sperm cells/45 mL). The studied reproductive parameters were abortion %, pregnancy %, farrowing %, litter size born  and fecundity index [Farrowing rate multiplied by the average number of total piglets born per litter ]. Additionally, pregnancy %, total and live piglets born were related with the length of the device protruding from the gilt and the number of oestrus [1 (n = 102) or 2 (n = 934)] prior to Dp-CAI application. Finally, the association between length of the device (outside the gilt) and litter size  was evaluated.A total number of 1296 gilts were divided into two groups: CAI   or percentage . A regression analysis was performed to determine the relationship between the length of the device protruding from the gilt and litter size .Statistical analyses were performed using IBM SPSS v.21 . Quantitative data  were analysed for normality using a Kolmogorov-Smirnov test and for homogeneity of variances using the Levene test. When neither of the tests was fulfilled, the non-parametric, Mann-Whitney test was used. Pregnancy, farrowing and abortion rates were compared using Pearson P = 0.068) towards a higher rate in gilts that previously had 2 oestrus cycles (1-oestrus 84.3% vs. 2-oestrus 89.4% of Dp-CAI success application)   for any parameter studied , no differences were found in any parameter evaluated (P > 0.05)    was close to 90%, which contrasts with the low level reached when PCAI is used in gilts, which ranges between 20% and 60% depending on the device and treatment used . One of rima vulvae to the last cervical cushion was evaluated by our group [The morphology of the female genital tract varies with age and the number of parturitions –26. Nullur group  it was ~The penetration length of the insemination device in gilts with two previous oestrus cycles was greater than in animals with only one cycle. The success rate of Dp-CAI tended to be greater when gilts had two previous oestrus cycles. The uterus varies in size in the transition from non-cyclic to cyclic gilts , thus faThe extent to which the success of Dp-CAI was associated with the length of the insemination device remaining outside the gilt during AI Fig. b was als9 fewer sperm cells per insemination (2 sessions of AI per oestrus cycle). This agrees with other reports that the deeper sperm can be deposited, the fewer sperm per AI dose are necessary without having a negative influence on the reproductive performance [One of the main goals that the swine industry hopes to achieve is a reduction in costs involved in pig production. One of the strategies to achieve this would be to decrease the number of spermatozoa inseminated per dose while maintaining the same reproductive efficiency . Indeed,formance –9. It haformance  and alsoformance . This inOne of the methodological differences between PCAI and Dp-CAI is the time necessary for sperm deposition. In contrast to the PCAI method, sperm deposition at the moment of insemination has to be performed slowly, more similarly to what is necessary with the CAI method. This is a consequence of not crossing the cervix barrier completely, and since the uterine part of the cervix is narrow more backflow can be observed if the insemination is performed quickly. The higher rate of backflow during insemination found in younger sows during CAI  is, probThe particular anatomy of the cervix in porcine females and the differences previously found in this anatomical part between nulliparous and multiparous animals have encouraged the development of new strategies and/or devices for deep AI in nulliparous females. The application of Dp-CAI using a new device specifically designed for gilts permits sperm to be deposited deep in the cervix with a high degree of success. Although additional studies are necessary into other aspects of Dp-CAI , this new method present itself as an alternative to PCAI and CAI in nulliparous females reducing the number of spermatozoa used per insemination without impairing the final reproductive performance, as demonstrated in a farm trial. The technique can be successfully used in gilts instead of the commonly used CAI method to reduce insemination costs.Additional file 1:9 sperm/45 mL) in gilts with 1 or 2 previously detected oestrus. Data show rate (%) or mean ± SD. No statistically significant differences were observed in any of the parameters studied (P > 0.05). (DOCX 19 kb)Reproductive parameters obtained after Dp-CAI . (JPG 15000 kb)Association between length of device protruding from the gilt at insemination (Dp-CAI) with total a, and live b, piglets born. The relationship between length and litter size  was not significant (


The quality check and determination of permitted and non-permitted additive colors in food products is very important for customer׳s right protection and health. This survey was undertaken to demonstrate the frequently use of additive colors and products targeted to color adulteration in Iranian foods and beverages. From the 1120 of the samples, 18.86% contained artificial colors, 11.89% contained natural colors and 69.25% of samples had no additive colors. Tartrazine (E102) was the only non-permitted artificial dye used in samples. Among products with additive colors, only 4.38% of samples failed to meet with national Iranian standard and 61.23% of non-compliance samples were from non-industrial sectors and mostly were saffron and food containing saffron such as saffron rock candy and saffron chicken. These places and products quality are main the concern to solve the color adulteration in Iranian food market. Specifications TableValue of the data•The dataset shows the amount of additive colors in local food and beverage products.•The dataset signifies the amount of permitted and non-permitted colors under Iranian laws.•The dataset of this article identifies products with non-authorized colors, which can be useful for other investigators working in area of quality assurance of foods.1The dataset provided here , Table 32The Iranian national standard organization only permits seven artificial dyes of Quinoline Yellow (E104), Sunset Yellow FCF (E110), Azorubine (E122), as well as Ponceau 4R (E124), Allura Red AC (E129), Indigotine (E132) and Brilliant Blue FCF (E133) in different type of products. The prevalence use of permitted and non-permitted artificial dyes has been reported from different states of Iran, previously. The required data were collected from the results documented in Kimia Test FAM Laboratory during the five years of 2011–2015. The additive color of 1120 samples, which represent 30% of samples analyzed in Tehran city for additive colors, was determined using thin layer chromatography methods (TLC) according to 2634 Iranian national standard and were compared to 740 Iranian national standard for permitted food additive colors. The data were entered into an Excel spreadsheet and were analyzed by descriptive statistics


Sepsis-associated encephalopathy induces cognitive dysfunction via mechanisms that commonly involve neuroinflammation and synaptic plasticity impairment of the hippocampus. The β2-adrenoceptor (β2-AR) is a G-protein coupled receptor that regulates immune response and synaptic plasticity, whereas its dysfunction has been implicated in various neurodegenerative diseases. Thus, we hypothesized abnormal β2-AR signaling is involved in sepsis-induced cognitive impairment. In the present study, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) to mimic the clinical human sepsis-associated encephalopathy. The levels of hippocampal β2-AR, proinflammatory cytokines tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), IL-6, cAMP-response element binding protein (CREB), brain derived neurotrophic factor (BDNF), post-synaptic density protein 95 (PSD95), and NMDA receptor 2 B subtypes (GluN2B) were determined at 6, 12, 24 h and 7 and 16 days after CLP. For the interventional study, mice were treated with β2-AR agonist clenbuterol in two ways: early treatment (immediately following CLP) and delayed treatment (on the 8th day following CLP). Neurobehavioral performances were assessed by open field and fear conditioning tests. Here, we found that hippocampal β2-AR expression was significantly decreased starting from 12 h and persisted until 16 days following CLP. Besides, sepsis mice also exhibited increasing neuroinflammation, down-regulated CREB/BDNF, decreasing PSD95 and GluN2B expression, and displayed hippocampus-dependent cognitive impairments. Notably, early clenbuterol treatment alleviated sepsis-induced cognitive deficits by polarizing microglia toward an anti-inflammatory phenotype, reducing proinflammatory cytokines including IL-1β, TNF-α, and up-regulating CREB/BDNF, PSD95, and GluN2B. Intriguingly, delayed clenbuterol treatment also improved cognitive impairments by normalization of hippocampal CREB/BDNF, PSD95, and GluN2B. In summary, our results support the beneficial effects of both early and delayed clenbuterol treatment, which suggests that activation of β2-AR has a translational value in sepsis-associated organ dysfunction including cognitive impairments. Sepsis is defined as the host’s reaction to infection and characterized by a systemic inflammatory response with important clinical implications . Sepsis-Sepsis-associated encephalopathy is a diffuse brain disorder involving multiple brain regions and the hippocampus is one of the most vulnerable parts, which is highly susceptible to ischemia, anoxia, and inflammation . The hipThe β2-adrenoceptor (β2-AR) is a G-protein coupled cell membrane receptor, which involves activation of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) cascade, resulting in phosphorylating cyclic AMP response element binding protein at Ser-133 (pCREB) and thus facilitating the transcription of key proteins necessary for synaptic plasticity such as brain-derived neurotrophic factor (BDNF) . The latBased on these findings, we aimed to investigated whether abnormal β2-AR signaling is involved in sepsis-induced cognitive impairments induced by cecal ligation and perforation (CLP). Furthermore, we hypothesized that the beneficial effects of β2-AR activation is related to its ability to reverse the neuroinflammation and synaptic abnormalities after sepsis.Two hundred and twenty male C57BL/6 mice  were obtained from the Animal Center of Jinling Hospital, Nanjing, China and efforts were made to minimize the number of mice involved and their suffering as possible as we can. All procedures were carried out based on the Guideline for the Care and Use of Laboratory Animals from the National Institutes of Health . Experiments were performed in accordance with protocols approved by the Ethics Committee of Jinling Hospital, Nanjing University, China. Mice were reared in groups of 3–5 individuals per cage under aseptic conditions maintained at 23 ± 1°C with stable humidity, and had free access to food and water. We begun the experiments after the mice acclimating to the environment for 2 weeks.i.p.) injection, followed by a 1-cm midline, under aseptic conditions. Subsequently, the cecum was exteriorized and ligated with a 4.0 silk suture, just below the ileocecal valve. Then, the cecum was gently perforated once with 14-gauge needle and squeezed to extrude little feces through the perforation site. Finally, the cecum was carefully returned to the peritoneal cavity and the laparotomy was closed with 4.0 silk sutures. Mice were subcutaneously resuscitated with normal saline (20 ml/kg) immediately after the surgery. All mice were returned to their cages with free access to food and water. For sham groups, mice were treated identically but without ligation or puncture of the cecum.Sepsis was induced by CLP as previously described . AnimalsTo determine the time course of hippocampal β2-AR, proinflamamtory mediators and synaptic proteins, the mice were euthanized at following time points: 6, 12, 24 h, 7 days and 16 days after CLP.i.p. immediately after operation and once daily for five consecutive days, followed by a 2-days off period, repeated weekly  at 0.02, 0.1, or 0.5 mg/kg dissolved in saline solution was injected d weekly . Animalsi.p. immediately after operation and until the end of the behavioral tests. The experimental design was shown in For the early treatment groups, mice received clenbuterol i.p. on the 8th day after operation and until the end of the behavioral tests. The experimental design was shown in For the delayed treatment groups, mice received clenbuterol All behavioral procedures were carried out at 2:00–5:00 p.m. in a sound-isolated room. Tests were recorded by the same investigator who was blinded to the grouping of mice as described by our previous studies .The spontaneous locomotor activity of the mice in the open field test was tracked and recorded by a photobeam activity system and software . The apparatus is made up of a white polyester resin chamber (40 cm × 60 cm × 50 cm high). The mice were placed in the center of the arena and allowed to explore for 5 min, and the total distance moved and the time spent in the center were recorded. The chamber was cleaned with 75% ethanol after each test.On the day of training, the animals were placed into an enclosed training chamber and allowed to explore for 180 s. Then, the mice were exposed to a tone , followed by a 2 s foot shock (0.75 mA). Afterward, the mice were left in the chamber for additional 30 s before being returned to their home cage. Twenty-four hours after the training session, the mice were reexposed in the same chamber for 5 min for the contextual fear conditioning test (a hippocampus-dependent task) . Two houi.p.) and perfused transcardially with 0.1M phosphate-buffered saline , followed by 4% phosphate-buffered paraformaldehyde (PFA). The brains were removed, post-fixed for 14 h in the same PFA solution, and dehydrated, sequentially embedded in paraffin. Coronal sections of 5 μm thickness were cut for the immunostaining. To quench endogenous peroxidase activity, brain slices were incubated with 3% H2O2 in methanol for 10 min. After being rinsed with phosphate-buffered saline (PBS), sections were blocked with 10% goat serum for 30 min, and incubated overnight at 4°C with a rabbit antibody against the β2-AR . After washing with PBS, sections were incubated with biotinylated goat anti-rabbit  for 1 h at room temperature. After washings, the freshly prepared 3,3′-diaminobenzidine (DAB) solution was added and control the reaction time properly. After dyed with hematoxylin, the sections were dipped in 1% hydrochloric acid in alcohol for differentiation. Then, the sections were washed in ammonia and stained blue, rinsed, and coverslipped following gradient dehydration in increasing concentrations of ethanol and cleared with xylene. All the sections were examined under the digital microscope  to record the intensity of antigen antibody reaction. The observation of the sections was carried out at magnification of 400X. For immunohistochemistry analysis, three sections per mouse and three mice per group were evaluated for hippocampal region (complete section). Morphometric analysis was carried to observe extent of immunoreactivity. Optical density (OD) was determined as measure of β2-AR expression in hippocampus by analyzing the images with ImageJ 1.51a software .The mice were deeply anesthetized with 2% sodium pentobarbital in saline , membranes were incubated overnight at 4°C in each primary antibody. The primary antibodies used were rabbit anti-β2-AR , rabbit anti-CD11b , goat anti-IL-1β , rabbit anti-TNF-α , mouse anti-IL-6 , mouse anti-CD16 , mouse anti-arginase1 , mouse anti-CD68 , rabbit anti-CD206 , rabbit anti-pCREB , rabbit anti-CREB , rabbit anti-BDNF , anti-pro BDNF (5H8) , rabbit anti-postsynaptic density 95 (PSD95) , rabbit anti-NMDAR2B (GluN2B) , rabbit anti-synaptophysin , rabbit anti-β-tubulin . After washing in TBST for three times, the membrane were incubated with appropriate secondary antibodies . Chemiluminesence method was used to detect the protein bands and the band intensity were analyzed with Image J Software .The hippocampus was harvested for Western blotting analysis at the indicated time points. The hippocampus was isolated as described previously . Each sat-tests, whereas differences among multiple means were assessed by one-way, two-way, repeated-measures of analysis of variance (ANOVA) followed by a Bonferroni test as appropriate. The interaction effect of operation  and treatment  has been indicated in the text as, e.g., the interaction between CLP and clenbuterol treatment and has been analyzed with a two-way ANOVA. Since IL-1β is not normally distributed, Kruskal–Wallis H and Mann–Whitney U-tests were used. The survival rate was analyzed by the Kaplan–Meier method and compared by the log-rank test. A p < 0.05 was regarded as statistically significant.Statistical analyses were performed by the Statistical Product for Social Sciences . All data are presented as mean ± SEM. Data were tested for normal distribution by the Shapiro–Wilk test. For data that are normally distributed, differences between two groups were compared by independent-samples F5,12= 17.384, p < 0.001; t = 5.177, p = 0.007; CA3: t = 2.918, p = 0.043; DG: t = 4.691, p = 0.009; t = 4.532, p = 0.011; CA3: t = 3.988, p = 0.016; DG: t = 3.976, p = 0.017; We utilized Western blotting analysis and immunohistochemistry to quantify β2-AR expression in the hippocampus following CLP. Western blotting analysis showed that β2-AR was significantly decreased starting from 12 h and persisted until 16 days following CLP  and the ratio of mBDNF/proBDNF  at 24 h and persisted until 16 days following CLP. As shown in F5,12= 13.08, p < 0.001) and GluN2B  decreased from 24 h and persisted until 16 days following CLP. However, the level of the presynaptic membrane protein synaptophysin was not altered .One of the typical signaling pathways downstream of the β2-AR activation involves pCREB at Ser-133 (pCREB) and facilitates the transcription of BDNF. BDNF is synthesized as a precursor molecule (proBDNF), and then cleaved into mature BDNF within endoplasmic reticulum, which plays an important role in synaptic plasticity and neuronal survival . As showF4,44= 4.463, p = 0.004; F4,44= 0.707, p = 0.600; time spent in the center of arena: F4,44= 0.219, p = 0.926; As revealed in F1,35= 0.177, p = 0.676; F1,35= 0.004, p = 0.949; Post hoc comparisons revealed that no difference in the total distance (p > 0.05) and time spent in the center of arena (p > 0.05) was observed among the four groups. As above, early clenbuterol treatment did not improve the survival rate and the weight of the sepsis , which were reversed by clenbuterol treatment (p < 0.05). However, there was no difference in interaction between CLP and clenbuterol treatment for the post-tone freezing time in the auditor-cued fear conditioning test  was observed among the four groups. These data suggested that early clenbuterol treatment reversed hippocampus-dependent cognitive impairments.To evaluate sepsis-induced cognitive impairments, the fear conditioning test was performed at 14 days after operation. The interaction between CLP and clenbuterol treatment was significant for contextual freezing time  were markedly increased in sepsis mice (p < 0.05), which were reversed by clenbuterol treatment . In addition, the interaction between CLP and clenbuterol treatment was significant for arginase1  were dramatically increased in CLP + clenbuterol group compared with CLP + vehicle group . The interaction between CLP and clenbuterol treatment was significant for IL-1β , whereas clenbuterol treatment markedly decreased these inflammatory mediators . There was no significant difference in IL-6 level at 24 h following CLP among the four groups. There was no difference in interaction between CLP and clenbuterol treatment for CD68, CD16, CD206, arginase1, IL-1β, TNF-α, and IL-6 at 16 days following CLP  was observed among the four groups. Interestingly, the interaction between CLP and clenbuterol treatment was significant for CD11b at 24 h , which could be decreased by clenbuterol treatment .We next investigated whether clenbuterol modulate sepsis-induced inflammatory responses in the hippocampus following CLP. The interaction between CLP and clenbuterol treatment was significant for CD68  and 16 days  following CLP, which were significantly reversed by clenbuterol treatment both at 24 h  and 16 days  following CLP.We next assessed whether clenbuterol upregulated CREB/BDNF signaling pathway in the hippocampus. The interaction between CLP and clenbuterol treatment was significant for pCREB, total CREB (CREB) and the ratio of mBDNF/proBDNF at 24 h following CLP  but not at 24 h . Post hoc comparisons showed that sepsis led to significantly decreased expressions of post-synaptic protein PSD95 and GluN2B at 24 h  and 16 days  following CLP in the hippocampus, which were significantly attenuated by clenbuterol treatment  and 16 days . As indicated in F1,8= 0.001, p = 0.996; F1,8= 0.059, p = 0.815; Post hoc comparisons revealed that no difference in synaptophysin at 24 h and 16 days following CLP  was observed among the four groups.Subsequently, we evaluated the effects of clenbuterol on the synaptic plasticity-related proteins. The interaction between CLP and clenbuterol treatment was significant for GluN2B at 24 h and 16 days following CLP in the hippocampus , which was reversed by clenbuterol treatment (p < 0.05). However, there was no difference in interaction between CLP and clenbuterol treatment for the post-tone freezing time in the cued fear conditioning test  was observed among the four groups.To be more clinically relevant, we determined whether clenbuterol initiated in late sepsis phase could also achieve analogous benefits. As shown in F1,8 = 14.96, p = 0.005; CREB: F1,8 = 27.98, p < 0.001; the ratio of mBDNF/proBDNF: F1,8 = 23.48, p = 0.001; Post hoc comparisons revealed that delayed clenbuterol treatment could rescue these downregulated protein expressions . In addition, the interaction between CLP and clenbuterol treatment was also significant for PSD95, but not for GluN2B . Likewise, there was no difference in interaction between CLP and clenbuterol treatment for the presynaptic protein synaptophysin  was observed among the four groups.As revealed in In the present study, we showed that the expression of β2-AR was significantly reduced in the hippocampus of sepsis mice. To the best of our knowledge, this was the first demonstration that sepsis induced downregulated expression of β2-AR in the hippocampus. Importantly, we found that early treatment with the brain penetrant β2-AR agonist, clenbuterol, had a neuroprotective effect but was not sufficient to improve survival rate. These data complement previous studies indicating that clenbuterol has neuroprotective actions in rodent models of ischemic stroke , Rett syThe noradrenergic transmission system is known to be involved in various forms of learning and memory . AbnormaThe pathophysiology of cognitive impairments induced by sepsis is likely to be multifactorial. Among these mechanisms, release of proinflammatory mediators within the brain play a vital role . Inflammin vitro study showed that β2-AR activation dampened the LPS-induced M1 polarization in macrophages  were significantly upregulated while M2-associated markers downregulated (CD206 and arginase1) at 24 h after sepsis, which were reversed by clenbuterol treatment. These data raise the possibilty that clenbuterol treatment suppressed inflammatory mediators IL-1β and TNF-α by promoting alternative microglia activation and exerting neuroprotective effects in sepsis-induced cognitive impairments, although we cannot exclude its possible peripheral anti-inflammatory effects.As resident macrophages of the CNS, microglia are well-known as a sensor of tissue injury or infection and are pivotal in the pathogenesis of neurodegenerative diseases . Activatrophages . Anotherrophages . In the i.p. injection of LPS induced synaptophysin loss lasted at least for 2 months after sepsis . Experiments were performed in accordance with protocols approved by the Ethics Committee of Jinling Hospital, Nanjing University, China.M-MZ carried out the behavioral study and drafted the manuscript. M-HJ, MJ, and HT performed the immunohistochemistry and Western blotting analysis. Z-QZ and M-HJ participated in the design of the study and statistical analysis. J-JY guided the study design and helped to draft the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.