The alphaherpesviruses are pathogens of the mammalian nervous system. Initial infection is commonly at mucosal epithelia, followed by spread to, and establishment of latency in, the peripheral nervous system. During productive infection, viral gene expression, replication of the dsDNA genome, capsid assembly and genome packaging take place in the infected cell nucleus, after which mature nucleocapsids emerge into the cytoplasm. Capsids must then travel to their site of envelopment at cytoplasmic organelles, and enveloped virions need to reach the cell surface for release and spread. Transport at each of these steps requires movement of alphaherpesvirus particles through a crowded and viscous cytoplasm, and for distances ranging from several microns in epithelial cells, to millimeters or even meters during egress from neurons. To solve this challenging problem alphaherpesviruses, and their assembly intermediates, exploit microtubule- and actin-dependent cellular motors. This review focuses upon the mechanisms used by alphaherpesviruses to recruit kinesin, myosin and dynein motors during assembly and egress. Alphaherpesvirinae include pathogens of the nervous system such as herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV) and the swine virus pseudorabies virus (PRV) [The us (PRV) ,4. Many us (PRV) ,6. In adus (PRV) . HSV-1 ius (PRV) ,9,10,11.us (PRV) ,15,16,17Assembly and egress of alphaherpesviruses requires a complex interplay between viral structural components and the host cell biosynthetic machinery, organelles and cytoskeleton ,12,15,17Alphaherpesvirus assembly and egress requires transport of viral particles from the vicinity of the infected cell nucleus to the cell periphery. It is therefore inevitable that kinesin motors play a major part in these events . NeverthThere are 45 known kinesins in the mammalian genome ,54,55,5640 [Kinesin-1 is a heterotetramer consisting of two kinesin heavy-chains (KHC) and two kinesin light-chains (KLC) ,54,55,5640 ,68,69. I40 ,71, a ph40 . Kinesin2) [−1) [Kinesin-3 motors ,54,55,562) ,54,55,562) [−1) , and exh2) [−1) . Kinesin2) [−1) . KIF1A mCytoplasmic dynein is a MT minus end-directed motor of approximately 1.5 mDa, containing multiple subunits . Two heaThe human genome contains 40 different myosin genes, encoding 20 distinct classes of myosin ,82. MyosNM-II, like M-II, comprises a myosin chain dimer in complex with additional polypeptides involved in regulation and stability . Dozens Myosin-V exists as three isoforms in mice and humans: Va, Vb and Vc. The former two are widely expressed in the CNS, whereas Vc appears to be epithelial cell-specific . The myo−1 radial motion [Following viral gene expression and DNA replication in the infected cell nucleus, the alphaherpesvirus genome is packaged into preassembled procapsids ,12,16. Ml motion  around sl motion ) and thil motion . Howeverl motion ,97, and l motion . Howeverl motion . Moreovel motion , jasplakl motion . Furtherl motion , inducedl motion . Immobill motion . Most rel motion . Primary enveloped virions in the perinuclear space undergo fusion of their envelopes with the outer nuclear membrane to deliver capsids into the cytoplasm. Capsids then utilize kinesin motors for transport to cytoplasmic organelles for secondary envelopment ,15,17,21UL36p is a ~330 kDa polypeptide anchored to the vertices of the icosahedral capsid ,17,103, −1 [Live-cell imaging of Vero cells infected by HSV-1 expressing a fluorescently tagged capsid, revealed that 10–30% of cytoplasmic capsids moved dynamically over long distances, with a mean velocity of ~2 µm s−1 . In cont−1 . Similar−1 ,119. Thi−1 ,120.−1 [These data suggest that capsid-associated UL36p and UL37p are essential for the binding, or at least the activity, of MT-dependent molecular motors. These presumably include MT plus end-directed kinesins, which would be expected to support anterograde capsid transport to the cell periphery during egress . The sim−1 . The MT-−1 . As seen−1 . This isRoles for UL36p and UL37p in kinesin attachment in vivo , are conThe binding of dynein and dynactin to purified capsids in vitro  might beAre there any clues in the sequence or structure of UL36p or UL37p that might point to the mechanism of kinesin motor recruitment? The Sodeik laboratory identified two “tryptophan-acidic” WD and WE motifs in UL36p that are conserved among all Alphaherpesvirinae. Alanine ablation of the motifs gave rise to HSV-1 strains with an approximately 3-log decrease in infectious titer and greatly impaired envelopment in the cytoplasm . CapsidsIf UL36p directly recruits kinesin-1, why does deletion of UL37p cause such a dramatic reduction in capsid anterograde transport for HSV-1 and (to a lesser extent) PRV? , resembling their behavior in the presence of nocodazole [Do capsids use UL36p to recruit dynein during egress, as they do during entry ,78,79,80−1 . Howevercodazole  or in thcodazole ,121. Whycodazole ,78,79,80Finally, although dynein recruitment during alphaherpesvirus anterograde transport is best understood in the context of the capsid, there is also evidence that dynein is present on PRV particles at later stages of export, following capsid envelopment in the cytoplasm  Section. The mectrans Golgi network (TGN) or endosomes [After MT-directed trafficking through the cytoplasm, the egressing alphaherpesvirus capsid associates with organelles derived from the ndosomes ,15,17. Andosomes ,14,15,17ndosomes ,17. ThesThe mechanism of motor recruitment to the alphaherpesvirus OEV is best understood in infected neurons, where the virally encoded membrane glycoproteins E and I (gE and gI), and a non-glycosylated membrane protein encoded by the US9 gene (US9p) play critical roles in MT-directed anterograde traffic into, or along, the axon ,142,143 Although gE/gI and US9p are important for anterograde spread of both HSV-1 and PRV, there are differences in the phenotypes of PRV and HSV-1 strains lacking these membrane proteins. For HSV-1, the gE/gI and US9p proteins appear to function redundantly in support of anterograde transport ,150,151.To investigate the mechanism responsible for the striking axon-entry/transport deficit of PRV US9-null mutants, Kramer and colleagues used mass spectrometry to identify proteins that copurified with a GFP-US9p fusion in extracts of PRV infected PC12 cells . Among tIf US9p is an essential component of a KIF1A recruitment apparatus on the PRV OEV, what is the role of gE/gI? The efficiency of pull-down of KIF1A by US9p was stimulated by the presence of gE/gI , suggestData described in Membrane-imbedded alphaherpesvirus envelope proteins traffic within neurons as constituents of OEVs, but are also carried separately from virions, within transport vesicles ,50,144. If gE/gI-US9p-KIF1A ensures delivery of OEVs into the axon, which kinesin motors transport them along axonal MTs, and to the nerve terminal? In the case of PRV it is possible that KIF1A also mediates this step, since KIF1A is present on PRV OEVs during their axonal transport . If thisCaenorhabditis elegans where it functions to remove KIF1A from its cargo after delivery of synaptic vesicles to the synapse [A recent study may shed additional light on how KIF1A and KIF5C motors might be exchanged during egress. Upon PRV infection of rat PC12 cells and SCG neurons, KIF1A becomes targeted for degradation by the proteasome, and gE/gI-US9p are sufficient to induce this accelerated turnover in the absence of other PRV proteins . As poin synapse . Presuma synapse . Huang a synapse . However synapse  , a type-1 membrane protein found in axonal transport vesicles and implicated in the kinesin-1-dependent transport of cargo proteins including presenilin 1, Growth associated protein 43 (GAP-43), synapsin-I and Tropomyosin receptor kinase A (TrkA) ,171. AntAny understanding of kinesin recruitment during alphaherpesvirus egress must account for the fact that, in some types of neurons, it has been proposed that non-enveloped HSV-1 capsids, rather than OEVs, traffic out of the cell body and along axons. These capsids eventually reach, and acquire their envelopes, at the nerve terminal. This has been termed the “Separate” model of transport, in contrast to the “Married” model where the capsid and envelope unite in the cell body then travel together in the context of the OEV . It is gThe alphaherpesvirus UL56p gene product  Table 1 bears soIn Vero cells, the UL56p of HSV-2 localizes to juxtanuclear vesicular structures resembling MVBs, late endosomes or lysosomes ,180, andMyosin motors have been implicated in the release of alphaherpesviruses from infected cells, and their spread to uninfected cells, though few molecular details are known . One mecIf TNTs facilitate alphaherpesvirus spread from infected to uninfected cells, how might viral particles move along them? TNTs extending from both uninfected and alphaherpesvirus-infected cells commonly contain F-actin ,192,193.Consistent with a role for myosins in TNT-mediated alphaherpesvirus egress, HSV-1 infection of Vero cells induces the formation of TNT-like structures that contain myosin NM-IIA  Section. This TNMyosin-Va has also been implicated in HSV-1 egress from infected cells, though whether this is mediated via TNTs remains to be determined. HSV-1 infection of HEp-2 and HeLa cells enhanced the immunoreactivity of a particular myosin-Va epitope without changing overall levels of the myosin-Va protein , suggestAlphaherpesvirus trafficking within and between cells is critical for viral replication and the spread of disease, and is accomplished by the conscription and exploitation of a variety of cellular motors. The best understood example of this, by far, is kinesin KIF1A recruitment by the gE/gI-US9p complex of PRV. However, even here we lack fundamental molecular details. Is the short cytoplasmic tail of US9p a stoichiometric receptor that directly contacts KIF1A, or does it act catalytically to stimulate KIF1A binding to, and dimerization on, the surface of the OEV bilayer? What is the role of gE/gI, and how is KIF1A recruitment coordinated with that of other kinesins, such as kinesin-1? As discussed above, the presence of dynein on egressing capsids and OEVs may enhance the processivity of transport during egress, but direct evidence for this is lacking. Furthermore, although there are some clues as to how dynein may bind to capsids during egress, the mechanism of dynein association with OEVs, and how egressing virions might coordinate dynein and kinesin activity to ensure processive anterograde transport, remain completely unknown. A fascinating area of study, still in its infancy, is the mechanism by which myosin is recruited by these viruses. It appears clear that alphaherpesvirus transport along TNTs is a significant route for alphaherpesvirus cell–cell spread, which may involve TNT actin filaments and virally associated myosin NM-IIA or Va. However, we know very little of how egressing alphaherpesviruses enter TNTs, and how they might select and bind myosins for transport. Other outstanding key questions are as follows.What is the advantage of selecting kinesin-1 and kinesin-3 for transport, and do these motors indeed traffic alphaherpesviruses at distinct stages of egress, as discussed above? How does the putative gE/gI-US9p-KIF1A complex deliver viral particles from the neuronal cell body to the privileged environment of the axon?Are the tryptophan acidic WD/WE motifs in UL36p required for kinesin-1 binding to capsids in vitro, and are they needed for processive, anterograde traffic of capsids in the cytoplasm prior to capsid envelopment?Does kinesin recruitment by gE/gI play a role in delivery of alphaherpesviruses to the lateral surfaces of polarized epithelial cells during cell–cell spread?Do gE/gI and US9p mediate traffic along TNTs that contain MTs?


In the last decades, the endoplasmic reticulum (ER) has emerged as a key coordinator of cellular homeostasis, thanks to its physical interconnection to almost all intracellular organelles. In particular, an intense and mutual crosstalk between the ER and mitochondria occurs at the mitochondria–ER contacts (MERCs). MERCs ensure a fine-tuned regulation of fundamental cellular processes, involving cell fate decision, mitochondria dynamics, metabolism, and proteostasis, which plays a pivotal role in the tumorigenesis and therapeutic response of cancer cells. Intriguingly, recent studies have shown that different components of the unfolded protein response (UPR) machinery, including PERK, IRE1α, and ER chaperones, localize at MERCs. These proteins appear to exhibit multifaceted roles that expand beyond protein folding and UPR transduction and are often related to the control of calcium fluxes to the mitochondria, thus acquiring relevance to cell survival and death. In this review, we highlight the novel functions played by PERK, IRE1α, and ER chaperones at MERCs focusing on their impact on tumor development. It is well known that the ER is the place where lipid and steroids are synthesized and, then, delivered to other endo-membranes throughout vesicular carriers or non-vesicular mechanisms that, if out of control, result in several pathologies including cancer . It is uartments . Remarkaartments .On the other hand, a large portion of the ER is associated to protein synthesis and translocation. The ER lumen is equipped with a molecular machinery that carries out a rigorous chaperone-mediated quality control (QC) of protein folding to ensure that proteins are delivered in a functional state to secretory compartments. QC is under the regulation of the unfolded protein response (UPR): a battery of signaling pathways that, when excessive protein unfolding occurs is able to choose between cell death and survival, this choice affecting either normal or cancer cells. This is why the participation of the UPR in tumorigenesis has long been studied. Moreover, the UPR has always been considered a strategic target for potential cancer therapy for its consequence on neoplastic cell proliferation and survival . BetweenThe control of lipid and protein flux toward the secretory pathway compartments is an essential function of the ER, which is the site of entrance, folding, and departure of luminal and integral membrane proteins destined to the ER itself, Golgi apparatus, endo-lysosomal compartment, the plasma membrane (PM), or the extracellular environment .Quantitatively, ER exit relies on an autoregulatory system consisting of a set of signals that, in case of overload of newly synthesized cargo proteins, modulate protein export by regulating their packaging at the ER exit sites (ERESs) into COPII anterograde vesicles and by attenuating protein synthesis to prevent traffic congestion of cargo proteins .Qualitatively, within the ER, a sophisticated machinery of enzymes and chaperones has evolved to retain unfunctional proteins within the organelle or to direct them to ubiquitin/proteasome degradation . QC of pAt the same time, to limit the amount of unfolded proteins entering the ER, PERK phosphorylates the eukaryotic initiation factor 2α (eIF2α) producing p-eIF2α that acts as a suppressor of protein translation initiation . This evOn the other hand, when ER stress is persistent, the UPR survival project turns into the cell death program  switchinA similar shift from pro-survival to pro-apoptotic activity happens to IRE1α transducer, which owns both kinase and RNAse activity . During It is universally acknowledged that the UPR plays critical roles in tumor progression, survival, and metastasis . MoreoveThe ER shows a complex membrane architecture consisting of plane sheets in continuity with the nuclear envelope (NE) and peripheral membranes made of tubules that owe their shape to endogenous, or also exogenous, proteins that form and stabilize tubules at the ER sheet boundaries . While EFindings on MCSs overturned the view of the cell seen like a complex of isolated compartments and acknowledged the ER, the role of intermembrane network that homeostatically controls exchange of several structural and bioactive molecules . Despite2+ flux from the ER to the mitochondria . As a consequence, PTP induction leads to the increase of IMM permeability and the loss of mitochondrial membrane potential, which, in turn, induces mitochondrial swelling, OMM break, cytochrome c release, and cell death  complex , known as S1T, which is located at MERCs . Nevertheless, it can be assumed that the role played by PERK at MERCs in the transmission of ROS-derived signals between ER and mitochondria is more significant for cancer cells in the mechanism of adaptation to oxidative stress and resistance to apoptosis. The modulation of PERK activity by Mfn2 at MERCs, in basal conditions, should be further investigated in cancer cells to understand whether Mfn2-dependent deregulation of PERK activity occurs at MERCs during carcinogenesis. At the same time, the non-canonical role performed by PERK at MERCs in the control of mitochondria bioenergetics could be strategic for cancer cells to overcome nutrient deprivation and adapt to hypoxic conditions. Altogether, increased knowledge about the contribution of MERC-localized PERK in cancer initiation and progression could considerably impact human studies and clinical therapies not only for cancer cell elimination, but for many other disorders correlated to PERK such as diabetes, neurodegenerative, or heart diseases .Since the discovery of the IRE1α presence at MERCs , it is cIn the light of these results, it was proposed that MERC stabilization limits IRE1α RNase activity that could lead to cell damage and apoptosis. It is assumed that this event could occur by a “timing mechanism” and throughout different ways. First, MERC reinstallation could destabilize IRE1α oligomers by modifying ER membrane fluidity; second, localization of IRE1α at MERCs could change its redox state and alter its endonuclease activity, and third, MERC-localized PPM1L and PP2A phosphatases could dephosphorylate and deactivate IRE1α .3Rs to support Ca2+ transfer and mitochondrial bioenergetics , which binds non-phosphorylated CLNX to the cytosolic cluster of acidic amino acids and places CLNX at MERCs  Myhill . MoreoveHowever, the task of MERCs-localized CLNX is to regulate the activity of SERCA2b pump . As show2+ concentrations and protects cells from apoptosis , promotes proper protein folding and the maintenance of the redox state.2+ fluxes toward the mitochondria (2+-transport is controlled by Ero1α oxidoreductase in concert with ERp44, another multitask chaperone, which interacts with IP3Rs in order to modulate its Ca2+-flux properties in a redox-sensitive manner (2+ homeostasis at MERCs (2O2. Indeed, ROS generated by either ER or mitochondria in the H2O2 nanodomains generated by cristae can also localize at MERCs and perturb calcium signaling (Instead, recent findings have shown that, at MERCs, Ero1-α regulates Cachondria  by interchondria . Furthere manner . Therefoat MERCs . Moreoveignaling . Moreoveignaling , in partignaling . Neverthignaling .2+ flux at MERCs.To conclude, the mechanisms by which Ero1-α expression affects the poor prognosis of tumors are still to be elucidated and should be explored in depth, particularly by evaluating the involvement of Ero1-α-mediated CaIn the last decades, an increasing number of studies have shed light on the role of ER MCS in coordinating key cellular functions and the maintenance of cell physiology. Along with the complex analysis of MCS composition and function, the study of MERCs has emerged as a fundamental topic in the cell biology of MCSs. Among the multiple aspects analyzed, the literature discussed hereby has unveiled an additional role played at MERCs by the UPR transducers and chaperones in the orchestration of ER-to-mitochondria calcium flux, mitochondria bioenergetics, and apoptosis, and how these functions are deregulated in specific cancer settings. Taken together, the localization of PERK, IRE1α, and ER chaperones at MERCs appears to be relevant for cancer initiation and progression, and we think that it may represent one of the aspects to be deeply explored in the upcoming cancer research. Ultimately, many outstanding questions have to be answered: Which of the effects showed by UPR transducers on cancer development are dependent on their localization at MERCs? In particular, can the dual role played by the UPR transducers as tumor suppressor versus tumor promoter be specifically attributed to the ER-localized actors rather than to the MERC-localized one? Are there any properties of the MERC-localized proteins that could be related to one specific type of cancer rather than another? How do the additional functions performed by the UPR transducers and ER chaperones at MERCs affect the strength and the outcome of UPR signaling?Future studies defining the significance of UPR protein residence at MERCs will allow to gain further insight into their role in cellular physiology and cancer development and, auspicably, to lead to breakthroughs in therapeutic strategies.2+ uniporter; MDR, mechanisms of multidrug resistance; MERCs, mitochondria–ER contacts; Mfn2, Mitofusin 2; MITOL, mitochondrial ubiquitin ligase; MRP1, multidrug resistance-associated protein 1; MTC, medullary thyroid cancer; mTOR, aammalian target of rapamycin; NE, nuclear envelope; Nrf2, nuclear factor erythroid 2-related factor 2; OMM, mitochondrial outer membrane; PACS-2, phosphofurin-acidic cluster sorting protein 2; PD-L1, programmed cell death-ligand 1; PDI, protein disulfide isomerase; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PKC, protein kinase C; PP2A, protein phosphatase 2; PPM1L, protein phosphatase, Mg2+/Mn2+-dependent 1L; PTP, permeability transition pore; PTPIP51, protein tyrosine phosphatase-interacting protein-51; PUMA, p53 upregulated modulator of apoptosis; QC, quality control; RIDD, regulated Ire1-dependent decay; ROS, reactive oxygen species; S1P, site-1 protease; S1T, SERCA1 truncated proteins; SCAF, super complex assembly factor; SERCA1, sarco/endoplasmic reticulum Ca2+ ATPase1; SERCA2b, sarco/endoplasmic reticulum Ca2+ ATPase2b; σ1R, sigma-1 receptor; SIMH, stress-induced mitochondrial hyperfusion; SK3, small conductance calcium-activated potassium channel 3; TNBC, triple negative breast cancer; TRAF2, TNF receptor-associated factor 2; Trb3, Tribbles ortholog 1; TXNIP, thioredoxin-interacting protein; UPR, unfolded protein response; VDAC, voltage-dependent anion channel; VEGF, vascular endothelial growth factor; XBP1, X-box binding protein 1.Nomenclature: ASK1, apoptosis signal-regulating kinase 1; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; BAP31, B-cell receptor-associated protein 31; BIM, B-cell lymphoma 2; BIP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; CLNX, calnexin; CLR, calreticulin; COPII, coat protein complex II; CREB3L1, CAMP responsive element binding protein 3 like 1; DR5, death receptor 5; eIF2α, eukaryotic initiation factor 2α; EMT, epithelial-to-mesenchymal transition; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; ERESs, ER exit sites; ERK, extracellular signal-regulated kinases; Ero1-α, ER oxidoreductin 1; ERp44, endoplasmic reticulum resident protein 44; ERp57, ER resident protein 57; Fis1, fission 1 protein; GADD153, growth arrest- and DNA damage-inducible gene 153; GADD34, growth arrest and DNA damage-inducible gene 34; GCN2, general control non-derepressible 2; GM, glioblastoma multiform; GRP75, glucose-regulated protein 75-kDa; GRP78, glucose-regulated protein 78-kDa; HCC, hepatocellular carcinoma cell; HIF1α, hypoxia-inducible factor 1α; HRI, heme-regulated eIF2α kinase inhibitor; IMM, inner mitochondrial membrane; IP3Rs, inositol trisphosphate receptor; IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinases; LDs, lipid droplets; MAMs, mitochondria-associated membranes; MCS, membrane contact sites; MCU, mitochondrial CaGA and VP performed the literature searches and wrote the manuscript. OM gave suggestions and significantly refined the manuscript. PR contributed to the conceptual idea, supervised the writing, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.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.


Importantly, we systematically summarized the diverse functions of Pti1s in tomato, rice, Arabidopsis, potato, apple, and cucumber. The potential cis-elements in promoters of Pti1s decide their functional diversity in response to various biotic and abiotic stresses. The protein kinase Pti1 was phosphorylated by Pto and then modulated the downstream signaling pathways for PTI and ETI in the disease insistence process. In addition, some transcription factors have been defined as Ptis  originally, which actually were ethylene-response factors (ERFs). Pti4, Pti5, and Pti6 were modulated by salicylic acid (SA), jasmonate (JA), and ethylene signaling pathways and regulated diverse defense-related gene expression to cope with Pst infection and insect wounding.Pto interaction (Pti) proteins are a group of proteins that can be phosphorylated by serine/threonine protein kinase Pto, which have diverse functions in plant development and stress response. In this study, we analyzed the phylogenetic relationship, gene structure, and conserved motifs of Pti1s and predicted the potential  Pseudomonas syringae pv. Tomato (Pst)  genes (Pto interaction protein (Pti) refers to the proteins that can directly interact with the resistance (R) protein Pto . Pti maito (Pst) . In Arabo SlPti1 , all of o SlPti1 . Additioo SlPti1 . ERF is R) genes .It has been reported that Pti proteins were critical for plant disease resistance, ROS homeostasis, and diverse abiotic stress responses . In thisArabidopsis thaliana, soybean (Glycine max), rice (Oryza sativa), maize (Zea mays), apple , and cucumber (Cucumis sativus), the protein sequence of SlPti1a in tomato (Solanum lycopersicum) was used as a query and submitted to the BlastP program for searching NCBI database.1 A total of 11 Arabidopsis AtPti1s, four maize ZmPti1s, two rice OsPti1s, three soybean GmPti1s, eight apple MdPti1s, and five cucumber CsPti1s were obtained with default parameters. Taken together with the two tomato SlPti1s, a total of 34 Pti1s were used to perform protein sequence alignment and phylogenetic analyses ; four members of apple MdPti1s, namely, MdPti1-2, MdPti1-4, MdPti1-6, and MdPti1-7; and rice OsPti1b were classified into group I. All cucumber CsPti1s belong to group I except CsPti1-1. GmPti1 belongs to group I, while the other two Pti1s of soybean, namely, sPti1a and sPti1b, belong to group II. Three Pti1s, namely, ZmPti1b, ZmPti1c, and ZmPti1d, belong to group I, while only one Pti1, namely, ZmPti1b, belong to group II. Group II also contained AtPti1-4, AtPti1-8, AtPti1-9, AtPti1-10, and AtPti1-11 of Arabidopsis; MdPti1-1, MdPti1-3, MdPti1-5, and MdPti1-8 of apple; and OsPti1a of rice. Tomato SlPti1a and SlPti1b were distributed in group II.To identify the putative Pti1s in analyses . The phyanalyses . Among t233 in tomato SlPti1 abolished the Pti1-Pto interaction in the yeast two-hybrid system , gibberellin, salicylic acid (SA), auxin, and methyl jasmonate (MeJA)] were overrepresented in the promoters . The subcellular localizations of 11 Ptis from tomato, maize, rice, cucumber, and apple have been detected using the transient expression system in tobacco leaves, onion epidermal cells, tobacco suspension cells, and protoplasts of rice and maize .P. syringae infection  and/or cell periphery, which were involved in ROS generation and resistance in response to nfection . Howevernfection . Interesizations . In addiizations .2, Cys3, Gly2/Cys3, and Cys6/Cys7 in ZmPti1a would affect its localization. The site-directed mutagenesis of the conserved Gly and Cys residues resulted in the migration of PM/cell periphery-localized kinase into the cytoplasm and/or nucleus  gene of the pathogen and the resistance protein encoded by the corresponding resistance (R) gene of plants  using map-based cloning and was identified as a disease resistance gene  plants. Under flg22 and flgII-28 infection, the early ROS production in hpPti1 plants was reduced when compared with the wild-type tomato. The ROS was restored to the same level as it is in wild-type plants after SlPti1a and SlPti1b genes were transiently complemented to hpPti1 plants , OSA7, vacuolar ATP synthase subunit d, and phytochrome-associated phosphatase type 2C (PAPP2C)  was a required downstream factor for most known R proteins  . The resresponse . Importaresponse . When thlization . Moreove(PAPP2C) . HoweverThe infection of pathogenic bacteria usually triggered two relatively independent signals, namely, ROS signal and phosphatidic acid (PA) signal, in plant cells . In the Arabidopsis (GST6 and HSP18.2) in response to oxidative stress , and co-immunoprecipitation (co-IP) detection and was phosphorylated by OXI1 (M. domestica), the MdOXI1-MdPTI1L module was supposed as a “stop” signal upon the pollen tube growth in S-RNase-mediated gametophytic self-incompatibility  and osmotin (Osm)  containing SA-responsive element (SARE) and JA/ethylene-regulated PR genes  containing GCC-box  that do not contain GCC-box, indicating SlPti4 indirectly regulates the downstream gene expression together with other transcription factors (Solanum tuberosum) StPti4 was recruited by and interacted with the repressor silencer element-binding factor (SEBF) to form an inhibitory complex, which binds to the promoter of PR-10a to inhibit its expression  regulated pathways , 2002. Sin (Osm) . Importaresponse . Besideswounding . The het GCC-box . Thus, S GCC-box . Moreove factors . Interespression . This supression .SlPti5 was involved in the resistance to wounding of tomato aphids and Pst infection   . Moreoveatalase) . HoweverSlPti4 RNAi transgenic fruits showed increased ABA level caused by the upregulation of SlNCED1 in ABA biosynthesis at mature green (MG) and breaker (B) stages and the downregulation of SlCYP707A2 in ABA degradation throughout fruits ripening process. Meanwhile, the expression patterns of the ABA receptor family SlPYL genes were varied. Besides, the expression of PYL1, PYL2, PYL5, PYL6, and PYL9 was upregulated during this process except in the B stage, while the expression of PYL4, PYL7, and PYL10 was downregulated in SlPti4-RNAi fruits during ripening. However, the molecular mechanisms of SlPti4-mediated ABA signaling and metabolism upon fruit ripening are still unclear (Tomato SlPti4 participates in fruit ripening and seed germination by regulating ABA metabolism.  unclear .SlPti4-RNAi seeds germinated earlier than seeds of wild type (WT) with ABA treatments. The ABA content and expression of SlNCED1/2 in dry seeds were markedly lower than that in the WT seeds. Moreover, the expression of several SlPYLs  in SlPti4-RNAi seeds was significantly lower than that in the WT seeds. Additionally, the expressions of two representative ABA response genes, namely, SlABI3 and SlABI5, were downregulated in transgenic seeds compared with those in WT seeds under ABA treatments. These indicate that the downregulation of SlPti4 reduced the ABA sensitivity in seeds  were lower than that in the wild type, which indicated that SlPti4 may enhance the drought resistance by regulating ABA content and perception  and ripening-related transcription regulator CNR (colorless non-ripening) increased in the mature green stage of the fruits of SlPti4/5/6-overexpression lines  were phosphorylated by Pto for PTI and ETI in tomato under Pst infection or by OXI1 for the modulation of downstream gene expression in rice and MS and LQ wrote the manuscript with suggestions by ZQ and SD. YL, HZ, YZ, YQ, YM, MZ, and XD performed the bioinformatics analysis. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mycobacterium tuberculosis CAS1-Kili strains that belong to lineage 3  were previously implicated in the spread of multidrug-resistant (MDR)-TB in Lusaka, Zambia. Thus, we investigated recent transmission of those strains by whole-genome sequencing (WGS) with Illumina MiSeq platform. Twelve MDR CAS1-Kili isolates clustered by traditional methods (MIRU-VNTR and spoligotyping) were used. A total of 92% (11/12) of isolates belonged to a cluster (≤12 SNPs) while 50% (6/12) were involved in recent transmission events, as they differed by ≤5 SNPs. All the isolates had KatG Ser315Thr (isoniazid resistance), EmbB Met306 substitutions (ethambutol resistance) and several kinds of rpoB mutations (rifampicin resistance). WGS also revealed compensatory mutations including a novel deletion in embA regulatory region (−35A > del). Several strains shared the same combinations of drug-resistance-associated mutations indicating transmission of MDR strains. Zambian strains belonged to the same clade as Tanzanian, Malawian and European strains, although most of those were pan-drug-susceptible. Hence, complimentary use of WGS to traditional epidemiological methods provides an in-depth insight on transmission and drug resistance patterns which can guide targeted control measures to stop the spread of MDR-TB.Globally, tuberculosis (TB) is a major cause of death due to antimicrobial resistance.  Worldwide, close to 4000 lives in a day are lost due to tuberculosis (TB). It is a major cause of death ascribed to antimicrobial resistance . In ZambMycobacterium tuberculosis, the cause of TB, has seven major human-adapted lineages. One of these is lineage 3 (L3) which is predominant in East Africa, the Middle East and South Asia [uth Asia . Centraluth Asia .6110-based restriction fragment–length polymorphism (RFLP) [M. tuberculosis transmission based on genotypic clustering [M. tuberculosis genome and have limited discriminatory power [Molecular epidemiological methods such as mycobacterial interspersed repetitive units-variable number of tandem repeats   have beeustering ,10,11. Nry power ,13,14. Try power . Furtherry power . Recentlry power ,17.M. tuberculosis strains in Zambia when compared to other southern African countries [M. tuberculosis strains, 24-loci MIRU-VNTR and spoligotyping showed clonal expansion of the SIT21/CAS1-Kili strains [M. tuberculosis SIT21/CAS1-Kili strains in Lusaka, Zambia, by WGS.Previously, we reported a relatively higher percentage of lineage 3 Spoligotype International Type (SIT) 21/CAS1-Kili strains among multidrug-resistant (MDR) ountries . Despiteountries ,19. Amon strains . The finAmong our Zambian strains, 11/12 (92%) differed by not more than 12 SNPs, while 6 (50%) strains differed with at least one other strain by 5 or fewer SNPs. Strain 7 was closely related to strains 4, 5, and 11. Additionally, strain 7 had the lowest average SNP difference of 9 SNPs to other strains, as shown in embA, and three of these were phenotypically resistant to ethambutol. Additionally, all the 12 strains had resistance-associated mutations to streptomycin. Of these twelve strains, four and one had mutations in rpsL and rrs genes, respectively, and all were resistant to streptomycin, while 7/12 had mutations in gid and only two out of those seven were resistant to streptomycin. Overall, strains had different mutations towards rifampicin resistance while all RpoB Ser450Leu mutants had compensatory mutations in RpoC Val483 . With the exception of one strain with EmbB Met306Leu  (Most of the lineage 3.1.1 (SIT21/CAS1-Kili) strains were from Europe, 52% (250/480), followed by Africa, 48% (228/480) . Zambian< 0.000) .M. tuberculosis strains were previously shown to be associated with the clonal spread of MDR-TB in the Lusaka District [The SIT21/CAS1-Kili District ,19. In tOf the 12 evaluated SIT21/CAS1-Kili strains, 11 belonged to a cluster, as they differed by 12 or fewer single nucleotide polymorphisms (SNPs) in their core genomes, while 6 strains were involved in recent transmission events as they differed by ≤5 SNPs to at least one other strain. Therefore, WGS revealed that the SIT21/CAS1-Kili strains were closely related as they exhibited comparatively low variabilities in SNPs. In addition, several strains shared the same combinations of drug-resistance-associated mutations to isoniazid, ethambutol, rifampicin, and other anti-TB drugs. This provided more evidence on the clonal spread of MDR SIT21/CAS1-Kili strains in this region, calling for interventions to stop this possible outbreak.rpoB, of which 33% (4/12) of strains with RpoB Ser450Leu mutation also had compensatory mutations in RpoC, Val483Ala (2/4) and Val483Gly (2/4). These compensatory mutations have been implicated in mitigating the fitness defects caused by RpoB Ser450Leu substitution in M. tuberculosis [rpoB  and rpoC  which were suspected to be compensatory as they occurred in association with a known mutation, Asp435 deletion (4/12), in the rpoB rifampicin resistance determining region (RRDR). However, this can only be confirmed by allelic exchange experiments.Despite some reports indicating the reduced fitness as a result of drug resistance evolution ,21, otherculosis  which inembA gene regulatory region (−35 A > deletion), whilst one other strain (strain 3) had −16 C > T and −16 C > G nucleotide variants in embA, which was suggestive of ongoing evolution within the strain. All the 12 MDR strains had mutations in codon embB306 which has been associated with isoniazid resistance [embB306 mutations have been reported to reduce susceptibility of M. tuberculosis to other drugs used in the treatment regimen [embB306 as a marker for broader drug resistance than ethambutol resistance [EmbB306) then independently became resistant to other drugs, rifampicin, streptomycin, and pyrazinamide, while propagating to the next patients  of strains had mutations in more than a single resistance-conferring gene to ethambutol. The phenomenon has been reported to be associated with high resistance to ethambutol ,26,27,28sistance , MDR, an regimen . Hence, sistance . Therefopatients . As a restant TB ,32.embB306, which is associated with the majority of drug-resistant strains to ethambutol, have also been detected in susceptible strains [gid to the drug, which may result in susceptible phenotype [rrs and rpsL genes have been associated with intermediate to high [Even though some strains were resistant to all the first-line anti-TB drugs and streptomycin, none had resistance mutations to other second-line drugs. This at least left some treatment options for these patients, though on the other hand, calls for improved resistance detection and individualized treatment. The observed discrepancy in phenotypic and genotypic drug susceptibility patterns for ethambutol can be attributed to lack of consistency of phenotypic DST to ethambutol resistance determination as indicated by several reports ,30. Muta strains , whilst henotype . Mutatio to high  and high to high ,34,35,36 to high .Phylogeny showed that some strains involved in recent transmission events were from patients residing in different districts  and Lusaka City . This coFurther, some previously identical strains by MIRU-VNTR and spoligotyping were delineated as unique strains by WGS, indicating its supremacy to traditional genotyping methods ,38. ThouGenerally, the global SIT21/CAS1-Kili L3.1.1) strains were pan-susceptible to anti-TB drugs a. Theref.1.1 straThe study limitations included small sample size due to poor quality DNA for some samples and inadequate patient history to precisely infer the direction for person-to-person transmission. In addition, a larger sample size with a longer collection period of the strains would have facilitated the determination of the emergency of MDR-TB and its hotspots in Lusaka. Additionally, the sample size also affected the generalization of these results. Therefore, we recommend MDR-TB surveillance on the large scale, whenever possible, to provide more information on the spread of this strain and other genotypes in the community, which can inform policy.We used strains from the previous study conducted in Lusaka from 2013 to 2017 . In the Briefly, sub-culturing of the isolates was performed in BACTEC™ 960 MGIT™ (Mycobacteria Growth Indicator Tube) system  following the manufacturer’s instructions. The critical concentrations for isoniazid, rifampicin, streptomycin, and ethambutol, as prescribed by the kit manufacturer , of 0.1, 1.0, 1.0, and 5.0 g/mL were used, respectively.DNA was extracted by heating method as described previously ,19. The Library preparations were performed following Nextera XT DNA Library Preparation Kit  manufacturer’s instructions, and Illumina MiSeq 2500 platform was used for sequencing.https://www.geneious.com (accessed on 16 December 2021). The mean coverages of the analyzed reads for 12 CAS1-Kili strains ranged from 35.73 to 139.32 (WGS data analysis was carried out as previously described ,43. The o 139.32 . One strM. tuberculosis BED mask file included in Snippy-package. Further, Gubbins generated filtered polymorphic sites and a maximum likelihood phylogenetic tree rooted to M. canetti. The phylogenetic tree was trimmed of M. canetti but topology maintained, for better visualization using ggtree [Sub-lineage 3.1.1 strain’s accession numbers were obtained from TB-Profiler, projects PRJEB29435  and PRJEg ggtree ,53,54,55g ggtree  and WilcThe study revealed ongoing transmission of MDR SIT21/CAS1-Kili strains in Lusaka, Zambia, which is a public health concern and needs more evaluation. It also showed the high resolution of WGS in delineating closely related strains and determining antibiotic resistance. Further, the study supports the complimentary use of WGS with traditional methods. Intensified case finding, improved drug resistance detection and adherence to treatment can help interrupt the transmission chains.


Cervical cancer is a common female malignancy that is mainly caused by human papillomavirus (HPV) infection. However, the incidence of HPV-negative cervical cancer has shown an increasing trend in recent years. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative cervical cancer and verify the underlying potential mechanism. Differentially expressed genes were compared among HPV-positive cervical cancer, HPV-negative cervical cancer, and normal cervical tissues retrieved from TCGA. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical cancer tissues and HPV-negative cell lines were validated by qRT-PCR, western blotting, and immunohistochemical staining. We found seventeen highly expressed genes that were particularly associated with HPV-negative cervical cancer from analysis of TCGA database. Among the 17 novel genes, 7 genes  displayed significantly elevated expression in HPV-negative cervical cancer cells and HPV-negative cervical cancer tissues. Additionally, higher expression of MEX3A and TTYH3 was associated with a shorter overall survival of patients with HPV-negative cervical cancer. Our study implies that these seven genes are more likely to provide novel insights into the occurrence and progression of HPV-negative cervical cancer.  The mechanisms of HPV-negative cervical cancer are largely elusive. In this study, the authors discovered the multiple genes in HPV-negative cervical cancer, including PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, and TTYH3, which could contribute to HPV-negative cervical cancer development. Eleven genes showed significantly elevated mRNA expression in C33A cells compared to SiHa, Caski, MS751, HeLa, and Ect1 cells. The 11 significantly expressed genes were PRAME, HMGA2, SBK1, ETV4, MEX3A, PLS1, TM7SF2, PYCR1, SLC19A1, TTYH3, and NIPSNAP1 A. HoweveWe assessed the protein levels of these 11 genes  in HPV-negative and HPV-positive cervical cancer cell lines using western blotting. The protein levels of eight specific genes, PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, TTYH3, and NIPSNAP1, were higher in C33A cells than in SiHa, Caski, MS751, HeLa, and Ect1 cells . HoweverWe conducted IHC staining to further validate differences in the protein expression of 11 specific genes, PRAME, HMGA2, SBK1, ETV4, MEX3A, PLS1, TM7SF2, PYCR1, SLC19A1, TTYH3, and NIPSNAP1, in HPV-negative cervical cancer in this study. The mean IHC scores for these 11 genes in HPV-negative cervical cancer, HPV-positive cervical cancer, and normal cervical tissues were shown in Finally, the prognostic value of the 11 upregulated genes  specifically associated with HPV-negative cervical cancer was assessed by performing a Kaplan-Meier analysis of patients with HPV-negative cervical cancer, patients with HPV-positive cervical cancer, and all patients with cervical cancer. Higher expression of MEX3A was associated with shorter overall survival only in patients with HPV-negative cervical cancer (p = 0.04) but was not associated with the survival of patients with HPV-positive cervical cancer and all patients with cervical cancer . AdditioHPV DNA detection for cervical cancer screening is increasingly used as an early diagnostic guideline to prevent the progression of cervical cancer. Nevertheless, an ongoing need exists for studies investigating the molecular mechanisms related to prevent the development of cervical cancer. Recently, some lncRNAs were reported to be downregulated in HPV-negative cervical cancer. For example, the lncRNA snaR was downregulated in patients with HPV-negative cervical squamous cell carcinoma.In the present study, we first compared changes in gene expression between HPV-negative cervical cancer and normal cervical tissues, as well as between HPV-positive cervical cancer and normal cervical tissues to identify differentially expressed genes. Then, we further compared the genes that were expressed at significantly higher levels in HPV-negative cervical cancer tissues than in HPV-positive cervical cancer tissues to discover genes that were specifically expressed at higher levels in HPV-negative cervical cancer. Ultimately, 17 genes specifically upregulated in HPV-negative cervical cancer were found. A subsequent multiplatform integrative analysis revealed that 11 genes, PRAME, HMGA2, SBK1, ETV4, MEX3A, PLS1, TM7SF2, PYCR1, SLC19A1, TTYH3, and NIPSNAP1, displayed upregulated mRNA levels, and 8 specific genes, PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, TTYH3, and NIPSNAP1, displayed upregulated protein levels in HPV-negative cervical cancer cells compared to HPV-positive cervical cancer cells and normal cervical squamous cells. Consistent with these findings, PRAME, HMGA2, SBK1, MEX3A, TM7SF2, SLC19A1, ETV4, PYCR1, and TTYH3 showed higher expression in HPV-negative cervical cancer tissues than in HPV-positive and normal cervical tissues. Accordingly, 7 genes, PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, and TTYH3, displayed consistent results in terms of mRNA and protein levels obtained using qPCR, western blotting, and IHC methods, as they showed significantly higher expression in HPV-negative cervical cancer cells and tissues than in HPV-positive cervical cancer and normal cells or tissues, implying that these 7 genes are more likely to play pivotal roles in the development of HPV-negative cervical cancer. Furthermore, high expression of MEX3A and TTYH3 was also related to shorter overall survival of patients with HPV-negative cervical cancer, which confirmed the important prognostic value of MEX3A and TTYH3 expression in patients with HPV-negative cervical cancer.,PRAME, a testis-selective cancer testis antigen (CTA), is associated with cell proliferation, apoptosis, differentiation, and the outcomes and risk of metastasis in human cancers.The architectural transcription factor HMGA2 is a nonhistone DNA-binding factor that tightly binds to adenine and thymine (AT)-rich sequences in the minor groove of the DNA helix.,,in vitro and in vivo.ETV4, a member of the PEA3 subgroup of the ETS transcription factor family,TM7SF2 is a human sterol reductase with a function that remains mysterious in human cancers.,SLC19A1, a folate-organic phosphate antiporter, serves as the major transporter of cyclic dinucleotides, and its depletion and overexpression affect cyclic dinucleotide uptake and functional responses, which has implications for the immunotherapeutic treatment of cancer via cGAS-STING pathway.The RNA-binding ubiquitin ligase MEX3A is overexpressed in glioblastoma multiforme specimens, and it targets retinoic acid inducible gene-I (RIG-I) for ubiquitylation and degradation to alter the tumorigenesis of glioblastoma multiforme.+-activated chloride channel that acts as the major volume-regulated anion channel (VRAC) in astrocytes, together with TTYH1/2.,TTYH3 in gastric cancer was associated with shorter survival.The TTYH family, chloride-channel-responsive genes, is involved in cell adhesion, cell division, tumorigenesis, and regulation of calcium signaling.In conclusion, to our knowledge, this study is the first to systematically analyze, examine, and disclose differentially expressed genes that are specifically expressed in patients with HPV-negative cervical cancer. Our results firmly identified the significantly increased expression of 8 novel genes at the mRNA and protein levels, including PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, TTYH3, and NIPSNAP1, in HPV-negative cervical carcinoma cell lines compared to HPV-positive cell lines and a normal cell line. Furthermore, significantly higher expression of PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, and TTYH3 was also detected in HPV-negative cervical cancer tissues, implying that these seven genes may serve as new biomarkers for HPV-negative cervical cancer and play pivotal roles in the occurrence and development of HPV-negaive cervical cancer. In addition, MEX3A and TTYH3 correlated with shorter overall survival of patients with HPV-negative cervical cancer, suggesting that the upregulation of MEX3A and TTYH3 were not only associated with the development of HPV-negative cervical cancer but also specifically involved in the prognosis of HPV-negative cervical cancer. In the future, further studies and more analyses will be performed to extensively investigate the effects of changes in the expression of these genes, especially MEX3A and TTYH3, on the biological behavior, chemotherapy sensitivity, related internal mechanisms, and signaling pathways specifically associated with HPV-negative cervical carcinoma to provide new insights into the early screening and effective treatment of HPV-negative cervical carcinoma.https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga. Three hundred five samples of cervical cancer and 3 normal cervical tissues from healthy controls were analyzed; among the cervical cancer tissues, 282 were from patients with a known HPV infection and 23 were from HPV-negative patients.Publicly available data from the TCGA database were downloaded on March 15, 2018 from The gene expression data were obtained from TCGA public level 2 transcription profiles as raw count values. R packages (edgeR) were applied to the transcription profiles, and differentially expressed genes between HPV-positive cervical cancer and normal cervical tissues and between the HPV-negative cervical cancer and normal cervical samples were analyzed. Compared to the normal cervix, differentially expressed genes in HPV-negative or HPV-positive cervical cancer were divided into four separate groups, including upregulated genes expressed in HPV-negative cervical cancer, downregulated genes expressed in HPV-negative cervical cancer, upregulated genes expressed in HPV-positive cervical cancer, and downregulated genes expressed in HPV-positive cervical cancer. Then, we compared the upregulated genes expressed in HPV-negative and HPV-positive cervical cancer, and two non-intersecting groups and one intersecting group were obtained, which represent upregulated genes expressed specifically in HPV-negative or -positive cervical cancer and upregulated genes expressed in both HPV-negative and HPV-positive cervical cancer, respectively. Using the same method, we compared downregulated genes expressed in HPV-negative and HPV-positive cervical cancer, and two other non-intersecting groups  and an intersecting group  were obtained. Finally, upregulated differentially expressed genes specifically detected in HPV-negative cervical cancer were identified.2 for growth.The normal cervical squamous cell line Ect1 was used, in addition to the human cervical carcinoma cell lines, C33A (HPV-negative) and SiHa, Caski, MS751, and HeLa (HPV-positive). Cells were purchased from the European Collection of Authenticated Cell Cultures and cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum at 37°C in an atmosphere of 5% CO−ΔΔCt method. All PCR reactions were performed in triplicate. The primers for the tested genes are listed in TRIzol reagent  was used to completely lyse and extract total RNA from HPV-positive, HPV-negative, and normal cells. The total RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated deionized water and quantified with a spectrophotometer at 260 nm (A260). RNA gel electrophoresis was conducted to examine DNA contamination. 1 μg RNA was used for reverse transcription reactions by TransStart Top Green qPCR Supermix (TransGen Biotech). The cDNA templates were synthesized, and qPCR was conducted using the SYBR Green Real-Time PCR Master mix . The following thermocycling conditions were used: 95°C for 55 s followed by 40 cycles of 95°C for 10 s and 57°C for 30 s. Then, gene expression was determined using a LightCycler 480 II real-time PCR (Roche) instrument and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2The primary antibodies included PRAME, HMGA2, SBK1, ETV4, MEX3A, PLS1, TM7SF2, PYCR1, SLC19A1, TTYH3, NIPSNAP1 GAPDH, and tubulin . After the detection of HPV within 1 month before the surgery, 20 HPV-negative and 20 HPV-positive cervical cancer tissues were collected and identified. Furthermore, another 20 normal cervical tissues were obtained from patients who underwent radical hysterectomy due to uterine adenomyosis, which was pathologically confirmed as normal cervical tissue or chronic cervicitis. Patients did not receive chemotherapy or radiotherapy before surgery. IHC was performed in cancer tissues using a previously described method.https://cran.r-project.org/web/packages/survival/citation.html) to assess independent predictors of overall survival. Three groups were tested: 22 patients with HPV-negative cervical cancer; 281 patients with HPV-positive cervical cancer; and 304 patients with cervical cancer (one person did not have a determined HPV infection history). All survival information of cervical cancer patients was obtained from the TCGA database. The mRNA expression status separated the cases into “low” and “high” according to the comparison between expression values with median cutoffs values. The log rank test was applied to detect significant differences between groups.The Kaplan-Meier analysis was performed by the R package (The expression levels of differentially expressed genes obtained from gene expression analysis section were validated by qRT-PCR for transcriptional levels and by western blotting for translational levels in HPV-negative cervical cancer cell lines. Moreover, IHC was used to measure the protein expression of these genes in HPV-positive cervical cancer tissues compared with HPV-negative cervical cancer tissues and normal control tissues .Table 3UData were analyzed with GraphPad Prism 8.0 software. The significance of differences was determined using unpaired t tests with Welch’s correction for RT-PCR, western blotting, and IHC experiments. Data are presented as the means ± standard deviations. The log rank test was applied to detect significant differences between groups for Kaplan-Meier analysis. p <0.05 was considered statistically significant. It is important to note that less numbers of HPV-negative cervical cancer cases and only 3 normal cervical tissues of TCGA database may compromise the data in our study.


Long non-coding RNAs (lncRNAs) are single-stranded RNA biomolecules with a length of >200 nt, and they are currently considered to be master regulators of many pathological processes. Recent publications have shown that lncRNAs play important roles in the pathogenesis and progression of insulin resistance (IR) and glucose homeostasis by regulating inflammatory and lipogenic processes. lncRNAs regulate gene expression by binding to other non-coding RNAs, mRNAs, proteins, and DNA. In recent years, several mechanisms have been reported to explain the key roles of lncRNAs in the development of IR, including metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), imprinted maternal-ly expressed transcript (H19), maternally expressed gene 3 (MEG3), myocardial infarction-associated transcript (MIAT), and steroid receptor RNA activator (SRA), HOX transcript antisense RNA (HOTAIR), and downregulated Expression-Related Hexose/Glucose Transport Enhancer (DREH). LncRNAs participate in the regulation of lipid and carbohydrate metabolism, the inflammatory process, and oxidative stress through different pathways, such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), polypyrimidine tract-binding protein 1/element-binding transcription factor 1c (PTBP1/SREBP-1c), AKT/nitric oxide synthase (eNOS), AKT/forkhead box O1 (FoxO1), and tumor necrosis factor-alpha (TNF-α)/c-Jun-N-terminal kinases (JNK). On the other hand, the mechanisms linked to the molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR. Therefore, it is essential to elucidate the role of lncRNAs in the insulin signaling pathway and glucose and lipid metabolism. This review analyzes the function and molecular mechanisms of lncRNAs involved in the development of IR. Insulin resistance (IR) is a clinical condition defined as a reduced metabolic response to the action of insulin on target tissues, which are unable to coordinate a normal glucose-lowering response involving the suppression of endogenous glucose production, the suppression of lipolysis, cellular uptake of available plasma glucose, and glycogen synthesis. IR is a critical component of metabolic syndrome, obesity, and hyperlipidemia and is a risk factor for cardiovascular diseases (CVDs) and type 2 diabetes (T2D), especially when an insulin-resistant individual cannot secrete enough insulin from pancreatic β-cells ,2. SeverSeveral factors, such as proinflammatory cytokines, saturated fatty acids (SFAs), amino acids, endothelin 1, angiotensin II, and hyperinsulinemia, increase kinase activity ,4. CelluPPAR) gamma, Kruppel-like factor 14 (KLF14), insulin receptor substrate 1 (IRS1), glucokinase regulator (GCKR), transcription factor 7-like 2 (TCF7L2), N-acetyltransferase (NAT) 1 and 2, transmembrane protein 163 (TMEM163), insulin-like growth factor (IGF1) 1, melanocortin 4 receptor (MC4R), transcription elongation regulator 1-like (TCERG1L), and sterol-C4-methyl oxidase-like (SC4MOL) . Ho. Ho15]. H19-IGF2 locus, a highly conserved imprint located on human chromosome 11p15.5, where these two genes are reciprocally imprinted. The H19 gene comprises five exons separated by small introns and produces a spliced, capped, and polyadenylated RNA that mainly resides in the cytoplasm ..59].Non-alcoholic fatty liver disease, a consequence of the high production and accumulation of lipids in the liver, is considered a key factor in the development of IR and T2D . A mechaSREBP-1c, ACC1, and fatty acid synthase (FAS) ..74].PGC-1α, PEPCK, and G6PC through the CREB–CREB-regulated transcriptional coactivator (CRTC2) complex, which contributes to the pathogenesis of hepatic gluconeogenesis, suggesting that MEG3 acts as a ceRNA to upregulate CRTC2 via miR-302a-3p  mice. Moreover, high fat enhanced MEG3 expression in hepatocytes through histone acetylation. MEG3 overexpression significantly increased FoxO1, G6PC, and PCK1 expression and hepatic gluconeogenesis and suppressed insulin-stimulated glycogen synthesis in primary hepatocytes, which led to increased hepatic IR . Subsequ-302a-3p  77]..PGC-1α, Furthermore, in a study carried out in samples of SAT and VAT adipose tissue from women, MEG3 expression in SAT had a significant relationship with obesity indices. In addition, MEG3 expression in SAT was positively correlated with PPARγ and FAS expression , suggestAdditionally, significant MEG3 overexpression has been reported in patients with T2D, and this overexpression was positively correlated with poor glycemic control, IR, transcriptional markers of senescence, and inflammation . Liu et MIAT is also known as Gomafu or retinal non-coding RNA 2; its gene is located on the short arm of human chromosome 22q12.1. MIAT contains multiple spliced exons, with a final transcript size of approximately 10,000 nucleotides, which is polyadenylated; it is not exported to the cytoplasm and is concentrated in the cell nucleus . MIAT caAs in most lncRNAs, the ceRNA function is a molecular mechanism through which MIAT exerts its pathogenic effects. In this regard, MIAT decreases the levels of miR-29c, resulting in an increase in lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes the oxidative deamination of lysine and hydroxylysine residues in tropocollagen and tropoelastin . LOXL2 iYing et al. reported that a reduction in miR-150 increases the plasma levels of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, with the subsequent activation of JNK; these events could participate in the development of IR . HepaticSRA is a type of lncRNA that coordinates the functions of various transcription factors, enhances steroid receptor-dependent gene expression, and serves as a distinct scaffold for corepressor complexes. SRA functions as an RNA coactivator for several nuclear hormone receptors, including androgen, estrogen, progesterone, glucocorticoid, thyroid hormone, retinoic acid, and vitamin D receptors ,96,97, aInterestingly, SRA is highly expressed in white adipose tissue (WAT) and functions as a transcriptional coactivator of PPARγ in in vitro models, suggesting that adipogenesis and adipocyte function can be affected by the SRA/PPARγ axis. Moreover, SRA increases insulin-stimulated glucose uptake and the phosphorylation of the downstream targets AKT and FoxO1 in adipose tissue . FurtherHOX transcript antisense RNA (HOTAIR) is a 2.2 kb lncRNA that is generated from the transcription of the antisense strand of the HOXC gene located on human chromosome 12q13.13. HOTAIR induces gene silencing by interacting with gene silencing machinery. The 5’-end of HOTAIR interacts with PRC2, and its 3’-end interacts with lysine-specific demethylase 1 (LSD1). The recruits of PRC2 and LSD1 by HOX transcript antisense RNA (HOTAIR) result in the methylation of histone H3 on lysine 27 and demethylation of H3 on lysine 4, which leads to gene silencing ,102,103.Li et al. investigated the role of HOTAIR in hepatic IR; their results showed that HOTAIR was overexpressed in patients with T2D compared with normal subjects, and animals on a high-fat diet or with diabetes showed the same upregulation of HOTAIR. Moreover, HOTAIR overexpression in HepG2 cells was associated with the elevated expression of PCK and G6PC genes and the subsequent reduction in glycogen levels and an increase in glucose delivery. In addition, the overexpression of HOTAIR in HepG2 cells decreased the phosphorylation levels of AKT and glycogen synthase kinase 3 beta (GSK3β) after insulin stimulation. Finally, the author suggested that overexpression of HOTAIR decreases the levels of SIRT1, and the overexpression of SIRT1 decreases the insulin-resistant state originating in HepG2 cells by the overexpression of HOTAIR .HOTAIR overexpression in the serum of patients with diabetic retinopathy, along with a decrease in miR-20b expression, was reported. In addition, miR-120b was observed to be negatively correlated with poor glycemic control. HOTAIR was found to have complementary sequences to miR-20b . MajumdeIn contrast, Gao et al. found that HOTAIR was downregulated, whereas, as a target of HOTAIR, miR-34a was upregulated in the heart of diabetic mice and in high-glucose-treated H9c2 cells. Additionally, HOTAIR overexpression has been shown to protect against inflammation and oxidative stress induced by diabetes via the HOTAIR/miR-34a/SIRT1 axis . In anotGrowth arrest-specific 5 (GAS5), whose gene is located on chromosome 1q25.1, was first characterized as non-coding lncRNA by Coccia et al., followed by Raho et al. ,112. IntGm15622 was significantly enriched in the liver and overexpressed due to an HFD. This overexpression favored the accumulation of lipids and liver steatosis in obese mice as a consequence of feeding an HFD. This lncRNA plays a vital role in the expression of lipid synthesis genes such as FAS. Gm15622 regulates SREBP-1c expression by competing for the binding of miR-742-3p in alpha mouse liver 12 (AML-12) cells. In other words, Gm15622 can act as a molecular sponge or ceRNA for miR-742-3p. Interestingly, metformin inhibits FAS and SREBP-1c expression by targeting Gm15622. Thus, metformin exerts its effect by regulating a network of genes involving Gm15622, miR-742-3p, and SREBP-1c . HoweverRegulator of insulin sensitivity and autophagy (Risa) or AK044604 is a potential lncRNA identified in different tissues, including the heart, kidney, liver, lung, muscle, spleen, thyroid, and white adipose tissue. Risa is located at the 10qB4 region close to the locus of SIRT1, an essential regulator of insulin sensitivity, autophagy, and metabolism. Risa is a polyadenylated lncRNA, and its overexpression significantly reduced the insulin-induced phosphorylation level of the insulin receptor, AKT, and GSK3β, which suggests that the overexpression of Risa induces IR . AlthougDownregulated expression-related hexose/glucose transport enhancer (DREH) was initially named for its downregulated expression by hepatitis B virus X protein (HBx). DREH is an lncRNA in HBx transgenic mice . AlthougAs previously described, several mechanisms have been reported to explain the key role of lncRNAs in the development of IR. However, studies have identified several molecular targets for only a few lncRNAs described in this manuscript. On the other hand, the mechanisms linked to molecular, cellular, and biochemical actions of lncRNAs vary according to the tissue, biological species, and the severity of IR . Table 1Insulin resistance is a multifactorial disease associated with metabolic syndrome and is considered a risk factor for the development of T2D, dyslipidemias, obesity, and cardiovascular diseases. Environmental risk factors, such as a sedentary lifestyle, stress, and malnutrition, and genetic factors, such as single-nucleotide polymorphisms (SNPs) and copy number variation, can contribute to the development of IR. However, in recent years, non-coding RNAs, including miRNAs and lncRNAs, have acquired increased relevance, the latter being the most clinically important because they are attributed various functions, such as acting as master regulators of different genetic, epigenetic, and metabolic processes, under normal physiological conditions and in metabolic disorders related to diseases. Various authors have associated lncRNAs, such as MIAT, MALAT1, H19, MEG3, and HOTAIR, with inflammatory and lipogenic processes that can lead to IR development through interactions with mRNAs, miRNAs, transcription factors, and proteins. However, a direct relationship between lncRNAs and the insulin signaling pathway or glucose metabolism has not yet been identified. Most of the presented results are from in vivo and in vitro models, and studies in patients need to be conducted to demonstrate the utility of these molecules by generating and implementing panels of the lncRNAs described here. In conclusion, it is essential to continue conducting research that clarifies the direct involvement of lncRNAs in the molecular mechanisms that contribute to IR and their molecular interaction networks that regulate insulin signaling pathways. In addition, it is necessary to demonstrate the usefulness of these biomolecules as potential biomarkers for the diagnosis and prognosis of the development of IR, as well as their potential application as therapeutic targets.


Phenuiviridae in the order Bunyavirales and possess negative-sense single-stranded RNA genomes. In this study, the genome sequence of three ARWV-2 isolates  and a CiVA isolate (CiVA-P) infecting pear trees grown in China were characterized using high-throughput sequencing combined with conventional reverse-transcription PCR (RT-PCR) assays. The genome-wide nt sequence identities were above 93.6% among the ARWV-2 isolates and above 93% among CiVA isolates. Sequence comparisons showed that sequence diversity occurred in the 5′ untranslated region of the ARWV-2 genome and the intergenic region of the CiVA genome. For the first time, this study revealed that ARWV-2 proteins Ma and Mb displayed a plasmodesma subcellular localization, and the MP of CiVA locates in cell periphery and can interact with the viral NP in bimolecular fluorescence complementation assays. RT-PCR tests disclosed that ARWV-2 widely occurs, while CiVA has a low incidence in pear trees grown in China. This study presents the first complete genome sequences and incidences of ARWV-2 and CiVA from pear trees and the obtained results extend our knowledge of the viral pathogens of pear grown in China.Apple rubbery wood virus 2 (ARWV-2) and citrus virus A (CiVA) belong to a recently approved family  Bunyavirales order, the two viruses with species names Apple rubodvirus 1 and Apple rubodvirus 2 are assigned to the genus Rubodvirus in the recently approved family Phenuiviridae in the Bunyavirales order. In addition, the genus Rubodvirus contains two other virus species Grapevine rubodvirus 1 and Grapevine rubodvirus 2 identified from grapevine [Apple rubbery wood virus 1 (ARWV-1) together with apple rubbery wood virus 2 (ARWV-2) were discovered in apple trees showing rubbery wood disease symptoms in Germany and USA [rapevine ,4, and Arapevine  and applrapevine . ARWV-1 rapevine .Coguvirus eburi has been established for CiVA, and the virus belongs to genus Coguvirus in the recently approved family Phenuiviridae [Coguvirus contains three species, including the type species Citrus coguvirus (the species name of CCGaV), Coguvirus eburi, and Grapevine coguvirus  [Citrus virus A (CiVA) was described in a non-symptomatic, field-grown sweet orange tree in southern Italy . The CiViviridae . The genGaCLV-1) ,10. Now,GaCLV-1) , and is GaCLV-1) ,13. A reGaCLV-1) .Pyrus spp.) is widely grown in China. During field investigations, viral-disease-like symptoms are frequently observed on pear trees grown in China. In a recent study, we molecularly characterized a pathogenic emaravirus named pear chlorotic leaf spot associated virus (PCLSaV), which is associated with pear chlorotic leaf spot (PCLS) disease [Pear  from a eviously , was incsymptoms . These fhttp://www.ncbi.nlm.nih.gov, accessed on 16 October 2019) using BlastX and BlastN programs.The raw RNA reads from the Illumina platform were trimmed of adaptor sequences and filtered for low-quality reads using FASTP version 1.5.6 to remove adapter sequences and reads with more than 5% Ns or with 20% base quality values (Q20) less than 20. Then, the obtained RNA reads from each sample were de novo assembled into larger contigs using Velvet version 1.2.08  with a kFor the amplification of the CiVA genomic RNAs, cDNA templates were generated using random primer pd(N)6. Specific primers  were desThe terminal regions of the genomic RNAs of CiVA and ARWV-2 were determined by the RACE strategy using a commercial kit  according to the manufacturer’s instructions. For 3′ RACE, poly (A) tails were added to the 3′ ends of the total RNAs by using the poly (A) polymerase kit , and cDNA was generated using the oligo (dT) primer provided in the 3′ RACE kit. For 5′ RACE, specific reverse primers were designed based on the nucleotide sequences conserved at the 5′ termini of the genomic RNAs of each virus .RT-PCR solutions and conditions were similar to those reported previously , except https://www.ncbi.nlm.nih.gov/orffinder, accessed on 12 July 2021) with minimal ORF length of 75 nt. Multiple sequence alignments and identity analyses were performed using the Muscle method in MEGA 7.0 [Prediction of ORFs was performed using the NCBI ORFFinder program  were done as described above. For the efficient detection of ARWV-2, a nested RT-PCR (RT-nPCR) method was developed by using SaF1/SaR1 as an outer primer set and NP1-F/NP1-R as an inner primer set. The two sets of primers were designed in the sequence of ARWV-2 Sa segment . The RT-attB recombination sequence (Two ORFs (without stop codons) coding for Ma and Mb of ARWV-2 isolate LYXS, and ORFs (without stop codons) coding for NP and MP of CiVA were amplified using gene-specific primers flanked with an sequence . The conAgrobacterium tumefaciens strain GV3101 , and agro-infiltrated into leaves of Nicotiana benthamiana plants (5 weeks old). The CMV3a-mCherry and H2B-mCherry vectors were used as plasmodesma (PD) and nuclear markers, respectively. Fluorescence signals of fusion proteins transiently expressed in infiltrated leaves of N. benthamiana plants were viewed at 2 days post infiltration (dpi) using confocal laser scanning microscopy  with an HC PL APO CS2 63×/1.20 WATER objective.Recombinant plasmids were individually transformed into Totally, 87,218,776, 69,655,848, 76,050,182, 83,312,860, and 124,323,162 clean reads were obtained from samples S17E2, LYC2, LYXS, FJCG, and FJHH, respectively. BlastN and BlastX searches against the NCBI database using assembled contigs revealed plant viruses presenting in these samples. From samples S17E2 and LYC2, three contigs with lengths of 381–1499 bp and six contigs with lengths of 261–475 bp matched sequences of ARWV-2 genomic RNAs named L, Ma, and Sa. From sample LYXS, six contigs with lengths of 282–7367 bp matched sequences of genomic RNA segments L, Ma, Mb, Sa, and Sb of ARWV-2 isolates 982-11, R7, and R12. From sample FJCG, two contigs with lengths of 6703 bp and 2765 bp covering near 95% of CiVA genomic RNA1 and RNA2 were identified. From sample FJHH, five contigs with lengths of 406–4597 bp matched sequences of CiVA genomic RNA1 and RNA2. Meanwhile, two well-documented viruses, apple stem pitting virus (ASPV) and apple chlorotic leafspot virus (ACLSV), were identified in the sample S17E2, and apple stem grooving virus (ASGV) and ASPV were identified in the samples LYC2 and LYXS. The specimens FJCG and FJHH were infected with ASPV, ASGV, and ACLSV. Here, we considered only the two -ssRNA viruses ARWV-2 and CiVA for further analyzes. The identified contigs of ARWV-2 and CiVA are presented in Complete and near-complete sequences of ARWV-2 genomic RNAs from the three samples S17E2, LYC2, and LYXS, and complete genomic sequences (GenBank accession numbers: MZ819702–MZ819703) of CiVA from the sample FJCG were reconstructed by Sanger sequencing of RT-PCR products. The reconstructed genomic sequences showed over 99% identities with corresponding contig sequences generated from RNA assembly, indicating that the assembled contig sequences from RNA-Seq data were reliable.The genome structures of ARWV-2 determined from samples S17E2 and LYC2, here named isolates S17E2 and LYC2, were identical to those of reported isolates BR-Gala, 355-1, and R7, and consisted of three RNA segments L, Ma, and Sa. The genome structure of ARWV-2 from sample LYXS, here named isolate LYXS, was identical to that of reported isolate R12, and consisted of five RNA segments L, Ma, Mb, Sa, and Sb. Complete sequences of L, Ma, and Sa segments of isolate S17E2 were 7370 nt, 1594 nt, and 1486 nt (GenBank accession numbers: MN163133–MN163135). For isolate LYC2, a near full-length L sequence , a full-length Ma sequence , and a near full-length Sa sequence  (GenBank accession numbers: MZ819704–MZ819706) were determined. For isolate LYXS, a near full-length L sequence  and Ma sequence , a full-length Mb sequence (1602 nt), a full-length Sa sequence (1510 nt), and a near full-length Sb sequence  (GenBank accession numbers: MZ819707–MZ819711) were determined. The obtained sequences, together with the corresponding sequences of reported ARWV-2 isolates, were comparatively analyzed . The genNeighbor-joining (NJ) trees constructed using the nucleotide sequences of the viral RNA segment L showed that the three ARWV-2 isolates from Chinese pear samples clustered into a large clade with the previously reported seven ARWV-2 isolates, and one isolate H2803 (host: apple) from the USA formed a separated clade. In the trees of segments M and S, the three Chinese ARWV-2 isolates clustered into two clades, with Mb and Sb segments of LYXS separated from M and S segments of S17E2 and LYC2, respectively .de novo assembly, respectively. The overall nt identities between the isolate CiVA-P from a Chinese pear sample and each of the nine isolates available in GenBank were about 95% for their RNA1 and 93–94% for their RNA2. Among these CiVA isolates, the intergenic region (IGR) between ORF2a and ORF2b ranged from 301 nt to 313 nt, and CiVA-P determined here showed relatively low nt identities with other isolates reported from other countries, ranging from 83.9% to 88.0%  were determined. Moreover, an expected amplicon with a size 1973 bp was obtained by using a primer pair N155F/M240R designed based on the sequences of the covering partial sequences of the two ORFs in the viral RNA2. The result confirmed that the ORF2a and ORF2b were encoded in the same RNA strand. The nt sequences of CiVA-P RNA1 and RNA2 obtained by Sanger sequencing were 99.9% and 99.6% identical to the corresponding contig sequences generated by to 88.0% . The IGRBunyaviriles [The CiVA-FJCG RNA1 was 6690 nt in length with one ORF  in its complementary strand. The ORF1 encoded a putative RdRp consisting of 2184 aa with a molecular mass of 251 kDa, which contained six motifs conserved in the RdRp of viruses in the aviriles . ORF1 ofIn the NJ trees constructed using the nucleotide sequences of the viral RNA1, RNA2, and IGR, two Chinses isolates CiVA-P and CiVA-HH were always separated from other-CiVA isolates reported from other countries, and formed a distinct subclade, indicating genetic diverse of CiVA isolates CiVA-P and CiVA-HH, identified from pear grown in China, from other reported CiVA isolates .In total, 251 reads, 77 reads, and 12,911 reads matching ARWV-2 genomic RNAs were derived from samples S17E2, LYC2, and LYXS and 90,617 CiVA RNA reads were derived from sample FJCG. The high RNA read productivities of ARWV-2 from LYXS and CiVA from FJCG made it possible to evaluate the distribution of RNA reads on each segment of the two viral isolates. It was found that ARWV-2 RNA reads deriving from sample LYXS covered the full lengths of the five RNA segments  of the viral genome . On eachCiVA RNA reads derived from sample FJCG also covered the full lengths of the viral RNAs 1 and 2. The overall read abundance on the viral genomic RNA1 was much lower than that of the viral genomic RNA2 . RNA rea−12) and Mb  of ARWV-2 isolate LYXS contained the plant viral MP family (pfam01107) domains  marker CMV-3a-mCherry  grown in Hubei, Sichuan, and Fujian provinces in China were subjected to RT-PCR assay for CiVA using primer set R2-F/R2-R . From sample S17E2, near full-length sequences of Sa and Ma segments were assembled, but only one short contig of 148 bp of the viral L segment was identified. CiVA reads had high cover depth and near full-length sequences of CiVA genomic RNA1 and RNA2 were assembled from FJCG library. HTS technologies have been proved to be a powerful tool for the identification of known and novel viruses ,27. SincThe overall genomic structures of ARWV-2 and CiVA determined from pear plants grown in China were the same as those of the corresponding viruses infecting apple  and citrP. communis, P. bretschneideri, P. pyrifolia, and pear hybrids grown in several Chinese provinces. Although ARWV-2 was first detected by HTS in three pear trees showing leaf mosaic symptom, the virus also presented in some asymptomatic trees as detected by using RT-nPCR. Our previous bioassays demonstrated the infectious nature of ARWV-2 in pear plants, but did not find the associated symptoms on pear A20 [P. pyrifolia trees grown in Fujian and Sichuan provinces, indicating a lower incidence. Although CiVA was identified in pear trees showing viral-disease-like symptoms (one leaf mottle and two chlorotic leafspot), we couldn’t conclude the association of the virus with pear disease since the most tested samples showing similar symptoms were negative to the virus. In addition, we did not find the co-infection of the two viruses.ARWV-2 was originally described in apple trees with rubbery wood disease . In recepear A20 . The leapear A20 , but likpear A20 . Howeverpear A20 . Recentlpear A20 . In thisConsidering the close taxon position of apple and pear belonging to the family Rosaceae, and the common infection of previously characterized viruses and viroids, including ASGV, ASPV, ACLSV, and apple scar skin viroid (ASSVd) , togetheThe 30K family MPs can form MP–RNA complexes to facilitate cell to cell movement of viral genome RNA via the PD . Here, fIn conclusion, ARWV-2 and CiVA were identified in pear trees grown in China using HTS. The genome of ARWV-2 pear isolates is divergent, with three RNA segments or five RNA segments. ARWV-2 occurs widely, but CiVA has low incidence in pear trees grown in China. ARWV-2 isolate LYXS codes two movement proteins Ma and Mb, which display PD localization. The MP of CiVA is located in cell periphery and can interact with the viral NP.


Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2), which is responsible for the coronavirus disease 2019 (COVID-19), has hit the world as a global pandemic at an unparalleled scale [In this Special Issue, Thakur et al.  showed t2/FiO2 ratio was >150 mmHg [Convalescent plasma (CP) from recovered patients is believed to provide passive immunity against viral infections, and it has resurfaced again as a potential treatment of many viral illnesses, including SARS, MERS, with inconclusive results ,8. Howev150 mmHg . The eff150 mmHg .In this Special Issue, Ghosn et al. found that severe acute kidney injury (AKI) was common in COVID-19 critically ill patients and was not linked to inflammatory or thrombotic markers. Additionally, in these patients, severe AKI was independently associated with hospital mortality and hospital length of stay . UsuallySeveral other interesting findings were also published in this Special Issue ,18,19. A


LIS1 (PAFAH1B1) gene that has been implicated in regulating the microtubule motor protein cytoplasmic dynein. Although this class of diseases has recently received considerable attention, the roles of non-synonymous polymorphisms (nsSNPs) in LIS1 on lissencephaly progression remain elusive. Therefore, the present study employed combined bioinformatics and molecular modeling approach to identify potential damaging nsSNPs in the LIS1 gene and provide atomic insight into their roles in LIS1 loss of function. Using this approach, we identified three high-risk nsSNPs, including rs121434486 (F31S), rs587784254 (W55R), and rs757993270 (W55L) in the LIS1 gene, which are located on the N-terminal domain of LIS1. Molecular dynamics simulation highlighted that all variants decreased helical conformation, increased the intermonomeric distance, and thus disrupted intermonomeric contacts in the LIS1 dimer. Furthermore, the presence of variants also caused a loss of positive electrostatic potential and reduced dimer binding potential. Since self-dimerization is an essential aspect of LIS1 to recruit interacting partners, thus these variants are associated with the loss of LIS1 functions. As a corollary, these findings may further provide critical insights on the roles of LIS1 variants in brain malformation.Disruptive neuronal migration during early brain development causes severe brain malformation. Characterized by mislocalization of cortical neurons, this condition is a result of the loss of function of migration regulating genes. One known neuronal migration disorder is lissencephaly (LIS), which is caused by deletions or mutations of the  LIS1 (PAFAH1B1), located in chromosome 17p13.3, is one of the major genes associated with neural migration [LIS1 contains 11 exons, and 10 of that encode the protein of 150 kDa [The lissencephaly 1 gene, igration , a funda 150 kDa , which i 150 kDa ,7.LIS1 gene was first disclosed in Aspergillus nidulans regulating microtubules formation and microtubules-based motors [LIS1 gene [LIS1 in non-neuronal cells increased retrograde movement of cytoplasmic dynein and promoted the peripheral accumulation of microtubules, reflecting the acquisition of neuron-like dynein behaviors [LIS1 has been linked to different stages of neuronal development, especially in neuronal migration and neurite outgrowth . Functiod motors . Sapir eIS1 gene . Interesehaviors . LIS1 inehaviors , NDEL1 [ehaviors , CLIP1 [ehaviors , NUDC [1ehaviors , TUBA1A ehaviors , NAGK [1ehaviors ,17, and ehaviors , and thuehaviors .LIS1 gene attributes the opposite effect, responsible for a developmental disorder called lissencephaly, manifesting typical facial dysmorphology with more severe brain malformation [LIS1 mutations are pronounced in about 60–75% of patients with isolated lissencephaly sequence (ILS) [LIS1 knock-out mice display disorganization of cortical, hippocampal, and olfactory organs, delay neural migration [LIS1 gene causes Type I lissencephaly (1), resulting in haploinsufficiency 2 [Accumulating studies suggested that deficiency or mutation of the ormation ,20,21. Lce (ILS) ,23, wherigration . Up to 2igration . Sporadiciency 2 .LIS1 gene, and it is expected that there is exist nsSNP in LIS1 gene that has a considerable impact on its functions. Therefore, we aimed to investigate the significant roles of the targeted gene-coded protein with the structural and functional consequence of nsSNPs of LIS1 protein using a wide range of bioinformatics tools and molecular dynamics (MD) simulation.Single-nucleotide polymorphism (SNP) is a common type of nucleotide modification or genetic variant in a single genetic code , occurriLIS1 gene, which were retrieved from the dbSNP database, have been subjected for risk prediction, using twenty deleterious predicting state-of-art-tools including SIFT, Polyphen, Condel, CADD, DANN, FATHMM, M-CAP, MetaLR, MutPred, MutationAssessor, PROVEAN, VEST3, fathmm-MKL, iMutant3.0, iStable, MuPro, PhD-SNP, PANTHER, SNPs & GO, and SNAP2  repeat protein family . LIS1 coHomodimerization of LIS1 is an essential aspect of performing the biological function, mediated mainly through the N-terminal region. Furthermore, Heterozygous animals deleting exon 1 (residues 1–63) exhibit a phenotype consistent with lissencephaly and result in no interaction between the mutant protein and PAFAH catalytic subunits . TherefoAll predicted high-risk variants, such as F31S, W55L, and W55R, were subjected to MD simulation along with wild-type. Three separate MD runs of 500 ns  for each case were performed. Root mean square deviation (RMSD) analysis was conducted to estimate the equilibrated trajectories, considering the initial structure of the simulation. The RMSD analysis suggests that subsequent stable trajectories were retained within the 200 ns of starting simulation  and mainThe root mean square deviation (RMSD) of Cα atoms of protein was calculated based on the equilibrated trajectories to explain the structural change during the simulations quantitatively. The change of RMSD was identified through comparing with wild-type in both dimer and monomeric forms, which denotes structural similarities ,47, reprThe influence of single amino acid substitution on Cα-RMSF of each residue was assessed using relative residue fluctuations, measured through RMSF , which dThe inter-and intra-monomeric correlation motion pattern in all systems was investigated using dynamic cross-correlation matrix (DCCM) analysis . As showTo characterize the dominating movement by overall conformations, principal component analysis (PCA) was considered, which compresses the ensembles into a sequence of eigenvectors ,50. EachThe result was projected onto a 2D plane consisting of the two most typical principal components (PC1 and PC2) to depict the overall conformational landscapes. The introduction of variants induced evident variations in dominant conformational distributions and affected the direction of projected dominant clusters . InteresA porcupine plot was created in The total occupancy of essential secondary structures, including helix and strand formation, contributed by each residue in the equilibrated trajectories, had been investigated by the tool called define secondary structure of proteins (DSSP) since RMSF, DCCM, and PCA analyses suggested significant variants induced changes in the LIS1 dynamics. Consistent with the findings from the previous analyses, DSSP showed a reduced secondary structure formation in the residue 50 to 60 in both monomers of the LIS1 dimers A. A signCOM) of two monomers is used to determine the overall tightness of the dimer. Wild-type structure showed DCOM averaging 9.081 ± 0.003 Å, while variants have a DCOM of 9.168 ± 0.004, 10.27 ± 0.005, and 10.04 ± 0.007, respectively, for F31S, W55L, and W55R . FEL provides a more accurate depiction of the protein conformational space in terms of energy and time, distinguishing the kinetic and thermodynamic characteristics of wild and variants containing protein structure during the simulation. In the free energy contour map, the energy minima and energetically preferred protein conformations are shown by dark blue patches, whereas yellow spots represent the unfavorable conformations A. The frThe electrostatic potential surface map for each system was created using the lowest energy structure received from the FEL, which also depicts the modest shift of the electrostatic potential in the variants, including structure relative to wild-type A. AlthouLIS1 gene using several bioinformatics techniques and verified their deleterious phenotypic consequences using MD simulation. As a result of consensus prediction, three nsSNP, including F31S, W55L, and W55R, were categorized as high-risk SNPs. It is worth noting that single-tool prediction of harmful SNPs is not error-free; hence, various techniques, including both sequence- and structure-based techniques, were used to enhance prediction accuracy [The current work unveiled three potentially harmful SNPs in the accuracy .The current work used microsecond-scale MD simulations to examine the atomic intricacies of the LIS1 dimerization and deduce the mechanism by which the identified variants F31S, W55L, and W55R impair LIS1 dimer formation. During the simulation, a substantial difference was noticed between the conformational change of monomers A and B in all systems, where a high difference was observed in the variant containing structures . Inter-mThe result of MD simulation demonstrated that although F31S containing structure showed a degree of fluctuation relative to wild-type structure , it induLIS1 gene have been retrieved from the NCBI dbSNP database with respective rsIDs. 270 SNPs have been retrieved and screened using 20 widely accepted tools for predicting the most deleterious SNPs. In silico bioinformatics tools, including SIFT, polyphen, condel, CADD, DANN, FATHMM, M-CAP, MetalR, MutPred, MutationAssessor, PROVEAN, VEST3, fathmm-MKL, I mutant3.0, iSTABLE, MuPro, PhD-SNP, PANTHER, SNPs and GO, and SNAP2 have been associated for this study. SIFT [For this study, the single nucleotide polymorphism (SNPs) related to dy. SIFT  is a PSIdy. SIFT . The predy. SIFT  is a webdy. SIFT  is a comdy. SIFT . DANN [6dy. SIFT  uses an dy. SIFT  is softwdy. SIFT . Clinicady. SIFT  can idendy. SIFT . PROVEANdy. SIFT  to predidy. SIFT ,69. Mutady. SIFT . fathmm-dy. SIFT . I mutandy. SIFT . PhD-SNPdy. SIFT . PANTHERdy. SIFT . SNPs andy. SIFT . SNAP2 idy. SIFT . iStabledy. SIFT , and I-Mdy. SIFT . Whereasdy. SIFT . A web-bdy. SIFT .All variants containing LIS1 structure had been created based on the LIS1 N-terminal crystal structure (PDB ID: 1UUJ), which had been downloaded from the protein databank and generated in Schrödinger 2021-2 , as previously reported ,83,84,85+Cl−) during the solvation. As mentioned in our previous protocol, the desmond trajectory followed eight stages [Desmond program of Schrödinger software 2021-2  ,88 was ut stages ,95. Conct stages  was usedt stages  with a rt stages  with a rt stages . Consinet stages .ijC) between the skeleton carbon atoms i and j.The dynamic cross-correlation maps were generated to explain the time-correlated motions in the protein atoms during simulation, which were assessed by Bio3D (an R package software) . The folith and jth residues was represented by ∆ir and ∆jr, respectively, and the angled brackets “〈〉” indicate the mean time of the whole trajectory. The ranged values to illustrate DCCM were from −1 to +1, where positive values represented a positively-correlated motion, and negative values denoted the negatively-correlated motion.Here, the average location of the Further, using principal component analysis (PCA), the flexibility and collective motions of wild-LIS1 and variants  were eluA conformational sampling method was used to obtain FEL, which provided all possible macromolecular structural conformations . In FEL B indicates Boltzmann’s constant, and Gi represents the Gibbs free energy of state, kB. Temperature set at 300 k is denoted by the letter T. The population bin I and the most inhabited population bin are determined by Ni and Nmax, respectively. The bin without population is assigned as minimum provability by an artificial scale where the energy of different levels was represented using a color-coded model. The lowest energy conformer from FEL was subjected to MM-GBSA calculation using HawkDock web server [Here, kb server ; the detb server .t-test and single-factor ANOVA tests were utilized for comparison. Student’s t-test with a 95% confidence interval was performed to compare MD trajectory data, which were resampled by bootstrapping the R software package to remove autocorrelation [SPSS v19 software was used to detect correlations across all used bioinformatics tools. In order to compare the most significant combinations, the relation ,108.LIS1 gene, which caused substantial structural modifications in the LIS1 structure, and thus disrupted dimer stability, as shown by MD simulation. Although further experimental validation into the precise function of the regions affected by these variants is needed, this study establishes a solid foundation to understand the molecular basis of LIS1 contribution in lissencephaly such as brain disorders, which can be a solid foundation for further research into targeting the LIS1 with therapeutics as a possible treatment for early brain developmental genetic disorders.Using a comprehensive bioinformatics approach, the current studies revealed three nsSNPs in the