Auxin plays crucial roles in multiple developmental processes, such as embryogenesis, organogenesis, cell determination and division, as well as tropic responses. These processes are finely coordinated by the auxin, which requires the polar distribution of auxin within tissues and cells. The intercellular directionality of auxin flow is closely related to the asymmetric subcellular location of PIN-FORMED (PIN) auxin efflux transporters. All PIN proteins have a conserved structure with a central hydrophilic loop domain, which harbors several phosphosites targeted by a set of protein kinases. The activities of PIN proteins are finely regulated by diverse endogenous and exogenous stimuli at multiple layers—including transcriptional and epigenetic levels, post-transcriptional modifications, subcellular trafficking, as well as PINs’ recycling and turnover—to facilitate the developmental processes in an auxin gradient-dependent manner. Here, the recent advances in the structure, evolution, regulation and functions of PIN proteins in plants will be discussed. The information provided by this review will shed new light on the asymmetric auxin-distribution-dependent development processes mediated by PIN transporters in plants. Auxin is a universal hormone in plants, which participates in many aspects of plant developmental and growth processes . The actAmong the mentioned auxin carriers above, the polar localizations of PIN proteins finely correspond to the directionality of auxin movement, which highlights that PIN proteins are mainly responsible for the asymmetric distributions of auxin in plants ,8,20. ThPIN1 in Arabidopsis was the first identified PIN gene, afterwards, seven additional PIN genes were found in the genome of Arabidopsis [PIN genes have been identified in 31 plant species by genome-wide approaches, including Brassica rapa, Glycine max, Medicago truncatula, Oryza sativa, Populus trichocarpa and Zea mays  and other databases, the information about PIN genes in these gymnosperm plants is still very limited at a genome-wide scale  depending on the phosphorylation states [cis-acting regulators to regulate PIN2 polar localization [The PIN carriers in plants participate in auxin transport at both intercellular and intracellular levels. The differences in function are dependent on the subcellular localizations of different PIN proteins with distinct molecular structures . All theproteins ,29,59. Tproteins . Some lon states ,61,62. Tn states ,59,63. Tn states . The HL n states . Addition states ,65,66. Tlization .In addition to the terms “long” and “short” PIN proteins, another pair of more precise terms, “canonical” and “noncanonical” PIN proteins is proposed based on the existing highly-conserved HC1–HC4 regions in the central loop domain . The PINPIN genes have been suggested to evolve from a single ancestral gene, since PIN genes from higher plants shared relatively high sequence similarities with each other compared to the homologues of the closest bacteria [The bacteria . The PINbacteria ,68. The bacteria ,60,69. Hbacteria ,36. The bacteria ,36. The bacteria . Furtherbacteria ,36. Thusbacteria ,36.PIN co-expression network was studied in Arabidopsis using an online database CressExpress  with data from 8941 arrays [PIN genes  were co-expressed with another 141 genes via 303 correlations  and three IAA) identified in this network all belonged to the PIN3-PIN4-PIN7 module, suggesting that the PIN3-PIN4-PIN7 co-expression module plays crucial roles in auxin transport and signaling processes and that the two processes were finely co-regulated at the transcriptional level. Additionally, two genes encoding the basic helix-loop-helix (bHLH) protein, HOMOLOG OF BEE2 INTERACTING WITH IBH 1 (HBI1) and BR ENHANCED EXPRESSION 2 (BEE2), were found in the core PIN3-PIN4-PIN7 module. These are known as regulators of cell elongation via targeting their downstream auxin metabolism relative genes [PIN3, PIN4 and PIN7, it is of interest to check the specific roles of these two genes in PIN-mediated auxin efflux. Overall, the information obtained from the co-expression network conforms to the current knowledge on the regulation of PIN genes and also provides the possible routes to dissect the new candidates involved in PIN-mediated auxin efflux.Genes involved in the same pathways are highly co-expressed compared to genes from different pathways . Thus, t1 arrays . In the elations A. Intereve genes ,75. GiveArabidopsis, it has become possible to identify a large set of physical interactors of PIN proteins , type-1 protein phosphatase 4 (TOPP4), phytochrome-associated serine/threonine-protein phosphatase 1 (FyPP1), FyPP3, ROOTS CURL IN NPA1 (RCN1) and SAPS-domain-LIKE like 1 (SAL1) [In addition to the co-expression network, by taking advantage of large-scale interactome analyses with diverse experimental approaches in proteins . The comproteins . About 3proteins . Many kn1 (SAL1) ,77,78,791 (SAL1) ,81,82. T1 (SAL1) ,83. The 1 (SAL1) ,85. The Overall, these resources provided a range of candidates potentially involved in regulating PIN proteins at both transcriptional and protein levels. These candidates participate in multiple processes beyond plant development and growth. Thus, new roles of PIN proteins in these aspects may be uncovered by digging this valuable mine of information.Auxin polar transport facilitated by PIN proteins plays a pivotal role in determining the auxin maxima and minima within cells via stitching auxin metabolism together with signal transduction processes. The activities of PIN proteins are turned in multiple dimensions to convert the internal and external stimuli into down-stream gene reprogramming events in a rapid and precise manner. Much attention has been paid to dissecting the regulation of PIN proteins. These processes are involved in regulating PIN gene expression levels, post-transcriptional modifications of PIN proteins to facilitate their cellular trafficking, PIN proteins recycling and turnover ,91,92,93PIN genes is finely regulated by diverse hormonal and environmental stimuli [PIN genes are induced by auxin itself, except PIN5, the transcription of which is downregulated by auxin [PIN genes to regulate their expression levels in response to changes in endogenous and exogenous signals [XAANTAL2 (XAL2/AGL14), belonging to the MADS-box gene family, was the first identified transcriptional factor [PIN1 and PIN4 to upregulate their expression and is involved in auxin-mediated root developmental processes in a PIN4-dependent manner [PIN1 has been shown to be one target of INDETERMINATE-DOMAIN 16 (IDD16) [YUCCA5 (YUC5) and PIN1 [PIN2 regulator by the yeast one-hybrid methodology. In silico analysis revealed that parts of PPP1 DNA-binding sites commonly exist in the promoter regions of most PIN genes [PIN4 and PIN5 and cause reduced expression levels of some PIN genes  in the wri1-1 mutant background compared to wildtype specimens [PIN gene expression and fatty acid synthesis.The expression of  stimuli ,21. Mostby auxin ,60. Addi signals ,91. XAANl factor . It diret manner . Further (IDD16) . IDD14–Iand PIN1 . A plantIN genes . Recentlpecimens . This stArabidopsis R2R3-MYB transcription factor FOUR LIPS , in cooperation with MYB88, targets the promoters of PIN3 and PIN7 genes to complementarily establish gravitropic set-point angles of lateral roots [FLP and PIN3 are targets of auxin response factor 7 (ARF7) and that lateral root development is mediated by PIN3, in cooperation with both ARF7 and FLP [PIN genes  by targeting the auxin response elements (AuxRE) in the promoter regions of these genes to accomplish the patterning processes in both shoots and roots in an auxin-dependent way [PIN1/3/7 were also shown to be the direct targets of another ARF transcriptional factor ARF3/ETTIN [PIN1 and PIN7 via the specific PIN cytokinin response element (PCRE) located on the promoter regions of these two PIN genes [CRF2 itself is the target of MP/ARF5, which also binds to the promoters of CRF2′s targets, PIN1 and PIN7 [PIN3 to regulate young gynoecium growth [The al roots . Further and FLP . Anotherdent way . InteresF3/ETTIN . These rF3/ETTIN . In addiIN genes . Interesand PIN7 ,102. Addm growth . Taken tPIN loci , to facilitate the formation of the root stem cell niche [PIN genes  are targeted by MP/ARF5 [PIN gene expression by BRM. These data indicate that chromatin remodeling of PIN genes plays a crucial role in regulating PIN expression levels.In addition, the SWI/SNF chromatin remodeling ATPase BRAHMA (BRM) acts as the upstream of the PLETHORA (PLT) pathway by directly targeting the chromatin of several ll niche . The recll niche ,105, tog MP/ARF5 . Thus, tGhL1L1, specifically targeted and activated GhPP2AA2 to regulate the activity of GhPIN1 during embryonic development [Arabidopsis [Solanum tuberosum, calcium-dependent protein kinase 1 (CDPK1), which is regulated by miRNA 390 at the post-transcriptional level, phosphorylates StPIN4 to regulate potato development [In recent years, much progress has been made in understanding the activation and regulation of PIN proteins by phosphorylation, as well as the possible mechanisms of phosphorylation-mediated PIN subcellular trafficking ,108,109.elopment . In tomaelopment . Anotherelopment . D6PK anelopment . PIDs anelopment . D6PK haelopment . It locaelopment ,92. Howeelopment . Recentlelopment . CRK5, wbidopsis . In Solaelopment . The MAPelopment . Besideselopment . Recentlelopment .2+ and the Ca2+-dependent proteins TOUCH3 (TCH3), PID-binding protein 1 (PBP1) and the CRK5 pathway; ENP/NPY/MAB4; and peptidyl-prolyl cis/trans isomerases (Pin1At) together with the MKK/MPK cascade [For a long time, it was proposed that the phosphorylation of PIN proteins at phosphosites S1–S3 controls the PIN polarity, and the unphosphorylated PIN proteins localize at the basal PM of root cells in the model of PID/WAG-dependent PIN polarity ,120,121. cascade .trans-Golgi network (TGN) subdomains mediating PIN2 polar sorting to the apical membrane of root epithelial cells in Arabidopsis [The polar distribution of PINs is maintained by continuously, dynamically cycling PIN proteins between the PM and endosomal compartments . ADP-ribbidopsis . Phosphobidopsis . Furtherbidopsis . In addibidopsis . A recenbidopsis . More rebidopsis .Arabidopsis [S-nitrosoglutathione reductase 1–3 (gsnor1–3) mutant, suggesting that NO signaling has negative effects on the internalization of PIN2 [ADP1 inhibited the PIN1 internalization processes [The clathrin-mediated endocytosis (CME) plays a crucial role in the internalization of PIN proteins from the PM . Two PI4bidopsis , suggestbidopsis , and thebidopsis . In addibidopsis ,143,144. of PIN2 . More re of PIN2 . Anotherrocesses . In addirocesses ,148.WRKY gene member, WRKY23 and a phosphatidylinositol transfer PATELLINS (PATL) [Aside from polar recycling and internalization via CME, the regulation of PIN proteins’ turnover in a ubiquitination-dependent manner in the vacuole also contributes to the PIN polarity at the PM . AUXIN RS (PATL) ,159. HowArabidopsis, the eight PIN genes have diverse spatiotemporal dynamic gene expression profiles, differential posttranscriptional regulation manners, as well as invariant protein structures with different subcellular localizations, which result in distinguished functions in development and growth processes mediated by auxin signaling [In ignaling ,162,163.ignaling ,163,164.ignaling ,166,167.ignaling ,168,169.ignaling ,171. Simignaling ,172. PINignaling . PIN5, wignaling . PIN6 haignaling . PIN6 alignaling ,174. PINignaling . PIN8 isignaling ,177.Arabidopsis, some progress addressing the important roles of these auxin efflux carriers in auxin-mediated processes of development and growth has been made in some other plant species. In a plant close to Arabidopsis, Cardamine hirsuta, PIN1 was involved in promoting leaflet initiation [Solanum lycopersicum, SlPIN1 negatively controlled follower abscission by regulating auxin accumulation at the ovary and abscission zone [SlPIN 4 and 5 changes the shoot architecture to have a large angle between the base and the shoot apical meristem, but did not affect fruit development [Nicotiana tabacum, NtPIN4 was induced by auxin and involved in auxin-dependent branching by negatively regulating the growth of axillary bud [Medicago truncatula, PINs have a role in forming the root nodules in an auxin-dependent manner by silencing MtPIN2/3/4, which reduced the number of nodules [GhPIN1a_Dt, GhPIN6_At and GhPIN8_At in Arabidopsis changed the number and size of leaf trichomes [OsPIN2 was expressed in epidermal and cortex cells of roots, and it regulated the gravitropic responses and root system architecture [OsPIN2 led to increased aluminum internalization by enhancing the endocytic vesicular trafficking capacity in root apex [In addition to itiation . In maizitiation . In Solaion zone . The co-elopment . In Nicolary bud . In Medi nodules ,45,179. richomes . In riceitecture . Overexpoot apex .PtPIN9 from Populus tremula × Populus alba promoted lateral root formation [PtoPIN3a led to the observation of a shrunken leaf phenotype, suggesting that this gene is involved in Populus leaf morphogenesis [MdPIN1b were proposed to be responsible for the dwarfing tree architecture phenotype in Malling 9 rootstock [Malus × domestic Royal Gala, heterologous overexpression of MdPIN1 in Arabidopsis led to changes in root architecture with enhanced phototropic and geotropic responses [In woody plants, overexpression of ormation . Meanwhiogenesis . In applootstock . In Maluesponses .WRKY23 and PATL as the regulators of PIN polarity rearrangements from transcriptome datasets has shown the power of this approach [Arabidopsis, the knowledge gained from this could facilitate the discovery of these processes in other plants with new features.To date, much progress has been made in understanding the roles of PIN proteins on developmental and growth processes. The activities of PIN proteins are fine-tuned at multiple layers, including transcriptional regulations, post-transcriptional modifications for subcellular trafficking, recycling, endocytosis and vacuolar trafficking for degradation. The identification of PIN proteins in diverse plant species led us to update our understanding of the evolutionary history of PIN proteins in the plant kingdom. However, the evolutionary history of PIN proteins is still in debate. Extensive work has been done to draw a comprehensive regulatory network of PIN proteins, and posttranslational modifications beyond phosphorylation that regulate PIN proteins and their distribution might be brought under the spotlight soon, since information regarding these aspects has barely been reported so far. Although three possible mechanisms interpreting the different effects of PID/WAGs on the PIN polarity have been proposed, the exact mechanisms still remain unclear. To achieve this goal, genetic methods combined with the computational analysis of large-scale datasets, such as transcriptome, proteomics and metabolome, may contribute to identifying new regulatory layers and the associated candidates. The successful identification of approach ,159. Thu


The genome replication process is challenged at many levels. Replication must proceed through different problematic sites and obstacles, some of which can pause or even reverse the replication fork (RF). In addition, replication of DNA within chromosomes must deal with their topological constraints and spatial organization. One of the most important factors organizing DNA into higher-order structures are Structural Maintenance of Chromosome (SMC) complexes. In prokaryotes, SMC complexes ensure proper chromosomal partitioning during replication. In eukaryotes, cohesin and SMC5/6 complexes assist in replication. Interestingly, the SMC5/6 complexes seem to be involved in replication in many ways. They stabilize stalled RFs, restrain RF regression, participate in the restart of collapsed RFs, and buffer topological constraints during RF progression. In this (mini) review, I present an overview of these replication-related functions of SMC5/6. Replication in any organism is constantly challenged at many levels. A deficit (or inhibition) in the deoxynucleoside triphosphate (dNTP) pool can slow or pause the replication fork (RF), damaged or modified DNA can block RF progression (and must be either repaired first or trans-replicated through the damaged site), some highly transcribed sequences must be replicated in the same direction as transcription (to avoid head-on collisions), the RF must “pause” at certain sites  sequences), or a stalled RF can even collapse and must be restarted. Furthermore, the genome replication involves not only a physical duplication of DNA molecules, but also its spatial organization, which must be tightly coupled with the duplication process. DNA must be specifically packed to fit into the nuclear space, especially when the DNA mass is doubled during its duplication. One of the most important factors organizing DNA into higher-ordered structures are Structural Maintenance of Chromosome (SMC) complexes.In prokaryotes, the SMC/ScpAB complexes (Scp stands for segregation and condensation protein) play key roles in the bacterial chromosome partitioning during replication and the successful segregation of the bacterial nucleoid ,2. The eAll SMC complexes are primarily built of long coiled-coil SMC proteins that use ATP hydrolysis to drive changes in DNA topology. The SMC proteins make dimers via their hinge domains  15,16,1,18,19. ISaccharomyces cerevisiae and Schizosaccharomyces pombe organisms [In addition, all kleisin proteins recruit supplementary protein subunits that are either composed of HEAT repeat motif  proteins, as in the case of cohesin and condensin, or tandem winged helix domain (WHD)  proteins, as in the case of prokaryotic SMC/ScpAB and eukaryotic SMC5/6 complexes  37,38,3,44,45,46rganisms ,51. FuncThe circular shape and the size of the SMC rings allow them to encircle two DNA strands. It was demonstrated that the SMC complexes can entrap DNA in a topological way a 52,53,,53,54. FIn this (mini) review, I first briefly summarize the SMC5/6 data related to the genome integrity, and then I focus on the SMC5/6 role(s) in the normal progression of replication.Thanks to numerous studies, SMC5/6 was implicated in homologous recombination, stabilization of stalled RFs, restarting of collapsed RFs, maintenance of ribosomal DNA (rDNA) and heterochromatin, telomerase-independent telomeres elongation, and the regulation of chromosomal topology . smc6 mutation  was identified in the yeast S. pombe where it conferred hypersensitivity to ultraviolet (UV), infrared (IR), and chemical agents causing DNA damage or affecting replication [smc6 mutations are genetically epistatic with S. pombe rad51/rhp51, indicating a function in homologous recombination (HR) downstream of rad51/rhp51 [S. cerevisiae, scSMC5/6 and cohesin are recruited to double-strand breaks (DSBs) in a RAD50/MRE11-dependent manner in gap 2/mitosis (G2/M) when sister chromatids are present and HR is the major DNA repair pathway (Homo sapiens (hs)SMC5/6 seems to be dependent on RAD18 and SLF1/SLF2 dimer . It 6 dimer; ,74, Arabbidopsis ,76, and bidopsis . SMC5/6 bidopsis . Like cobidopsis . Howeverotes SCR ,43,79.nse2/mms21 SUMO-deficient mutant [S. cerevisiae or their recruitment to ALT-associated promyelocyctic leukemia (PML) bodies in humans (due to the SUMOylation of telomeric proteins) [smc5/6 mutants can be ascribed to aberrant HR, as the segregation defects at rDNA seem to be HR-independent and relocalization does not suppress them; instead, incomplete replication of rDNA causes segregation defects (see below) [Yet, SMC5/6 may also regulate HR negatively—at repetitive sequences—as HR at repetitive sequences must be tightly controlled to avoid loss of genetic information. SMC5/6 is enriched in repetitive regions , centromeres, and telomeres ,83,84,85t mutant . Directlt mutant ,87,88,89t mutant ,91. Noteroteins) ,44,92. Ne below) .rad51 deletion, smc5/6 hypomorphic mutants are synthetically lethal with mutations in genes promoting recovery from stalled replication , suggesting a role for SMC5/6 in recovery of stalled RFs by recombination [S. cerevisiae and its mutants show arrest at the state of X-shaped joint molecules, suggesting a problem with their resolution  improves rDNA replication in cells without SMC5/6. At highly transcribed rDNA, the RNA–DNA hybrids can block RF progression, and Mph1 must be controlled by SMC5/6 to avoid toxic RF regression [However, in y scMph1 ,104,105.y scMph1 ,107. Fury scMph1 ) and thagression ,110. In gression ,106. Whigression .smc6 rrm3∆ double mutant is rescued by individual deletions of Tof1-Csm3 (a fork protection complex that enforces pausing at RPS). This indicates that prolonged pausing and unfinished replication at rDNA, which represents 10% of the yeast genome, could be the major contributor to the observed phenotypes in S. cerevisiae [smc5/6 mutations, causing combined defects in DNA damage tolerance and pausing site replication, lead to recombination-mediated DNA lesions that are, however, invisible to the checkpoints. This results in premature mitosis with interconnected chromatids (that are cut during cytokinesis) and chromosome breakages [Another piece of data also points to natural RPSs, as SMC5/6 is enriched at RPSs  and co-localizes with Rrm3 helicase, which facilitates fork passage through these sites ,112,113.revisiae ,93. Genereakages ,114,115.The SMC5/6 functions described above become indispensable when cells are exposed to genotoxic stresses or when the replication process is challenged at specific sites (such as repetitive rDNA). However, localization and phenotypic data suggest that SMC5/6 may also play a role during unchallenged replication.S. cerevisiae, the scSMC5/6 complex is present on chromosomes at sites of replication initiation ) during the synthesis (S) phase [S. cerevisiae [S. pombe, spSMC5/6 sites also significantly overlap with cohesin distribution, and spSMC5/6 is required for timely removal of cohesin from chromosome arms, suggesting the intimate relationship of these two SMC complexes for chromosomal structure maintenance [In S) phase ,116,117.revisiae . In S. pntenance ,119,120.A change in scSMC5/6 localization from ARS to cohesin-like distribution during cell cycle may indicate that the complex is associated with the fork and follows fork progression (and then it dissociates during mitosis). Furthermore, scSMC5/6 binding along chromosome arms is enhanced in a replication-dependent manner after Top2 inactivation. This increased chromosomal association of scSMC5/6 is independent of recombination, DNA breaks, and replication fork stalling (and no increase in X-shaped structures is observed ), suggesXenopus laevis egg extracts, the chromatin binding of xlSMC5/6 is induced by origin firing and elongation [In higher eukaryotes, SMC5/6 is loaded onto chromatin before or during DNA replication in a manner dependent on the initiation of DNA synthesis, and it dissociates from chromatin during mitosis ,131. Forongation . When thConsistent with the (non-repair) chromatin structure  function, depletion of hsSmc5 and hsSmc6 results in aberrant mitotic (curly) chromosome phenotypes that are accompanied by the abnormal distribution of topoisomerase IIα and condensins, and by chromosome segregation errors ,132. Cursmc6-56 and smc6-56 rad51∆ mutant cells are equally delayed [smc6-56 top2-4 double mutant, there is a threefold increase in missegregation, which may indicate that the SMC5/6 complexes recruited upon Top2 inhibition facilitate resolution of SCIs. Altogether, SMC5/6 is intimately involved in the normal progression of replication, most likely as a specific organizer of chromatin structure.In accordance with the hsSMC5/6 data, inhibition of yeast scSMC5/6 during the S phase causes replication and mitotic delays that are unrelated to aberrant homologous recombination (as  delayed ). In con delayed ,116. In Despite many efforts to discover the “main function” of SMC5/6 and give the complex an appropriate name, we are still “gathering” data on its multiple functions to obtain a clearer picture. In this review, I summarized only those data that enlightened larger parts of the SMC5/6 puzzle: its role in HR, stabilization of stalled RFs, restart of collapsed RFs, restraining of RF regression, and RF progression in a topological context. Interestingly, all these SMC5/6 functions are related to replication in eukaryotes and may have developed in a very complex way from the role of bacterial SMCs in replication. This assumption is strongly supported by our recent work that suggested striking similarity in the architecture of bacterial SMCs and eukaryotic SMC5/6 complexes . Future


Congenital long-QT syndrome (LQTS) is an inherited cardiac disorder characterized by the prolongation of ventricular repolarization, susceptibility to Torsades de Pointes (TdP), and a risk for sudden death. Various types of congenital LQTS exist, all due to specific defects in ion channel-related genes. Interestingly, almost all of the ion channels affected by the various types of LQTS gene mutations are also expressed in the human sinoatrial node (SAN). It is therefore not surprising that LQTS is frequently associated with a change in basal heart rate (HR). However, current data on how the LQTS-associated ion channel defects result in impaired human SAN pacemaker activity are limited. In this mini-review, we provide an overview of known LQTS mutations with effects on HR and the underlying changes in expression and kinetics of ion channels. Sinus bradycardia has been reported in relation to a large number of LQTS mutations. However, the occurrence of both QT prolongation and sinus bradycardia on a family basis is almost completely limited to LQTS types 3 and 4 . Furthermore, a clear causative role of this sinus bradycardia in cardiac events seems reserved to mutations underlying LQT3. Theews, see . For exaews, see . Modulatews, see , and treews, see , 9.KCNQ1 may result in sinus bradycardia  . It is tdycardia , but maydycardia .Because LQTS-related rhythm disorders can be triggered by slow or high HR and sinus pauses , 11, detIn Tables KCNQ1, the gene encoding the pore-forming α-subunit of the IKs channel (KV7.1). A decrease in IKs will result in a prolongation of the ventricular action potential (AP) and a prolongation of the QT interval on the ECG , but at the same time shorten the considerably longer , will inhibit diastolic depolarization and substantially increase cycle length, despite an accompanying decrease in APD, as observed in simulations by Fabbri et al. . Observation of sinus bradycardia in LQT2 patients seems rare . Sinus bradycardia is indeed observed in SQT1 patients, although being less common than in SQT2 patients . Unlike LQT1 and LQT2, the occurrence of sinus bradycardia is not limited to isolated cases. Several families, including the large Dutch family with the 1795insD founder mutation  during diastole  is a prerequisite for QT prolongation, but not for sinus bradycardia. A sole decrease in window current (“loss-of-function”), as for example observed in case of the R376C and D1275N mutations in SCN5A , 32, is ANK2, encoding the widely distributed ankyrin-B adaptor protein. Loss of ankyrin-B results in Ca2+ homeostasis dysfunction by reduced Na+-Ca2+ exchange current (INCX), L-type Ca2+ current , Na+-K+-ATPase, and IP3 receptor expression   , the latCa,L (f) . Despiteed Table . It is cE2 on If .KCNJ2, the gene encoding the Kir2.1 protein, which assembles in tetramers to build the channels that carry the inward rectifier K+ current (IK1) (Andersen-Tawil syndrome (“LQT7”) is a multisystem disorder due to loss-of-function mutations in nt (IK1) . Given tnt (IK1) , it is nnt (IK1) .CACNA1C, encoding the pore-forming α-subunit of the ICa,L channel (CaV1.2), and results in bradycardia in almost all patients known, but caused by 2:1 atrioventricular block rather than sinus bradycardia  is a severe multisystem disorder due to gain-of-function mutations in on Table .CAV3, encoding caveolin-3, an important structural component of caveolae membrane in muscle cells , and which the latter may underlie LQT13, seems not involved in pathogenesis of SAN dysfunction (To the best of our knowledge, no sinus bradycardia has been reported in relation to the rare LQTS types LQT11–LQT13 (see footnote to Table function . Howeverfunction . Thus, tCALM1–CALM3 genes, each encoding the ubiquitous Ca2+ sensing protein calmodulin, in relation to LQTS and sinus bradycardia (Table 2+-related processes in the cardiomyocyte (Ks channel (CALM1 and CALM2 may impair Ca2+-dependent inactivation of ICa,L (Ca,L in case of LQT8 (Table Several reports exist of mutations in the ia Table . Calmoduomyocyte , includi channel . Mutatioof ICa,L , 51, funT8 Table .CALM1–CALM3. We are, however, well aware that the naming LQT16 has been used in other review articles (SCN1B (TRDN (TRDN, encoding triadin, associated with LQTS, and themselves proposed that “triadin knockout syndrome” or “TRDN-mediated autosomal-recessive LQTS” should be used rather than “LQT17,” because of the atypical phenotype that was observed (In Table articles , 55 in rs (SCN1B  and in T1B (TRDN . Altmannobserved .2+ clock” mechanisms . Whi2+ sensing protein like calmodulin, but also for mutations in the α-subunit of a specific ion channel. The LQT1-related T587M mutation in KCNQ1 for example does not only reduce IKs, but also fails to increase membrane localization of the KCNH2-encoded KV11.1 protein, as opposed to wild-type KCNQ1, thus also reducing IKr (It is important to realize that a mutation in a single LQTS-related gene may affect several ion currents. This does not only hold for mutations in a Cacing IKr . Furthercing IKr , Yeh et cing IKr , and D'Scing IKr .LQT2 and LQT3, but not LQT1, patients have a more pronounced risk for arrhythmias at slower HR . LQT2 anAs shown in Tables We conclude that, although sinus bradycardia has been reported in relation to a large number of LQTS mutations, a causative role of this sinus bradycardia in cardiac events is limited to mutations underlying LQTS type 3.RW and AV: experimental design, data acquisition, analysis and interpretation of data, drafting manuscript, editing manuscript, and approval.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.


Silencing CD47 significantly suppressed cell viability and orosphere formation, accompanied by a downregulated expression of CD133, SRY-Box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and c-Myc. In addition, CD47-silenced OSCC cells showed reduced EMT, migration, and clonogenicity reflected by increased E-cadherin and decreased vimentin, Slug, Snail, and N-cadherin expression. Conclusion: Of therapeutic relevance, CD47 knockdown enhanced the anti-OSCC effect of radiotherapy. Collectively, we showed an increased CD47 expression promoted the generation of CSCs and malignant OSCC phenotypes. Silencing CD47, in combination with radiation, could provide an alternative and improved therapeutic efficacy for OSCC patients. Background: Oral squamous cell carcinoma (OSCC), with high mortality rates, is one of the most diagnosed head and neck cancers. Epithelial-to-mesenchymal transition (EMT) and the generation of cancer stem cells (CSCs) are two keys for therapy-resistance, relapse, and distant metastasis. Accumulating evidence indicates that aberrantly expressed cluster of differentiation (CD)47 is associated with cell-death evasion and metastasis; however, the role of CD47 in the generation of CSCs in OSCC is not clear. Methods: We investigated the functional roles of CD47 in OSCC cell lines SAS, TW2.6, HSC-3, and FaDu using the bioinformatics approach, immunoblotting, immunofluorescence staining, and assays for cellular migration, invasion, colony, and orosphere formation, as well as radiosensitivity. Results: We demonstrated increased expression of CD47 in OSCC patients was associated with an estimated poorly survival disadvantage ( Oral cancer is one of the most diagnosed malignancies, with a global annual incidence of 300,373 cases . RegardlCurrently, the treatment of choice remains surgery, radiation therapy, and/or chemotherapy, which consists of cisplatin (CDDP), docetaxel (DTX), and 5-fluorouracil (5-FU), especially for advanced-stage (III and IV) malignancies; however, this is often associated with increased risk of severe drug-induced adverse effects and toxicities, high rates of treatment failure and disease recurrence, as well as low median survival rates for OSCC patients , with anThe last two decades have been characterized by accumulating evidence, implicating cancer stem cells (CSCs) in the initiation and sustenance of tumor, resistance to chemo- and/or radiation therapy, disease recurrence, and metastases ,7.Previously, we demonstrated that the direct targeting of CSCs or so-called tumor-initiating cells (TICs) in OSCC by gene loss of function  or theraThe ubiquitously-expressed transmembrane glycoprotein CD47, also known as integrin-associated signal transducer or protein (IAST or IAP), belongs to the immunoglobulin (Ig) superfamily and is an antigenic surface determinant protein that provides a “self” or “do not eat” signal by interacting with the N-terminus of its ligand, signal regulatory protein alpha (SIRPα) on immune cells to suppress or inhibit macrophage phagocytosis . In factIn this study, we investigated and provided evidence for the feasibility of a CD47-mediated attenuation of OSCC cell viability, suppression of OSCC-SCs-like attributes and associated pluripotency, deregulation of the constitutive CSCs-EMT loop in OSCC, and enhancement of OSCC cell sensitivity to radiation therapy.We evaluated the prognostic relevance of CD47 expression in a cohort of OSCC patients (n = 71) aged between 29 and 72, with a median age of 48 years, who had undergone definitive treatment with curative intent in National Defense Medical Center from October 2000 to March 2013. The present study was reviewed and approved by the institutional review board (TSGHIRB 2–102-05–125), and all participants provided written informed consent. Patient distribution based on American Joint Committee on Cancer (AJCC) staging, 7th edition, was as follows - 9 stage I disease (13%), 22 stage II (31%), 13 stage III (18%), and 27 stage IV (38%), as well as normal non-tumor (2), mild dysplasia (1), moderate dysplasia (1), and severe dysplasia (3) from peritumoral tissue. Location-wise, buccal mucosa  was the most common affected site, followed by tongue , gingival , and others  . None of the specimens had received radiation or a chemotherapeutic regimen. The clinicopathological characteristic of our cohort is shown in For the CD47 IHC staining analysis of OSCC (n = 71) and non-tumor oral tissues (n = 7), 1% bovine serum albumin (BSA) was used for blocking the formalin-fixed paraffin-embedded (FFPE) tissue sections before they were incubated with CD47 antibody  at 4 °C overnight. The sections were then incubated with goat anti-mouse IgG  for 1 h. Tissue staining was scored by two independent pathologists. The staining index (SI) was calculated based on the formula.Gibco® RPMI 1640, trypsin/EDTA, dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), sulforhodamine B (SRB) medium, acetic acid, and TRIS base were also purchased from Sigma Aldrich Co. .The human OSCC cell lines HSC-3 and FaDu were obtained from the American Type Culture Collection , while the SAS and TW2.6 cells were kindly provided by Prof. Hsiao Michael . Both cell lines were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin  and incubated at 37 °C in 5% humidified CO2 incubator. The OSCC cells were passaged at 98% confluence, and the medium was changed every 72 h before exposure to 5 Gy–15 Gy of radiotherapy. 4 SAS or TW2.6 cells plated in 24-well plates were primed with 8 μg/mL hexadimethrine bromide, and viral particles were added to the culture medium at a multiplicity of infection (MOI) of 6. After transfection for 12 h, the virus-containing medium was replaced with fresh culture medium. The selection of viable OSCC cells stably transfected with shCD47 was performed with 2 μg/mL puromycin the next day, and the surviving colonies were expanded for further experiments.The SAS, TW2.6, HSC-3, or FaDu cells were then transfected with shRNA specifically targeting CD47 or control/scramble shRNA purchased from Shanghai GenePharma Co., Ltd . The shRNA sequences were as follows: shCD47-1 - 5’-CGTCACAGGCAGGACCCACTGCCCA-3’; shCD47-2 - 5’-CCACAGATG TACAAGGGATGACCACAGTGTCATT-3’; and scramble shCD47 - 5’-CGTGACAGCCACGACCGACTGCGCA-3’. The OSCC cells were transfected with packed and harvested shRNA-coding lentiviruses following the manufacturer’s instruction. Briefly, 5 × 104 cells/well in 6-well non-adherent plates  in serum-free DMEM/F12 medium  supplemented with basic fibroblast growth factor (bFGF) , B27 supplement , 5 μg/mL insulin ), and epidermal growth factor (EGF) . The cells were then incubated at 37 °C in a 5% humidified CO2 incubator for 12–15 days, and the formed orospheres were observed and counted using an inverted phase-contrast microscope. After 12 days of culture, primary orospheres consisting of ≥50 μm were counted, and images were taken under the microscope. Secondary orospheres were generated by dissociating primary orospheres using the trypsinization method, and the dissociated primary orospheres were then pipetted through a 22G needle to obtain a single-cell suspension . After dissociation of the primary orospheres, cell seeding was done as for primary tumorspheres. After 12–15 days of culture, secondary orospheres consisting of ≥50 µm were counted, and the images were taken under the microscope.For generation of orospheres, the OSCC cells were seeded at 5 × 104 cells per well in 6-well plates and cultured for 12 days. After the colonies formed attained the cluster size of ≥50 cells, they were washed with PBS two times, fixed with 95% methanol for 15 min, and then stained with crystal violet at room temperature for 15 min. This was followed by colony visualization and counting under the microscope. The size and number of colonies formed were estimated with the ChemiDoc-XRS imager from the QuantityOne software package .WT or shCD47 SAS, TW2.6, HSC-3, or FaDu cells were plated in triplicate at 2 × 104 WT or shCD47 SAS, TW2.6, HSC-3, or FaDu cells were seeded and cultivated per plate in triplicate in 96-well plates. After 24 h, both adherent and floating cells were harvested, baseline cell-count was carried out, then triplicate wells were irradiated with 5 Gy–15 Gy or left without irradiation for another 24 h. The irradiated or unexposed OSCC cells were then harvested, fixed with 10% trichloroacetic acid (TCA), washed with ddH2O, and stained with 0.4% SRB (w/v) in 1% acetic acid. The unbound SRB dye was carefully washed off with 1% acetic acid several times, and then plated air-dried. The bound SRB dye was solubilized in 10 mM Trizma base, and absorbance was read using a microplate reader at 570 nm wavelength. Variation in cell numbers was relative to the baseline number of viable cells before irradiation. 3 × 10https://imagej.nih.gov/ij/). Target protein expression was normalized to that of GAPDH, and assays were repeated four times in triplicates.For western blotting, cells were lysed using RIPA buffer  with a cocktail of protease inhibitors . Ten micrograms of protein samples were separated using 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using the Bio-Rad Mini-Protein electro-transfer system . Then, non-specific binding was blocked by incubating the membranes in 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature, and then the membranes were incubated overnight at 4 °C with the antibodies against CD47 , OCT4 , SOX2 , CD133 , vimentin , Slug , Snail , N-cadherin , E-cadherin , and GAPDH , listed in For the wound-healing migration assay, OSCC cells were seeded in 6-well plates and cultured to 100% confluence. At full confluence, a 1 mm wound was made along the median axis of the well using a yellow pipette tip. Cell migration into the wound area was then observed at 0, 3, 6, and 12 h time-points under the microscope. The assay was performed three times in triplicates.4 irradiated or un-irradiated WT, shCD47-1, or shCD47-2 OSCC cells in serum-free culture medium were plated in the upper chambers, while the lower chambers contained complete culture medium with 10% FBS as a chemoattractant. After overnight incubation, the un-invaded OSCC cells on the upper side of the filter were carefully removed with a cotton bud, while the invaded cells on the lower side of the membrane were washed, fixed in 95% ethanol and then stained with 10% Giemsa dye. The number of invaded cells was counted in 4 randomly selected fields of each membrane and averaged to obtain a representative number of the invaded cells. For the Boyden chamber migration assay, the porous membranes without the Matrigel were used.Cell invasion assays were performed in Boyden chambers (pore size = 8 μm) with the upper side of the filter covered with 0.2% Matrigel diluted in DMEM. The 1 × 10https://imagej.nih.gov/ij/).For the immunofluorescence analysis, the OSCC cells were plated in 6-well chamber slides  for 24 h, fixed in 2% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 in 0.01 M PBS (pH 7.4) containing 0.2% BSA, air-dried, and rehydrated in PBS. The cells were then incubated with antibody against CD47, Oct4, Sox2, c-Myc, vimentin, or E-cadherin diluted 1:200 in PBS containing 3% normal goat serum at room temperature for 2 h, followed by incubation with anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated secondary antibody . The cells were allowed to rest at room temperature for 1 h, washed in PBS, and mounted using Vectashield mounting medium while counterstaining with 4′,6-diamidino-2-phenylindole . Images were captured using a Zeiss Axiophot  fluorescence microscope, and the microphotographs were analyzed using ImageJ software v. 1.46  was used for comparison between 3 or more groups. The association between the differential expression of CD47 and overall survival (OS) in patients with OSCC was determined using univariate Cox proportional regression of covariates, including the age, gender, AJCC stage, pathological grade, local recurrence, and lymph node involvement. Variables for which p < 0.05 were identified as significantly associated with prognosis, and Cox multivariate analysis was subsequently performed for these variables. Hazard ratios (HRs) and 95% confidence intervals (CIs) for multivariate analyses were computed using the Cox proportional hazards regression. p-value <0.05 was considered statistically significant.Each experiment was performed at least 3 times in triplicates. All statistical analyses were carried out using IBM SPSS Statistics for Windows, Version 25.0 . All data represent means ± standard deviation (SD). Comparison between two groups was estimated using the 2-sided Student’s https://xena.ucsc.edu/) was used to further characterize CD47 expression in patients with OSCC; our results indicated that CD47 was overexpressed in the keratinizing, non-keratinizing, and ‘not otherwise specified’ (NOS) squamous cell carcinoma of the oral cavity, which constituted over 75% of HNSCC (p = 0.0009) showed that compared to expression in the ‘normal’ oral epithelium (n = 32), CD47 was significantly more expressed in the OSCC tissue samples (n = 380) , including head and neck squamous cell carcinoma (HNSCC) matched with corresponding normal samples from the cancer genome atlas (TCGA) and genotype-tissue expression (GTEx) (n = 8587 non-tumors) datasets, using the analysis of variance for the evaluation of differential expression. Results of these analyses indicated that CD47 was overexpressed in tumors compared to adjacent non-tumor oral tissues and was mostly expressed in HNSCC tissues with ~ 125 transcripts per million (TPM) after the ovarian  and lung adenocarcinoma  . Furtherof HNSCC B. Furthen = 380) C.p = 0.0391; We also demonstrated using downloaded and reanalyzed malignant OSCC data from the TCGA HNSCC cohort that high CD47 expression conferred a significant survival disadvantage in OSCC patients with high CD47 expression, compared to those with low CD47 expression , and strong CD47 staining in the late stage (III and IV) group , especially in the cytomembranous region (p = 0.001), presence of local recurrence , and late American Joint Committee on Cancer (AJCC) stage , high CD47 expression was strongly associated with worse survival ((HR (95%CI) = 6.83 (1.72 – 18.09), p = 0.01)) and multivariate analyses (cum higher risk of disease-specific death ((multivariate: HR(95%CI) = 5.18 (0.73 – 12.64), p = 0.019)), akin to local recurrence  and AJCC stage . Together these data did indicate the active role of CD47 in OSCC carcinogenesis and poor prognosis.Furthermore, consistent with earlier data, compared to the normal or dysplastic tissues, results of our immunohistochemical staining showed varying degrees of positive CD47 staining in all 71 OSCC cases; of which, 87.5% were membranous, 10.9% cytoplasmic, and 1.6% perinuclear staining. A strong positive correlation between enhanced CD47 protein expression and disease progression or tumor stage was established A. Interes region A–C. Thess region , which danalyses , indicatp = 0.00003), SOX2 , and CD133  in the OSCC tissues, compared to normal tissue group  or TW2.6  primary orospheres  and shCD47-2  SAS cells , we carried out a bivariate analysis of the transcript expression profiles of CD47, vimentin (VIM), or E-cadherin (CDH1) in the Roepman OSCC cohort (n = 220). Results of our analyses further validated earlier demonstrated direct and inverse correlation of CD47 with VIM and CDH1, respectively, with the significance of correlation r-value = 0.081 p-value = 0.23 T-value = 1.198 for CD47 versus VIM, and r-value = −0.061 p-value = 0.37 T-value = −0.898 for CD47 versus CDH1, with both sharing same degrees of freedom = 218  staining showed that compared to expression in the negative control shCD47 scramble cells, the SAS orospheres (SAS Sp) cells harbored enhanced expression of vimentin A but decom = 218 C,D. In aom = 218 E. These shCD47 and irradiation induced significantly greater cell-death in comparison to the radiation only group, as evidenced by ~52% vs. 46%, 24% vs. 19%, and 28% vs. 20% reduction in the viability of shCD47-1 vs. shCD47-2 transfected SAS cells treated with 5 Gy, 10 Gy, and 15 Gy, respectively (p < 0.001), un-irradiated shCD47 alone , and 5 Gy radiation alone SAS WT  (p < 0.01), 3-fold (p < 0.01), or 12-fold (p < 0.001) reduction in the number of invaded un-irradiated shCD47-transfected, 5 Gy radiation alone, or 5 Gy irradiated shCD47-transfected cells, respectively, was observed (p < 0.001) in comparison to un-irradiated SAS WT  C. Similaobserved . Consistobserved . Furtherd SAS WT . Our datDespite advances made in the diagnostic approach and therapeutic strategies, OSCC remains a clinical challenge with great financial and emotional implications for patients and caregivers alike. The failure of conventional anticancer therapy in OSCC is increasingly being associated with the presence and activities of CSCs, where these OSCC-SCs are constitutively resistant to chemotherapeutic agents and radiation therapy. The last two decades have been characterized by heightened interest in and increased exploration of the role of CSCs in the resistance or reduced sensitivity of OSCC cells to standard chemotherapeutics or radiation therapy, with most research focused on the discovery of new therapeutic targets and/or development of novel anticancer drugs that preferentially target CSCs-enrichment in oral cancer cells ,7,8,9,21In this present study, we hypothesized and provided evidence that the ”don’t eat me” signal—CD47—modulated radiosensitivity by CSCs regulation and EMT deactivation in OSCC cells. We demonstrated that CD47 was aberrantly expressed in human OSCC and influenced the survival rate of patients with OSCC ; Table 2In search of OSCC-relevant actionable molecular targets, our work for the first time, to the best of our knowledge, demonstrated that CD47 modulated the CSCs-like phenotype in OSCC, as evidenced by concomitant upregulation of known pluripotency transcription factors and stemness markers with CD47 expression, and co-suppression of SOX2, OCT4, c-MYC, and CD133 protein expression following shRNA-mediated downregulation of CD47 expression in wild-type adherent and anchorage-independent orospheres . As alreBy inference, extrinsic perturbations, such as CD47 blockage/knockdown and cytotoxic treatments, including radiation therapy, may mold oral tumor by selective targeting of the aggressive OSCC cells, including OSCC-SCs, which subsequently facilitate malignant growth; thus, any efficacious therapy must eradicate OSCC-SCs; however, there is mounting evidence, showing these cells are intrinsically less or in-sensitive to current OSCC anticancer therapy. Thus, interestingly, having shown the existence of a positive correlation of CD47 expression with CSC, EMT, and metastatic phenotypes, as well as an inverse correlation with OSCC patients’ overall survival, cell death; we finally demonstrated that the suppression of CD47 expression enhanced the sensitivity of OSCC-SCs to radiation therapy, as evidenced by the remarkable synergistic effect of concurrent CD47 knockdown and radiotherapy on cell viability, migration, invasiveness, clonogenic, and orospheric survival . This haHuman papillomavirus (HPV) infection has been implicated in over 25% of HNSCCs. In fact, many of the HPV positive HNSCCs are oropharynx cancer, including tongue base and tonsil ,36. ClinOur initial analysis of the TCGA-HNSCC cohort (n = 604) gene expression profile dataset showed it contained limited number of the HPV positive HNSCC as sorted by FISH or p16 testing; most of the tumor sites were located at the tongue base and oropharynx, which were excluded in our original TCGA analysis since our study focus was oral cancer instead. In addition, the expression level of CD47 in these samples was low or null. These findings suggested that the therapeutic potential of targeting CD47 was likely independent of the HPV status of the patients with OSCC. The mechanism underlying the response of CD47 to radiation in OSCC remained relatively underexplored, thus, necessitating a further investigation of the use of CD47 as a potential therapeutic target in anti-OSCC treatment.As with most studies of this nature, this present study had several limitations within which the findings presented herein should be interpreted carefully. This study was a retrospective, single-center investigation of the selected biomarker in a relatively small cohort of Taiwanese OSCC patients (n = 71), limiting the ‘generalizability’ of the findings reported, especially to other ethno-geographics and raises the question of probable predisposition to selection biases.In conclusion, as depicted in


TSPO forms a complex with the voltage-dependent anion channel (VDAC), a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, controls metabolism and apoptosis and interacts with many proteins. This review focuses on the two OMM proteins TSPO and VDAC1, addressing their structural interaction and associated functions. TSPO appears to be involved in the generation of reactive oxygen species, proposed to represent the link between TSPO activation and VDAC, thus playing a role in apoptotic cell death. In addition, expression of the two proteins in healthy brains and diseased states is considered, as is the relationship between TSPO and VDAC1 expression. Both proteins are over-expressed in in brains from Alzheimer’s disease patients. Finally, TSPO expression levels were proposed as a biomarker of some neuropathological settings, while TSPO-interacting ligands have been considered as a potential basis for drug development. The translocator protein (TSPO), located at the outer mitochondrial membrane (OMM), serves multiple functions and contributes to numerous processes, including cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca Rhodobacter sphaeroides, Q9RFC8) and eukaryotes , where it is predominantly located at the outer mitochondrial membrane (OMM), Recently, the 3D structure of TSPO in complex with (R)-PK11195 was obtained, revealing a complex structure characterized by the tight packing of its five α-helical transmembrane domains [The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR) or the isoquinoline-binding protein, is an evolutionarily-conserved protein complex that binds benzodiazepines, such as RO5-4864, and isoquinoline carboxamide derivatives, such as PK11195 ,3. TSPO 2+ signaling, oxidative stress regulation, porphyrin transport, and heme synthesis [TSPO is involved in a wide variety of functions. These include cholesterol import and regulation of the mitochondrial membrane potential, as well as mitochondrial metabolism, apoptosis, cell proliferation, immunomodulation, inflammation, Caynthesis ,10,11,12Tspo knockout mice, the absence of TSPO resulted in altered mitochondrial energy metabolism, together with lower oxygen consumption, membrane potential, and ATP levels [2+ signaling, resulting in increased levels of cytosolic Ca2+, leading to activation of the Ca2+-dependent NADPH oxidase (NOX), thereby increasing ROS levels [The influence of TSPO on gene expression and functional implications has been demonstrated by TSPO knockdown and by TSPO ligands, involving TSPO/VDAC interactions ,14,15. TP levels . TSPO alP levels . In addiP levels . It has S levels . FinallyS levels . It has been proposed that TSPO forms a complex with the OMM protein voltage-dependent anion channel (VDAC) and the inner mitochondrial membrane (IMM) protein adenine nucleotide transporter (ANT) . Of the 2+ signaling by transporting Ca2+. VDAC1 is a Ca2+ channel that also transports Mg2+, Zn2+ and other ions. Once transported across the OMM via VDAC1, Ca2+ is transported across the IMM into the matrix by a Ca2+-selective transporter, the mitochondrial Ca2+ uniporter (MCU), which is regulated by a calcium-sensing accessory subunit (MCU1). In the matrix, Ca2+ regulates energy production via activation of pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (α KGDH), leading to enhanced activity of the citric acid cycle; (c) participation in mitochondrial-mediated apoptosis. Via homo-oligomerization to form a protein-conducting channel, VDAC1 allows cytochrome c (Cyto c) release and apoptotic cell death; (d) involvement, both structurally and functionally, in mitochondrial association with the ER, mediating Ca2+ transport from the ER to mitochondria; (e) involvement in lipid metabolism, mediating the transfer of fatty acid acyl-CoA across the OMM into the intermembrane space (IMS), where it is converted into acylcarnitine by CPT1a for further processing by β-oxidation; and (f) involvement in cholesterol transport as a constituent of a multi-protein complex, the transduceosome, comprising Star, TSPO, and VDAC1. Located in the OMM, VDAC1 serves as a mitochondrial gatekeeper, controlling a wide variety of mitochondrial functions  23,24,2,224,25. Finally, VDAC1 is considered to be a hub protein, interacting with over 200 proteins  that regMany high-resolution 3D structures of integral membrane proteins have proven to be fundamental for better understanding of many biological processes . Neverthwww.rcsb.org). Eleven are crystal structures and two are solution NMR structures. These structures were obtained from three different organisms, namely Rhodobacter sphaeroides , BacillID: 4RYJ ), and MuID: 2N02  and 2MGYUC3 , BacID: 2N02 ,32,33,34147ATVLNYYVWRDNS159 at the carboxylic terminus of TM5 [Cholesterol has been shown to bind with nanomolar affinity to recombinant TSPO . This ins of TM5 ,37. The s of TM5 ,37 points of TM5 ,37. The s of TM5 .Rhodobacter sphaeroides (Rs) was the first structure to present an oligomeric structure for the protein [TSPO purified from  protein . The RsT protein ,39. Crys protein  was dete protein . All thr protein  and how  protein . TSPO al protein  (W/YxxL/http://lichtargelab.org/software/ETserver) analysis of mouse TSPO (PDB 2N02) and RsTSPO (PDB 4UC1) assigned high relevance to approximately 10% of the homologous residues. These amino acids, found in both the prokaryotic and eukaryotic structures, are associated either with the cholesterol binding domain, as compared to region identified in the TSPO-PK11195 structure [Homology between eukaryotic and prokaryotic TSPO (~33%) is insufficient to extrapolate findings regarding oligomeric status suggested by the RsTSPO structure . NeverthTSPO PDB UC1 assigtructure . Thus, eThe oligomeric state of TSPO in the OMM remains a matter of controversy. Blue native gels and electron microscopy  identifiVDAC1 is a channel protein comprising 19 transmembrane β-strands that form a β-barrel structure. A 26 residue-long N-terminal region usually lies inside the pore Ba, yet cVDAC1 is able to oligomerize, forming dimers, hexamers, and higher-order moieties Bb 51,52,59,60,61https://string-db.org/cgi/input.pl). The STRING database indicates that both RsTSPO and mammalian TSPOs interact with proteins related to ROS elimination, gene regulation and metabolism. RsTSPO binds proteins related to gene regulation, nutrients and ROS metabolism [Another piece of evidence suggesting a conserved function for TSPO between prokaryotes and eukaryotes is the annotated database of protein-protein interactions  contains three GxxxG domains in its TM1, TM2, and TM3 transmembrane domains, 18-GCFVG, 50-GPVWG, 83-GLYTG. Additionally, the C-terminal end of TSPO also contains 160-GWRGG, although very little information was obtained from the NMR structure (PDB ID: 2MGY), where it appears disordered [The GxxxG motif has been implicated in alpha helical structures of membrane protein dimer formation . The N-tsordered   [2+, acidification, and ROS involving TSPO regulate TSPO-VDAC1 interaction, the VDAC1 oligomeric state, autophagy and apoptosis [Another proposed link between TSPO activity and VDAC1 is related to the regulation of cytosolic Ca A (PKA) . Such TS A (PKA) . Thus, epoptosis ,71,88  opening. The PTP represents a high-conductance, non-specific pore activated by ROS, Ca cytosol . Initial cytosol ,91,92. T cytosol . However cytosol ,95 or VD cytosol  still sh cytosol . Yet, se cytosol , DIDS  also down-regulated TSPO expression . FollowiIt has also been shown that in peripheral mononuclear cells (PBMCs) of bipolar disorder (BD) patients, both TSPO and VDAC mRNA and protein expression levels were highly increased, relative to their levels in healthy controls . MoreoveTSPO and VDAC1 were also implicated in NLRP3 inflammasome formation . The TSPTaken together, these results indicate a relationship between TSPO and VDAC1 expression levels and several mitochondria-associated activities, such as ROS and ATP production, signaling and pathological conditions. The molecular mechanisms by which the interaction of TSPO and VDAC modulates these activities is not well-defined. While TSPO is expressed in many organs, the highest levels are seen in tissues containing steroid-synthesizing cells, such as adrenal glands, gonads and placenta ,111. TSPTSPO was found to be over-expressed in a variety of human diseases . TSPO isImmunohistochemical staining of the brain from wild-type and an AD-like mouse model, APP/PS1dE9 transgenic mice, with anti-Aβ, anti-TSPO, and anti-Iba-1 antibodies (marker for microglia/macrophage), showed that plaque-associated Iba-1 was observed in cortex, hippocampus, cerebellum striatum, and thalamus, while plaque-associated TSPO was seen at all sites other than the thalamus .Immunofluorescent staining of brain cortex sections for TSPO and VDAC1 they show punctuated and co-localized staining, as expected for two mitochondrial proteins A. ImmunoAlongside the classical pathological hallmarks of AD, such as misfolded and aggregated proteins, neuro-inflammation is thought to be a major driver in progression of the disease ,131. RecThe mechanism(s) of regulation of altered TSPO expression under various conditions is not clear. The broadly expressed transcription factors (TFs) acting on the TSPO promoter, such as specificity protein 1/specificity protein 3 (Sp1/Sp3), the v-ets erythroblastosis virus E26 oncogene homologue (Ets), and activator protein 1 (AP1), were proposed to regulate TSPO expression ,137. ForAs TSPO is a multi-functional protein, expressed by different tissues and implicated in many pathological conditions ,118,123,In addition, due to the proposed function of TSPO in apoptosis, potentially via its interactions with VDAC1, interfering with the TSPO-VDAC1 interaction can offer a target for the development of drugs directed at neurodegenerative diseases ,143. In summary, these two outer mitochondrial membrane proteins, TSPO and VDAC1, are associated by direct interaction, and demonstrate functional and regulatory cross-talk. The link between the activation of TSPO and VDAC is proposed to play a role in cell proliferation and apoptotic cell death. In addition, there is a close relationship between TSPO and VDAC1 expression in healthy and disease states. Finally, as suggested for VDAC1 , TSPO ex


A large part of these alterations is due to dysfunctional Ca2+-storing organelles, including the endoplasmic reticulum (ER) and mitochondria. Very recently, lysosomal Ca2+ dysfunction has emerged as an important pathological change leading to neuronal loss in ALS. Remarkably, the Ca2+-storing organelles are interacting with each other at specialized domains controlling mitochondrial dynamics, ER/lysosomal function, and autophagy. This occurs as a result of interaction between specific ionic channels and Ca2+-dependent proteins located in each structure. Therefore, the dysregulation of these ionic mechanisms could be considered as a key element in the neurodegenerative process. This review will focus on the possible role of lysosomal Ca2+ dysfunction in the pathogenesis of several neurodegenerative diseases, including ALS and shed light on the possibility that specific lysosomal Ca2+ channels might represent new promising targets for preventing or at least delaying neurodegeneration in ALS.Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alteration in calcium (Ca Accordingly, increased activity of P/Q-type voltage-gated Ca2+ channels i) are used as specific signals regulating various biological outputs such as metabolic activity, cell growth and differentiation, transmitter release, and long-term modification of synaptic efficiency. On the other hand, alteration in [Ca2+]i is even implicated in neuronal damaging occurring in the neurodegenerative process.Furthermore, transient increases in the intracellular Ca2+ signals are initiated by the occurrence of action potentials opening voltage-dependent Ca2+ channels on plasma membrane at the level of specialized domains. Moreover, [Ca2+]i increase also depends on release from intracellular Ca2+-storing organelles through specific ion channels, pumps, and exchangers. A further level of complexity is due to an integrated cellular network among these intracellular Ca2+-storing organelles. Interestingly, they interact with each other on various cellular tasks through specialized membrane contact sites rather than acting as isolated entities [2+ signals within the cell [In excitable cells such as neurons and cardiomyocytes, these Caentities ,17. In tentities . It has the cell . For insthe cell . Indeed,the cell ,22,23.2+-storing compartments that participate in the regulation of global intracellular Ca2+ homeostasis under both physiological and pathological conditions [2+ signals requiring the intervention of different intracellular receptors [2+ concentration within their lumen is approximately 500 μM [2+ concentration reached in the ER [Lysosomes, tiny acidic organelles traditionally studied for their catabolic activity , have renditions ,25. Accoeceptors . Indeed,y 500 μM , which in the ER .2+ could be released by intracellular cues, including nicotinic acid adenine dinucleotide phosphate (NAADP) [2, mainly involved in membrane trafficking [2+ release is crucial for lysosome function, but may be also involved in global Ca2+ signaling by interacting with ER Ca2+ signaling [2+ ions. Very recently, the ER Ca2+-sensor STIM1  has been identified as a fine regulation mechanism of lysosome/ER coupling [Lysosomal Ca (NAADP)  and lysofficking . This loignaling ,18,30,312+-release channels are localized to the lysosomal membrane (see 2+ channels (VGCCs); and the non-selective cation channel belonging to the ankyrin subfamily of TRPs, TRPA1. Different Carane see , includi2+-releasing channel used as lysosomal receptor by PIP2. Interestingly PIP2 levels are significantly impaired in some ALS forms [Unfortunately, for some of these proteins, direct measurement of their activity is still lacking. The most studied among them is the transient receptor potential mucolipin subfamily 1 (TRPML1) that belongs to the mucolipin subgroup of TRP ion channel family . MoreoveLS forms ,34. 2+ and participates in the regulation of several Ca2+-dependent functions within the cell [2+ release may fulfill lysosomal exocytosis [2+ release from lysosomes [2+ permeability of TPC2 has been questioned by recent studies, thus considering TPC2 as Na+-selective channel with a low Ca2+ permeability [+ selectivity in TPC2 structure [Loss-of-function mutations in TRPML1 cause mucolipidosis type IV (MLIV), a devastating lysosomal storage disorder characterized by neurodegeneration . TRPML1 the cell . Therefoocytosis ,39, membocytosis , autophaocytosis , nutrienocytosis , oxidatiocytosis , lysosomocytosis . Anotherocytosis  and is pysosomes ,46,47,48ysosomes . Howevereability ,50. In ttructure .2+ must be refilled to ensure all lysosomal functions. Recently Atakpa et al. [2+ concentration presented to lysosomes by the ER IP3  receptor (IP3R) may facilitate lysosomal Ca2+ uptake. However, how lysosomal Ca2+ is taken up after its release remains to be answered. Two different refilling mechanisms have been proposed: (a) a putative Ca2+–H+ exchanger transporting Ca2+ ions into lysosomal lumen in an H+-dependent manner [2+ transporter at the level of ER-lysosome membrane contact sites activated by ER Ca2+ release [2+ within the ER is essential for nascent protein folding and cell survival [2+/Akt/ERK1/2 neuroprotective pathway in a model of ALS/Parkinson-dementia complex (ALS/PDC) [Once released, Caa et al.  proposedt manner   .2+-storing properties, other functions are controlled by ER in motor neurons. During neuronal circuit development, the spatial localization of ER controls the growth cone cytoskeleton to direct motility of motor neurons [2+ ATPase) inhibitor thapsigargin, by depleting the ER Ca2+ store, influences lysosomal Ca2+ content [2+ homeostasis in one of these organelles has a dramatic repercussion on the other store [2+ refilling in other cellular systems [Besides the Ca neurons . This is neurons , thus su content , thus hier store . In fact systems .2+ release [2+ release [Many proteins control organellar interplay and are controlled in turn by store functions. For instance, in addition to mitochondria, the calcium-sensitive GTPase dynamin is also associated with lysosomal membrane , where i release . In part release . It has  release , mitocho release  and lyso release .2+ ions between these organelles is essential for lysosomal function and global cellular homeostasis. Interestingly, the functional ER/lysosome interplay is not pH-sensitive, since the v-ATPase inhibitors bafilomycin-A or concanamycin-A did not abolish the refilling, but it depends on IP3R activity [2+ ions and controlling lysosomal-related functions.Since lysosome size is dynamically regulated by lysosomal membrane fusion and fission , the pasactivity ,23,64. OAutophagy is an evolutionarily conserved cellular process through which cytoplasmic materials such as proteins or even complete organelles are directed to lysosomes for degradation. This process allows the clearance of protein aggregates, long-lived proteins, misfolded proteins, and dysfunctional and damaged organelles, and ensures the recycling of their constituents ,66. In m2+-dependent manner, forming a fusion-hybrid organelle called autolysosome. Within the autolysosome, lysosomal enzymes degrade cargoes and the resulting macromolecules are transported back to cytosol for recycling [In mammalian cells, three major subtypes of autophagy have been described, based on the mechanism by which substrates reach lysosomal lumen: Chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy. Only soluble proteins, but not complete organelles, can be delivered to lysosomes via CMA , whereasecycling ,72,73,74ecycling .2+-dependent process, during which a crucial role is played by the lysosomal channel, TRPML1 [-/-mcoln1 mouse, a murine model of MLIV. This finding suggested an accumulation of protein aggregates and a defect in autophagy which could account for the neurodegeneration occurring in MLIV [Drosophila trpml mutant and Caenorhabditis elegans cup-5 null mutant. In particular, autophagy disruption, observed in trpml mutant flies exhibiting an MLIV-like phenotype, was attributable to a reduced degradation of cargoes following fusion between autophagosomes and lysosomes [C. elegans, mutations in cup-5 were responsible for defects in the autophagic pathway. Indeed, in cup-5 null mutant worms, a variety of autophagic substrates accumulated in enlarged vacuoles similar to late endosomes and lysosomes, thus indicating a dysfunction in autolysosomes proteolytic degradation [Several studies have shown that autophagy induction is a Ca, TRPML1 . Indeed,, TRPML1 . Further, TRPML1 . A simil in MLIV . Autophaysosomes . Similarradation .2+ release through the channel, by activating Ca2+/calmodulin-dependent Ser/Thr phosphatase calcineurin, promoted the dephosphorylation and subsequent nuclear translocation of TFEB (transcription factor EB) [The molecular mechanisms underlying TRPML1-mediated regulation of autophagy have been recently described. In particular, lysosomal Cactor EB) , a lysosctor EB) ,81. Oncector EB) ,81. Intector EB) .2+ release through TRPML1 in response to oxidative stress conditions. Indeed, following an elevation in ROS production or an increase in exogenous oxidants, TRPML1-mediated Ca2+ release promoted calcineurin-dependent TFEB nuclear translocation, thus enhancing autophagy and lysosome biogenesis and mitigating oxidative stress. In fact, the subsequent increase in the autophagic flux facilitated the elimination of damaged mitochondria that produce excessive ROS and restored a balanced redox homeostasis [Furthermore, it has been shown that autophagy can also be activated by lysosomal Caeostasis . Therefo2+ handling and release have been indicated as the main causes of the lysosomal storage disorders (LSDs), of which neurodegeneration constitutes an important component. This is mainly due to defects in the key components of lysosomal Ca2+ machinery leading to Ca2+ dysregulation. In example, defects in lysosomal Ca2+ release play a fundamental role in the pathogenesis of MLIV, an autosomal recessive LSD characterized by severe neurodegeneration and caused by loss-of-function mutations in the gene encoding for TRPML1 [Defects in lysosomal Car TRPML1 ,82,83. 2, such as FIG4, are associated with other neurological disorders [2+ channel can also be activated by ROS [Moreover, mutations in key proteins involved in the synthesis of TRPML1 endogenous agonist PIPisorders , includiisorders ,34. Furtd by ROS , whose pd by ROS .Besides its well-established role in the neurodegenerative processes underlying MLIV, TRPML1 function is compromised also in other neurodegenerative diseases, including the LSD Niemann–Pick type C (NPC) disease  and HIV-2+ levels within the acidic stores, caused by defective lysosomal Ca2+ uptake and the subsequent reduction in lysosomal Ca2+ release, led to an impairment in late endosomes/lysosomes fusion and to a defective lipid trafficking [In particular, in NPC, the low Cafficking ,89. The fficking .Similarly, in cellular models of HIV-dementia, the pharmacological activation of TRPML1 was able to promote the clearance of amyloid-β peptides and sphingomyelin at lysosomal level .2+ channels could be involved in the pathogenesis of some neurological diseases. For instance, the inhibition of the voltage-gated Ca2+ channel CACNA1A function at lysosomal but not at plasma membrane level led to lysosomal fusion defects [Other lysosomal Ca defects . Mutatio defects .On the other hand, P2X4 channel is involved in epilepsy and in some forms of ALS , althoug2+ dysfunction also in the most frequent neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) [A nascent literature has emerged on the role of lysosomal Caase (HD) ,94,95,96-/-) significantly altered lysosomal Ca2+ storage and release, thus causing an impairment in lysosomal fusion processes [2+ observed in PSEN1-/- cells was dependent on the elevation of lysosomal pH, which caused an abnormal Ca2+ efflux from lysosomes via a hyperactivated TRPML1 [2+ dysregulation and cell death induced by Aβ1-42 exposure [Moreover, in a mouse model of familial AD, loss-of-function mutations in presenilins  and sporadic (sALS) ALS forms .G85R mice [G93A animals in which p62 and LC3-II levels were elevated [Indeed, almost 7 out of 25 genes related to Mendelian inheritance of ALS are involved in endosomal/vesicular biogenesis, maturation, and trafficking. In virtue of this defective clearing mechanism, the main pathological hallmark and potential triggering mechanism of ALS is represented by the accumulation of misfolded proteins in motor neurons. Ubiquitin-positive inclusions linked to SOD1, transactive response DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS) protein, and C9orf72 have been reported in fALS . However85R mice  that, inelevated . Furthermore, C9orf72 insufficiency negatively modulated lysosomal exocytosis process , while TMoreover, exposure of motor neurons to cerebrospinal fluid from sALS patients led to a significant dysfunction of the lysosomal proteins hexosaminidase, sialidase, and aryl sulfatase . The sam2+-storing organelles, Vollrath et al. showed that depletion of sigma-1 receptor (SigR1), an ER chaperone located at the mitochondria/ER interface, led to an impairment of calcium mobilization, mitochondrial and ER defects, accumulation of undegraded substrates, and altered endosomal trafficking in NSC-34 motor neurons [2+ dyshomeostasis in ALS [In support of the neurobeneficial role of a balanced interplay among Ca neurons . Further neurons , thus ins in ALS .2+ homeostasis dysfunction. In accordance with this idea, Rusmini and coauthors [2+-dependent lysosomal clearance of neurotoxic misfolded proteins in motor neurons in a TFEB-dependent manner.All these findings are indicative of the occurrence of defects in cellular cleansing mechanisms in ALS that, in part, are dependent on Caoauthors  demonstr2+ homeostasis in the correct modulation of autophagic flux useful in cleansing the cell of defective organelles and misfolded proteins, the involvement of TRPML1 in the pathogenesis of the Guamanian form of ALS (ALS/PDC) has been recently characterized [Given the relevance of a balanced lysosomal Cacterized . In this2+ homeostasis occurred in neurons exposed to L-BMAA , most likely depending on the disruption of the lysosomal/ER interplay. This suggests that ER store is acting as a major source of lysosomal Ca2+ in motor neurons and that dysfunctional Ca2+ homeostasis in one of these organelles could have a dramatic repercussion on the other store. Importantly, in this study, early pharmacological stimulation of lysosomal TRPML1, by boosting autophagy, efficiently rescued motor neurons from L-BMAA toxicity and prevented ER stress, a hallmark of several forms of ALS [Furthermore, a concomitant defect of ER Cas of ALS ,106.2+ content, and impairment in organellar interplay. In fact, a concomitant dysfunction in ER and mitochondrial Ca2+ content has been described together with lysosomal dysfunction. This results in the occurrence of defects in cellular cleansing mechanisms and global Ca2+ dyshomeostasis in ALS. In accordance with this evidence, dysfunction of the lysosomal channel TRPML1 is emerging as a pathogenetic mechanism of the disease. Indeed, TRPML1 downregulation determined autophagy engulfment, disruption of lysosomal/ER interplay, and ER stress. These pathognomonic signs are all overcome by boosting autophagy through TRPML1 stimulation. Due to the complexity of the mechanisms involved in lysosomal Ca2+ regulation and function, many other efforts are needed to identify new targets within the lysosomal machinery whose modulation could impact Ca2+ dyshomeostasis in ALS.The impact of endolysosomal system defects on autophagic process is now considered one of the most important pathogenetic mechanisms of several neurological diseases. However, this concept is starting to emerge also for ALS pathogenesis. The exact mechanism underlying lysosomal dysfunction remains to be established. Many studies documented a downregulation of expression and activity of lysosomal enzymes, reduction of lysosomal Ca


Infection of cattle by bovine herpesvirus 1 (BoHV-1) can culminate in upper respiratory tract disorders, conjunctivitis, or genital disorders. Infection also consistently leads to transient immune-suppression. BoHV-1 is the number one infectious agent in cattle that is associated with abortions in cattle. BoHV-1, as other α-herpesvirinae subfamily members, establishes latency in sensory neurons. Stressful stimuli, mimicked by the synthetic corticosteroid dexamethasone, consistently induce reactivation from latency in latently infected calves and rabbits. Increased corticosteroid levels due to stress have a two-pronged effect on reactivation from latency by: (1) directly stimulating viral gene expression and replication, and (2) impairing antiviral immune responses, thus enhancing virus spread and transmission. BoHV-1 encodes several proteins, bICP0, bICP27, gG, UL49.5, and VP8, which interfere with key antiviral innate immune responses in the absence of other viral genes. Furthermore, the ability of BoHV-1 to infect lymphocytes and induce apoptosis, in particular CD4+ T cells, has negative impacts on immune responses during acute infection. BoHV-1 induced immune-suppression can initiate the poly-microbial disorder known as bovine respiratory disease complex, which costs the US cattle industry more than one billion dollars annually. Furthermore, interfering with antiviral responses may promote viral spread to ovaries and the developing fetus, thus enhancing reproductive issues associated with BoHV-1 infection of cows or pregnant cows. The focus of this review is to describe the known mechanisms, direct and indirect, by which BoHV-1 interferes with antiviral immune responses during the course of infection. Bovine herpesvirus 1 (BoHV-1) is an α-herpesvirinae subfamily member that causes significant economical losses to the cattle industry . SubtypeBoHV-1 is the most frequently diagnosed cause of viral abortion in North American cattle . ExposurThe incubation period for the genital forms of BoHV-1 is 2–6 day and initial clinical signs are frequent urination and a mild vaginal infection . It is aMannheimia haemolytica (MH), exists in the upper respiratory tract of healthy ruminants , a poly-microbial disease initiated by stress and/or virus infection, is the most economically important disease that affects beef and dairy cattle. Annual BRDC losses in the U.S. are ~$1 billion –18. A gruminants , 20. Foluminants , this coDC cases –25. BoHVDC cases , 27, higry tract , 23, 25,trophils , 29. Co-neumonia . Finallyneumonia  confirmide novo protein synthesis. Two IE transcription units exist: IE transcription unit 1 (IEtu1) and IEtu2. IE transcription unit 1 (IEtu1) encodes two transcriptional regulatory proteins, bICP0 and bICP4, because a single IE transcript is differentially spliced and then translated into bICP0 or bICP4 . Viral gene expression  and infeAbundant expression of the viral encoded latency related (LR) gene occurs in latently infected neurons, but infectious virus is not readily detected (maintenance of latency) , 45–48. The synthetic corticosteroid dexamethasone (DEX) initiates reactivation from latency in latently infected calves or rabbits 100% of the time , 47, 59.The IEtu1 promoter that drives bICP0 and bICP4 expression is stimulated by DEX and contains two consensus GR binding sites that are bound by the activated GR , 68. TheCattle acutely infected with BoHV-1 develop an innate immune response –76; howeAlthough the host immune response clears virus after acute infection, viral infection impairs immune-recognition on several levels impairs: (1) cell-mediated immunity –86, (2) 3HC4 zinc RING finger that is conserved in all ICP0 proteins  necessary for efficient transcriptional activation , and a Cproteins , 101. Popromoter , stimulapromoter , 102, anpromoter –95. bICPpromoter , 101. PMpromoter . Many DNpromoter , 105 andpromoter .bICP0 inhibits IFN-β promoter activity in transient transfection studies , 94 by rBos Taurus encodes three functional IFN-β genes; all have anti-viral activity but each gene contains a unique promoter  promotes cell to cell spread  and main+ T cells , is a 96 amino acid protein . The BoH T cells , 124–126 T cells .de novo viral protein synthesis.VP8, the most abundant tegument protein in the virion, enhances growth in cultured cells and is required for pathogenesis in calves . VP8 intBoHV-1 is a very successful pathogen because it encodes several genes that impair intrinsic and innate immune responses throughout productive infection see . VP8 is The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ARGs confering resistance to 12 classes of antibiotics (ARG types) persisted through the treatment stages, which included genes that confer resistance to aminoglycoside , beta-lactams , chloramphenicol , fosmidomycin (rosAB), macrolide-lincosamide-streptogramin , multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide , and tetracycline . Although the ARG subtypes in sludge and MBR effluents reduced in diversity relative to the influent, clinically relevant beta lactamases  were detected, casting light on other potential point sources of ARG dissemination within the wastewater treatment process. To gain a deeper insight into the types of bacteria that may survive the MBR removal process, genome bins were recovered from metagenomic data of MBR effluents. A total of 101 close to complete draft genomes were assembled and annotated to reveal a variety of bacteria bearing metal resistance genes and ARGs in the MBR effluent. Three bins in particular were affiliated to Mycobacterium smegmatis, Acinetobacter Iwoffii, and Flavobacterium psychrophila, and carried aquired ARGs aac(2′)-Ib, blaOXA−278, and tet36 respectively. In terms of indicator organisms, cumulative log removal values (LRV) of Escherichia coli, Enterococci, and P. aeruginosa from influent to conventional treated effluent was lower (0–2.4), compared to MBR effluent (5.3–7.4). We conclude that MBR is an effective treatment method for reducing fecal indicators and ARGs; however, incomplete removal of P. aeruginosa in MBR treated effluents (<8 MPN/100 mL) and the presence of ARGs and intl1 underscores the need to establish if further treatment should be applied prior to reuse.Reclaimed water provides a water supply alternative to address problems of scarcity in urbanized cities with high living densities and limited natural water resources. In this study, wastewater metagenomes from 6 stages of a wastewater treatment plant (WWTP) integrating conventional and membrane bioreactor (MBR) treatment were evaluated for diversity of antibiotic resistance genes (ARGs) and bacteria, and relative abundance of class 1 integron integrases ( Wastewater treatment plants (WWTP) receive large volumes of sewage that are enriched in nutrients, chemicals, and bacteria originating from human and animal wastes ] and sul genes, suggesting that the presence of heavy metals in WWTP play a role in co-selecting for ARGs , primary settling tank (PST), secondary settling tank (SST), membrane bioreactor (MBR), and wet well (WW). Also, recycled activated solids (SLUDGE) was collected from a Modified Ludzack-Ettinger (MLE) bioreactor. Samples were collected at six different time points: October 2016, November 2016, January 2017, March 2017, May 2017, August 2017. The water reclamation process and sampling locations are indicated in Grab samples were collected in sterile Nalgene carboys and initially passed through a filter on-site at the facility prior to further concentration in the laboratory. Turbid samples (INF and PST) were collected in small volumes (1–5 L), while 60 L were collected for clear samples . Recycled SLUDGE (50 mL) was collected in a sterile tube directly from the “return” piping. Samples were concentrated to a final volume of 400 mL using a Hemoflow dialyzer  via standard bloodline tubing . Prior to concentration, the ultrafilter was rinsed with nanopure water for 5 min, and subsequently pre-treated with 500 mL of blocking solution [0.1 g of Sodium Pyrophosphate  in 1 L of nanopure water] through continuous recirculation for 15 min. After the pre-treatment step, wastewater samples were pumped through the ultrafilter and the retentate tube was blocked. The filtrate was discarded and the biomass trapped on the ultrafilter was eluted with 400 mL of elution buffer  with the retentate tube unblocked. The concentrates that were eluted from the hollow fiber ultrafiltration unit were then filtered through 0.22 μm cellulose nitrate membrane filters  to further concentrate the microbial biomass. SLUDGE was centrifuged at 15,000 g for 15 min and the pellet was kept for further analysis.E. coli, Enterococcus spp., P. aeruginosa) were assayed via Colilert, Enterolert, and Pseudalert kits  following the manufacturer's instructions with slight modification. Briefly, 100 mL of wastewater sample and 10-fold serially diluted samples were prepared by mixing the respective kit reagents in provided sterile bags and poured into a multi-well tray (Quanti-Tray 2000). All kits were incubated over 24 h at temperatures of 37°C, for Colilert and Pseudalert samples, and 44°C for Enterolert samples. The trays were visualized under long-wave ultraviolet light (365 nm) and positive wells producing a fluorescence was counted and expressed as the most probable number (MPN) per 100 mL of water sample (MPN/100 mL).To estimate the level of fecal contamination and determine whether wastewaters met microbiological criteria for recreational purposes  calculated using the following formula:CIN represents the concentration in the inlet wastewater source and Cout represents the concentration in the effluent of the treatment stage. For effluent samples that did not contain detectable concentrations of indicators (<1 MPN/100 mL), a “zero” value was substituted in order to calculate LRV representative of the actual removal.where Nucleic acids were extracted from total wastewater biomass concentrated on 0.22 μm cellulose nitrate membrane filters  and 0.5 g (wet weight) of SLUDGE samples using the PowerSoil DNA Isolation Kit  according to the manufacturer's recommendations. The quality and quantity of DNA extracted were determined using a Qubit 3.0 Fluorometer  and extracted DNA was run on a 1% agarose gel to ensure that samples were not degraded.PRJNA438174  where library preparation and sequencing was performed on the Illumina HiSeq2500 sequencer as stated in Ng et al. . MetagenNA438174 .https://sourceforge.net/projects/bbmap/). Taxonomic assignment of clean reads was done using Metaxa2 2.2 beta 10 , an online analysis pipeline developed by Yang et al.  and types  with relative abundances measured by normalizing the number of assigned ARG reads to 16S rRNA genes in the metagenomic dataset for each sample. Cleaned reads were interrogated against integron-like sequences extracted from the INTEGRALL database  between the different treatment steps.All paired end raw reads were removed of sequence adaptors and low quality reads using bbmaps of the BBTools packages . Primer v7  transformation and plotted using the online web tool ClustVis (https://cge.cbs.dtu.dk/services/ResFinder/) at a selected threshold of 90% and minimum query length of 60%. Gene calling and functional annotation of genome bins were performed using RAST .All clean metagenomics reads from the MBR effluent were assembled into contigs using Megahit , sedimentation during PST stages resulted in only slight reduction (<1.00) of E. coli (2.3 × 107 ± 2.3 × 107 MPN/100 mL) and Enterococci (6.8 × 105 ± 2.2 × 105 MPN/100 mL), as well as P. aeruginosa (4.7 × 106 ± 4.8 × 106 MPN/100 mL) , but contained P. aeruginosa at a mean concentration of 4.0 ± 2.8 MPN/100 mL.Although mean concentrations for each indicator were abundant in raw sewage (/100 mL) . SuspendbacA), sulfonamide (sul1), aminoglycoside (aad), beta-lactam (class A beta-lactamase), multidrug resistance , tetracycline (tetX), and macrolide-lincosamide-streptogramin  , bacitracin (bacA), beta lactams , chloramphenicol , fosmidomycin (rosAB), MLS , polymyxin (arnA), quinolone (qepA), rifamycin (arr), sulfonamide , tetracycline , trimethoprim (dfrAB) and vancomycin (vanRSX) were detected in the MBR effluent.The average diversity of ARG subtypes was higher in the INF (450 subtypes), PST (460 subtypes), and WW (325 subtypes), compared to the SST (292 subtypes), SLUDGE (195 subtypes), and MBR (181 subtypes) . The aveS, ermF) . A heatpaadA, aph(6)-I, aph(3′)-I, aac(6′)-I, aac(6′)-II, ant(2″)-I], beta-lactams , chloramphenicol , fosmidomycin (rosAB), MLS , multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide , and tetracycline   showed that ARG subtypes, class A beta lactamase, cAMP regulatory proteins, and kasugamycin resistance protein (ksgA) correlated with the INF, SST, and WW cluster, while mutidrug efflux pumps  correlated with the MBR samples.Wastewaters from all six locations along the treatment train shared a core resistome of 100 ARG subtypes belonging to 12 ARG types . There wQ, tetX) . A PrincintI1 genes was highest in INF (33 ± 9) and PST (34 ± 8), compared to the other points further down the treatment train  (P < 0.05) reduction in relative abundance values from PST to SST, and SST to SLUDGE (intI1 genes from SST to MBR.The relative abundance of  14 ± 7) . There wo SLUDGE . There wR = 0.357, P = 0.01, Pseudomonas, Bacteroides, Aeromonas, Prevotella, and Cloacibacterium dominated the first cluster in wastewater samples. However, a higher prevalence of Alteromonadaceae, Pedobacter, Gemmatimonas, and Flexibacter differentiated SLUDGE from the rest of the samples , Acinetobacter (25%), Varivorax (12%), Comamonas (5%), Thermomonas (4%), Acidovorax (3%), and Delftia (3%) persisted in post-MBR treated effluent, while Cloacibacterium (24%), Pseudomonas (11%), Aeromonas (8%), Arcobacter (6%), Flavobacterium (5%), Bacteroides (5%), Acinetobacter (5%) persisted in SST treated effluent (Cloacibacterium (26%), Pseudomonas (8%), Flavobacterium (7%), Acinetobacter (6%), and Acidovorax (2%).The Shannon-Weaver and Simpson reciprocal indices were used to assess the microbial diversity for each sample. The average values showed SLUDGE had the greatest bacterial diversity followed by SST, WW, INF, PST, and MBR . A Princeffluent . The WW Magnetospirillum, Acidovorax, Nitrospira, Rhodospirillum, Sphingopyxis, Flavobacterium, Acinetobacteria, Chitinophage, Dechloromonas, Janthinobacterium, Mycobacterium, Novosphingobium  and metal resistance  were present in the genomes of bacteria in the MBR effluent (aac(2′)-Ib, blaOXA−278, tet36, respectively  from INF to post-SST was 0–2.4, and 5.3–7.4 post-MBR, which validates a greater removal capacity via MBR as evident in other studies  of nine different wastewater MBR systems in California  and Enterococci (6) were comparable to another study with a full scale MBR system in a municipal WWTP in Italy , which meets the U.S. EPA guidelines for recreational purposes, and within reported limits used for agriculture reuse for surface or spray irrigation of food crop intended for human consumption  chloramphenicol, fosmidomycin, MLS, polymyxin, quinolone, rifamycin (arr), sulfonamide, tetracycline, and intI1 genes . Detection of sulfonamide , tetracycline , MLS (ereA) resistant genes and intI1 in our study has also been observed in MBR effluents of municipal wastewater treatment plants in China  were detected across the treatment process, including in the sludge. A variety ARG subtypes belonging to 14 different ARG types  were detected in sludge samples which underscores problems of the application of sludge for organic farming purposes. Another study by Calero-Cáceres et al. , class B , and class C (blaPDC) beta lactamases were detected in INF and MBR effluents, which confer resistance to beta lactam antibiotics such as cephalosporin and carbapenems. These antibiotics are of clinical importance and considered as last resort antibiotics in the treatment of Gram-negative infections , and the presence of viable P. aeruginosa cells in MBR effluent is a basis for P.aeruginosa playing an important role in the carriage and dissemination of antimicrobial resistance in post MBR treated effluent. Although low concentrations of viable P.aeruginosa (<8 MPN/100 mL) were measured in the MBR effluent, their biofilm forming ability and antibiotic tolerant properties within a biofilm community  also observed in a 16S rRNA- based microbial classification study of municipal water effluent by Harb and Hong  and Chryseobacterium (Chryseobacterium gleum), known to cause infections in fish and occasionally humans  and phosphorus (Gemmatimonas), formation of bioflocs , and other taxa involved in degradation pathways  which are typically present in activated sludge , bacitracin (bacA), and multidrug resistance  genes.Shifts in community composition were observed at each treatment stage, with the SLUDGE samples having the highest diversity enriched in bacteria involved in the removal of nitrogen (P. aeruginosa was not as effective. In another study, Zhu et al. (intI1, did not show significant rates of removal with MBR treatment.The reduction in the diversity of ARG subtypes and concentrations of fecal indicators along the treatment train of a full-scale municipal wastewater system is a clear indication that MBR as a treatment strategy is effective at removing raw-sewage associated bacteria and most ARGs in the influent, although removal of u et al.  reportedu et al. . Apart fu et al. . AlthougCN, HC, BS, LH, FC, and KG conceptualized and designed the study. CN, HC, BS, LH, and FC processed samples and assisted with data collection. CN, BT, X-TJ, XG, and TZ provided technical expertise in sequence data analysis, assembly, and interpretation. CN and KG wrote the manuscript. KG and BT helped obtain funding support for the work.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.