Areca catechu, areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV). These two viruses share a distinct genomic organization of leader proteases and represent the only two species of the newly established genus Arepavirus of the family Potyviridae. The biological features of the two viruses are largely unknown. In this study, we investigated the pathological properties, functional compatibility of viral elements, and interspecies interactions in the model plant, Nicotiana benthamiana. Using a newly obtained infectious clone of ANRSV, we showed that this virus induces more severe symptoms compared with ANSSV and that this is related to a rapid virus multiplication in planta. A series of hybrid viruses were constructed via the substitution of multiple elements in the ANRSV infectious clone with the counterparts of ANSSV. The replacement of either 5′-UTR-HCPro1–HCPro2 or CI effectively supported replication and systemic infection of ANRSV, whereas individual substitution of P3-7K, 9K-NIa, and NIb-CP-3′-UTR abolished viral infectivity. Finally, we demonstrated that ANRSV confers effective exclusion of ANSSV both in coinfection and super-infection assays. These results advance our understanding of fundamental aspects of these two distinct but closely related arepaviruses.Previously, our group characterized two closely related viruses from  Arepavirus is a newly established genus under the family Potyviridae and consists of two virus species, namely, areca palm necrotic spindle-spot virus (ANSSV) and areca palm necrotic ringspot virus (ANRSV) ( (ANRSV) . The two (ANRSV) . It has  (ANRSV) . In addi (ANRSV) . NeverthPotyviridae family (referred to as potyvirids) , with subsequent determination of the biological alterations, is a common approach to examine functional roles of viral factors in viral infection. This strategy has been widely used to investigate pathogenesis-related biological phenotypes for viruses in the yvirids) . For insyvirids) . The VPgeraction . NotablyIpomovirus, cassava brown streak virus and Ugandan cassava brown streak virus, synergistically cause cassava brown streak disease, a risk to food security and a major threat to the cassava value chain (Tritimovirus) and Triticum mosaic virus (Poacevirus) cause synergistic mixed infection in cultivar-specific wheat. Antagonistic interactions universally exist between different strains of the same virus or closely related viruses; these interactions have been utilized to develop powerful management strategies against plant-infecting viral pathogens (referred to as mild strain cross-protection)  unrelated or closely related viruses . Usuallyue chain . The whetection) . TherefoNicotiana benthamiana. First, we developed the infectious complementary DNA (cDNA) clone of an ANRSV and tested its pathogenicity in comparison with the closely related ANSSV. Furthermore, the functional compatibility of viral elements was assessed by individually replacing different genomic segments in ANRSV with their counterparts from ANSSV. Finally, co-infection and super-infection assays were used to examine virus interactions in planta.Given that the arepavirus–areca palm pathosystem has not yet been established, in this study, we investigated the pathological properties, functional compatibility of viral elements, and interspecies interactions in the model host—N. benthamiana was maintained in a growth cabinet set to 16-h light/25∘C and 8-h darkness/23∘C.Fresh leaf tissues from one ANRSV-infected areca palm tree at the 5-year-old stage in a commercial growing orchard in Dingan, Hainan, were sampled for cloning full-length genome of ANRSV and subsequent generation of infectious cDNA clone. This virus isolate was referred to as ANRSV-ZYZ. The experimental host The determination of the complete genome sequence of ANRSV-ZYZ was essentially the same as that of previously reported ANRSV-XC1 . In brieStuI-MluI-BamHI-EcoRI-SalI) was amplified from the plasmid pVPH-green fluorescence protein (GFP)//mCherry  position 2,566 into A during primers synthesis, but the mutation did not alter the encoded aa. The fragments S2, S3, S4, and S1 were integrated, step-by-step, into the modified pCB301 vector to generate the full-length cDNA clone of ANRSV-ZYZ (named pRS).A low-copy mini-binary T-DNA vector  was used/mCherry  using pr/mCherry . Four pa/mCherry  were desvia overlapping PCR method with corresponding primers listed in via a potential recombination event during viral replication, the corresponding nt sequences of the two peptides differ in seven nts. Similarly, a mCherry-tagged ANRSV clone (pRS-mCh) was created.To develop a GFP-tagged ANRSV clone (pRS-G), the GFP-coding region was amplified from pVPH-GFP/mCherry  and engiN. benthamiana , which was tested to be highly infectious in thamiana . To exammplified . Two PCRmplified . A mixtumplified . The obt3 Real-Time PCR Thermol Cycler  following the manufacturer’s instructions. In pathogenicity test assay, primer sets RS-9200F/RS-9350R (for ANRSV) and SS-8600F/SV-8700R (for ANSSV) (For reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR assays, total RNAs were extracted from newly expanded leaves using TRNzol Universal Reagents (TIANGEN) and treated with DNase I (Thermo Fisher Scientific). The first-strand cDNAs were synthesized by reverse-transcription reactions using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with random hexamer primer. The PCR reactions with primer sets RS8900F/RS9300R and SS9000F/SS9300R , targetir ANSSV)  were desr ANSSV)  were useN. benthamiana seedlings at an indicated stage were subjected to the infiltration of Agrobacterium tumefaciens (GV3101) cultures harboring the relevant plasmids. The cultures were adjusted to the desired optical densities at 600 nm (OD600) and infiltrated into leaf tissues of N. benthamiana plants essentially as described previously . All photographs showing fluorescence signals were taken in a dark chamber at the indicated time point. In co-infection assay, an inverted fluorescence microscope  was used to examine mCherry signals in leaf tissues indicative of the infection with ANRSV-mCh.in planta. The systemically diseased N. benthamiana plants, which were previously agroinfiltrated with pSS-I-G or pRS-mCh, were maintained in a growth cabinet as inoculum sources. Both infected leaf tissues with comparable weight and fluorescence areas  were, respectively, grounded in phosphate buffer in a ratio of 1:10 (g/ml). The homogenates mentioned earlier were mixed in equal volume, followed by immediate rub-inoculation into the third leaves of 30 N. benthamiana seedlings at 4- and 5-leaf-stage. As a control, two mixed homogenates, either pSS-I-G or pRS-mCh and virus-free one , were, respectively, rub-inoculated into 25 seedlings. For sap inoculation, a single leaf of each plant was rubbed two times from the leaf bottom to the top using an inoculum-dipped forefinger. In co-infection and super-infection assays, two specific primers, Spe-RS-1000R (for ANRSV) and Spe-SS-1900R (for ANSSV), are designed on the basis of nt sequence differences in the HCPro1–HCPro2 region  was performed. First, N. benthamiana plants were agroinfiltrated with pSS-I-G, and systemically infected leaves were used as inoculum sources. The fresh leaf tissues from diseased (pSS-I-G) and healthy plants were, respectively, grounded in phosphate buffer in a ratio of 1:10 (grams per milliliter). The resulting homogenates were mechanically inoculated into 100 N. benthamiana seedlings at 3- to 4-leaf-stage, including 60 for pSS-I-G and 40 for virus-free homogenate. These plants were equally divided into two groups. Ten days later, one group of plants were subjected to challenging rub-inoculation with ANRSV-containing homogenate, and the other group plants as control were inoculated with virus-free homogenate.To examine whether the ANRSV interferes with ANSSV MZ209276), followed by its annotation with reference to a previously documented isolate ANRSV-XC1 . We first determined the complete genome sequence of ANRSV-ZYZ  and severe rugosity and chlorosis at 10 dpi  containing corresponding cDNA clones of ANSSV (pSS-I) and ANRSV (pRS) were each infiltrated into 12 N. benthamiana seedlings at the 5- to 6-leaf-stage. All plants inoculated with pRS exhibited typical vein clearing symptoms in the top new leaves at 6 dpi and severe chlorosis and rugosity at 12 dpi  harboring corresponding GFP-tagged ANSSV (pSS-I-G)   and ANRSt 12 dpi . Howevert 12 dpi . This ret 12 dpi . Consistt 12 dpi .Agrobacterium-mediated transfection efficiency, homogenates were prepared at 30 dpi from leaves systemically infected with ANRSV-G (pRS-G) and ANSSV-G (pSS-I-G), followed by rub-inoculation into the third leaves of more than 10 N. benthamiana plants each at the 5- to 6-leaf-stage. Time-course observation results revealed a similar trend with the Agrobacterium-mediated transfection assay, either in symptom development or viral multiplication, as that obtained with the GFP signals  is caused by differential  signals . Collectvia individual substitution of viral elements in the ANRSV genome with their counterparts from ANSSV. For potyvirids, the first two viral proteins encoded by the genomic N-terminus (upstream P3-coding region) might confer certain biological relevance in a coordinated manner  were created  were referred to as ANRSV-G and ANSSV-G. Similar to ANSSV-G, all these five hybrid viruses did not trigger discernable symptomatic phenotype at 10, 15, and 22 dpi  did not show any distinguishable symptoms and fluorescence signals  of ANRSV function in a virus-specific manner.Remarkably, all plants inoculated with the other hybrid virus clones  (N. benthamiana (data not shown). The mixed homogenates of ANSSV-G and ANRSV-mCh were rub-inoculated into 30 N. benthamiana plants. As the control, the mixed homogenates, either ANSSV-G or ANRSV-mCh and virus-free (indicated by the symbol “−”) ones, were inoculated into 25 plants each.As ANRSV and ANSSV are two distinct but closely related viruses in the genus pRS-mCh) , and theFour days later, several bright green fluorescence foci were observed in the inoculated leaves of ANSSV-G/− control plants (48.8 foci on average per leaf) . In contin planta, an assay for specific RT-PCR detection of ANRSV and ANSSV was established , the typical ANRSV-induced symptoms such as vein clearing and rugosity in top leaves were observed in all plants treated with −/ANRSV. A similar symptom phenotype was observed in 26 of 30 plants treated with ANSSV-G/ANRSV (data not shown). The association of these symptoms with ANRSV infection was confirmed by specific RT-PCR detection of ANRSV . At 15 dIn contrast to ANSSV-G/− plants, the GFP signal in ANSSV-G/ANRSV plants gradually faded after 8 dpci. Only scattered fluorescence spots, indicative of the infection by ANSSV-G, were observed in the top leaves of ANSSV-G/ANRSV plants at 15 dpci, indicating the primarily infected ANSSV-G was greatly restricted . In compPotyviridae  a series of ANRSV-derived cDNA clones were successfully generated, which could serve as valuable tools to study fundamental aspects of arepaviruses; (ii) the viral factors  from a heterologous virus (ANSSV) are compatible for replication and systemic infection of ANRSV but not P3-7K, 9K-NIa, and NIb-CP-3′-UTR; and (iii) the ANRSV effectively excludes ANSSV both in co-infection and super-infection assays, indicating that the interactions between them in planta are antagonistic. These findings greatly advance our understanding of fundamental aspects of the two distinct but closely related viruses in the new genus Arepavirus of the family Potyviridae.Previously, our group identified two closely related arepaviruses (ANRSV and ANSSV) from areca palm trees, with the former being the prevalent virus in the field , 2019. Ayviridae . Hence, in planta. However, further testing in natural hosts is required, for which the following aspects should be considered critically: (i) the demarcation criteria for areca palm cultivars have not been established; (ii) the perennial areca palm is unyielding to genetic transformation, limiting the development of transfection methods of arepavirus; and (iii) although only one ANSSV isolate has been documented thus far  were non-infectious is likely because of the complex interactions between the notorious multifunctional potyvirid proteins, especially those involved in virus replication  facilitates the exploration of the functional roles of viral factors in viral infection. For potyviruses, the primary task executed by leader proteases (P1-HCPro) during viral compatible infection is defending the RNA silencing-mediated immune response. For example, the replacement of HCPro (or P1-HCPro) by other heterologous viral suppressors of RNA silencing in PPV generates viable viruses with different efficiencies in systemic infection . The VSRlication .Citrus tristeza virus  can be found in the article/HC, YW, WS, and ZD conserved and designed the project. YW, WS, and ZD carried out experiments. HC supervised the work. HC, YW, and ZD wrote the manuscript. All authors analyzed, discussed the data, reviewed, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Outbreaks of influenza, caused by the influenza A virus (IAV), occur almost every year in various regions worldwide, seriously endangering human health. Studies have shown that host non-coding RNA is an important regulator of host–virus interactions in the process of IAV infection. In this paper, we comprehensively analyzed the research progress on host non-coding RNAs with regard to the regulation of IAV replication. According to the regulation mode of host non-coding RNAs, the signal pathways involved, and the specific target genes, we found that a large number of host non-coding RNAs directly targeted the PB1 and PB2 proteins of IAV. Nonstructural protein 1 and other key genes regulate the replication of IAV and indirectly participate in the regulation of the retinoic acid-induced gene I-like receptor signaling pathway, toll-like receptor signaling pathway, Janus kinase signal transducer and activator of transcription signaling pathway, and other major intracellular viral response signaling pathways to regulate the replication of IAV. Based on the above findings, we mapped the regulatory network of host non-coding RNAs in the innate immune response to the influenza virus. These findings will provide a more comprehensive understanding of the function and mechanism of host non-coding RNAs in the cellular anti-virus response as well as clues to the mechanism of cell–virus interactions and the discovery of antiviral drug targets. Influenza viruses belong to the Orthomyxoviridae RNA virus family and is divided into genera A, B, C and D. Among them, genera A, B and C cause respiratory diseases in humans . InfluenNon-coding RNAs (ncRNAs) are a large class of RNA transcripts that are transcribed from the genome but lack the ability to encode proteins . At the MiRNAs are widely distributed in eukaryotes and some viral genomes. They are highly conserved non-coding single-stranded RNA fragments encoded by nuclear genes and play a role in the regulation of post-transcriptional gene expression ,23. In aFrom the perspective of IAV infection, cell miRNA expression is crucial for virus–host interactions such as virus entry, replication, translation, and transmission. When H9N2 or H5N1 infected avian-derived DCs, gga-miR6675 and gga-miR6616 were found to target of mRNA nuclear localization sequence (NLS) of viral PB1, triggering PB1 gene silencing and inhibiting H9N2 replication. , and initiate the antiviral immune response. Studies have shown that host miRNAs can be involved in regulating the activation of host PRR-mediated antiviral signaling pathways, thereby indirectly inhibiting the replication of influenza viruses. -mediated innate immune response signaling pathways. RLRs play a critical role in host innate immunity, and mice with either RIG-I or MDA5 deficiency have shown susceptibility to RNA viruses . When anHost miRNA induces interferon expression by regulating the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway and inhibits the replication and proliferation of IAV. The antiviral effect of IFN mainly occurs through the activation of the JAK/STAT signal, which induces the production of ISGs. IFN regulatory factor 5 (IRF-5) is a key transcription factor for maintaining the inflammatory phenotype of macrophages. IRF-5 can promote the replication of IAV, while miR-302a regulates the cytokine storm induced by IAV by binding to the 3′-UTR of IRF-5 and interferes with the replication of influenza virus ,58. IAVsHost miRNA inhibits IAV replication through other antiviral pathways. The antigen presentation ability of DCs plays an irreplaceable role in the recognition and removal of viruses. Host miRNA can inhibit the replication of IAV by regulating the antigen presentation of host DCs. Both active and inactive H9N2 avian influenza viruses can enhance the ability of DCs to present antigens and activate T lymphocytes. GGA-miR1644 enhances the ability of DCs to inhibit viral replication by muscle blind-like protein 2. This study provides new clues for the role of miRNAs in inducing DC antigen presentation and inhibiting virus replication .Host miRNA can inhibit the replication of IAV by regulating cell apoptosis. In the process of cross-species infection by the influenza virus, ssc-miR-221-3p and ssc-miR-222 can induce the apoptosis of positive allosteric modulators (PAM) cells by directly targeting the mRNA of HA and PA genes and the expression of the anti-apoptotic protein HMBOX1. Moreover, these two miRNAs inhibited the infection and replication of IAV in newborn pig trachea (NPTR) cells. Compared to Simian immunodeficiency virus (SIV) infection, P65 can be phosphorylated more efficiently after IAV infection, which is why the expression of ssc-miR-221-3p and ssc-miR-222 is significantly upregulated after viral infection . AccordiDuring the interactions between the influenza virus and host, host miRNAs inhibit virus replication directly and indirectly. However, some studies have shown that the miRNAs can negatively regulate the antiviral response pathway, reduce the expression of antiviral factors, and promote the replication of the influenza virus.Host miRNAs negatively regulate TLR and RLR-mediated innate immune response pathways to promote IAV replication. The expression of miR-21-3p was significantly reduced in A549 cells infected with H5N1, and miR-21-3p down-regulated the expression of basic fibroblast growth factors 2 (FGF2) recombinant proteins, accelerated the replication of H5N1, and inhibited the IFN response. The overexpression of miR-21-3p significantly increased the expression levels of viral genes M1 and NP and viral titers in influenza virus-infected cells and significantly decreased the expression of antiviral factors such as IFN, PKR, MxA, and OAS . In addiHost miRNA negatively regulates NF-κB and IRF-mediated innate immune response signaling pathways to promote IAV replication. As a pro-inflammatory cytokine, zinc finger protein A20 participates in the negative feedback regulation of IL-I/tumor necrosis factor and other types of signal transduction and plays a role in inhibiting the antiviral immune response in the NF-κB and IRF signaling pathways. Influenza virus infection induces the up-regulation of miR-29c expression and stabilizes the expression of A20, thus improving the capacity for viral replication . In IAV-These results indicate that some host miRNAs can directly regulate viral gene expression during host-influenza virus interaction. In addition, some host miR-RNAs indirectly regulate the replication of the influenza virus by regulating the antiviral immune response signal in host cells. These findings provide new ideas for a comprehensive understanding of the interaction mechanism between host cells and influenza virus and the discovery of effective targets for antiviral drugs.According to reports, lncRNA, as a new type of regulatory factor, can be induced by the influenza virus and expressed in the cytoplasm or nucleus ,84. ThesIt was found that the host lncRNA could directly target the virus gene and affect virus replication during the process of infection. IAV infection significantly induces two host lncRNAs (PAAN and IPAN) not involved in IFN regulation. LncRNA PAAN enhances the activity of viral RNA polymerase by promoting the assembly of the RNA polymerase complex of the influenza virus, and as a forward regulator of influenza virus replication, ensures the efficient synthesis of virus RNA. LncRNA PAAN synthesis is synchronized with IAV replication . It has Host lncRNAs inhibit IAV replication by regulating innate immune signaling pathways mediated by TLRs. The innate immune response mediated by TLRs includes several key factors, such as the expression and activation of PRRs, ISG expression, and IFN production . LncRNA-Host lncRNA inhibit the replication of IAV by regulating the innate immune response mediated by RLRs. The RLR-mediated innate immune response is an important pathway for lncRNAs to participate in the regulation and inhibition of IAV replication. When the virus invades, the intracellular “whistle” RIG-I recognizes the double-strand RNAs (dsRNAs), transmits antiviral signals through downstream mitochondrial antiviral-signaling (MAVS) proteins, and finally induces the expression of IFNs and proinflammatory cytokines. At the early stage of viral infection, lncRNA z3h7a in the cytoplasm binds to tripartite motif-containing protein 25 (TRIM25) and activates RIG-I. As a molecular scaffold, lncRNA z3h7a stabilizes the TRIM25/RIG-I interaction and enhances TRIM25-mediated K63 ubiquitylation of RIG-I. Thus, the downstream signaling of RIG-I and the antiviral innate immune response are promoted . LncRNA-Influenza virus negatively regulates the antiviral immune response mediated by TLRs by inducing the expression of intracellular lncRNA . A/WSN/1RLRs signaling pathway plays a key role in the escape process of the influenza virus. After the influenza virus invades the body, viral proteins will degrade or inactivate RIG-I, MDA5, MAVS, and other monitoring proteins, thus avoiding the immune mechanism of the body . The lncA new study found that the lncRNA ACOD1 promotes IAV replication by regulating cell metabolism . LncRNA The above studies showed that in addition to host lncRNAs participating in the innate immune response pathway to regulate the replication of IAV, influenza viruses can also use host-encoded lncRNAs to negatively regulate the innate immune response of the host.Vault RNA (vtRNA) is a class of non-coding RNAs in the Vault ribosome complex, which plays a key role in the process of influenza virus infection and replication . PKR is Virus–host interactions are complicated processes, and multiple cellular proteins promote or inhibit viral replication through different mechanisms. CircRNAs are a newly discovered class of endogenous regulatory RNAs that are widely expressed and characterized by a covalent closed-loop structure without a 5′ cap or 3′ tail. In recent years, a growing number of circRNAs have been reported to play important roles in a variety of human diseases, including in dynamic interactions between the virus and host ,112,113.Influenza B viruses have a limited host range compared to influenza A viruses, the mutation rate was low, and is not easy to cause a pandemic. In the past few decades, there have been many studies on the interaction between host and influenza B virus Host noncoding RNA plays an important role in directly or indirectly regulating the replication of influenza B virus.  . ReplicaInfluenza C virus is a common pathogen of acute respiratory diseases, and Children are susceptible to the influenza C virus. Influenza D is a newly discovered virus in cattle and pigs, which can cause mild to moderate respiratory diseases and target the upper and lower respiratory tract. IDV and ICV have about 50% homology. The current studies on these two influenza viruses focus on the interaction between virus and host proteins. For example, the NS1 of ICV and IDV, similar to the NS1 of IBV and IAV, can be overexpressed in the infected host cells and inhibit the IFN function of the host cells; ANP32A-mediated influenza virus replicase assembly ; TMPRSS2In recent years, non-coding RNAs have attracted extensive attention from researchers. In this paper, through a comprehensive analysis of relevant studies on the regulation of host non-coding RNAs involved in the replication of IAV, we found that host noncoding RNAs mainly regulate influenza virus replication directly and indirectly. Some of them play a role in inhibiting the replication and proliferation of the virus, while others assist the virus to escape the monitoring system of the host immune response and invade the host cells faster by interacting with the virus genes. This provides a new strategy for the development of anti-influenza drugs targeting key genes of the virus.In addition to directly regulating the expression of influenza virus genes, host non-coding RNA can also participate in the coordination of intracellular signaling and the expression of antiviral factors, and indirectly regulate the replication of the influenza virus. With further research on non-coding RNAs, how these host non-coding RNAs regulate the expression of antiviral factors in the process of innate immune response and play an indirect role in the regulation of the replication and proliferation of influenza viruses will become clearer, and these non-coding RNAs will hopefully become new candidate drugs for anti-influenza virus research.CeRNAs provide a new regulation mode for gene expression. Studies have shown that compared to simple miRNA-mRNA regulation, the ceRNA regulation network is more complex and generally consists of an integrated regulation network involving circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA. Currently, studies on ceRNAs are mainly focused on their role in the regulation of tumor genesis, and there are few reports on how ceRNAs regulate influenza virus replication. LncRNAs and circRNAs have abundant miRNA binding sites, so they can bind the MERS of miRNAs, like a sponge, to form ceRNAs, prevent miRNAs from binding with target gene mRNAs, and regulate the expression of the target genes (see In the face of cunning and changeable influenza virus, effectively activating the immune response to the conserved domains of the virus is an effective method to realize a broad-spectrum influenza vaccine . SpecifiIn conclusion, host non-coding RNAs affect the process of viral infection by regulating the expression of host or virus-related genes and play an important role in the complex relationship between the host and virus regulation, facilitating a state of balance between the virus and host. The relationship between non-coding RNAs and viral infection still has broad research prospects. However, the mechanism of non-coding RNAs in influenza virus infection remains to be further studied. On the one hand, the current research mainly focuses on the function of non-coding RNAs in influenza virus infection, and it is not clear whether non-coding RNAs play a role in the escape process of influenza virus. On the other hand, influenza virus infection can induce the differential expression of a large number of non-coding RNAs in host cells. Only a few studies have confirmed their function in the interaction between virus and host. The role of many non-coding RNAs in the process of influenza virus infection is still poorly understood. Further clarification of the interaction mechanism will help researchers better understand the precise regulatory mechanism of these host non-coding RNAs during the process of IAV infection. It will also help provide promising targets for the development of antiviral strategies by reducing key regulatory factors associated with viral infection and enhancing innate immune responses.


The trans species, more stable by about 7 kJ/molthan gauche-2-azabutadiene, has been experimentallyobserved, and its rotational and centrifugal distortion constantshave been obtained with remarkable accuracy, while theoretical estimatesof the spectroscopic parameters are reported for gauche-2-azabutadiene.Being N-substitutedunsaturated species, azabutadienes are moleculesof potential relevance in astrochemistry, ranging from the interstellarmedium to Titan’s atmosphere. 2-Azabutadiene and butadieneshare a similar conjugated π system, thus allowing investigationof the effects of heteroatom substitution. More interestingly, 2-azabutadienecan be used to proxy the abundance of interstellar butadiene. To enablefuture astronomical searches, the rotational spectrum of 2-azabutadienehas been investigated up to 330 GHz. The experimental work has beensupported and guided by accurate computational characterization ofthe molecular structure, energetics, and spectroscopic propertiesof the two possible forms,  Molecules that are isoelectronic to butadiene and sharea similar conjugated π system form an interesting family ofspecies. Indeed, they allow the investigation of the heteroatom effectsin the butadiene skeleton.1 Limiting ourselvesto the second row of the periodic table, there are three singly substitutedspecies isoelectronic to butadiene: acrolein , 1-aza-1,3-butadiene , and 2-aza-1,3-butadiene .In this context, high-resolution molecular spectroscopy and computationalchemistry are among the most powerful tools to elucidate the molecularstructure, internal dynamics, and conformational behavior of thesespecies, especially when the two approaches are combined synergistically.21,3-Butadiene  together with its small dipole moment value (0.09 D) haveprevented its nonplanarity from being established unambiguously untilrecently.8These butadiene-like species and butadieneitself are not easyto characterize experimentally. For example, due to the lack of apermanent electric dipole moment, the most stable trans and cis forms, respectively9), acrolein has beenwell characterized from a spectroscopic point of view, as demonstratedby the vast literature on this subject.16 On the other hand, spectroscopic study of 1- and 2-azabutadienehas been quite limited to date21 as both species are unstable and require an efficient and selectiveproduction method.Whenever a heteroatomis introduced into the butadiene skeleton,the molecular symmetry is noticeably reduced, the resulting asymmetrythen generating both an appreciable electric dipole moment and anincreased number of active infrared modes. For these reasons, acrolein,1-azabutadiene, and 2-azabutadiene represent more suitable spectroscopictargets. For example, owing to its large dipole moment values (about3 and 2.5 D for the 22 orLupus-1A23), thus making butadiene andheterodienes good candidates for astronomical searches. However, becauseof its centrosymmetric nature, trans-butadiene cannotbe detected in space through rotational emission and observation ofthe more energetic gauche form in cold molecularclouds is very unlikely. The abundance of centrosymmetric species,such as molecular nitrogen, cyanogen, or benzene, has been indirectlyestimated through observation of their protonated,24 isomeric,25 or functionalizedforms.26 Analogously, we suggest that thepresence of interstellar butadiene can be proxied by the detectionof its isoelectronic heterodienes. This strategy represents a promisingroute given the fact that acrolein has been recently discovered inthe ISM27 and the detection of 1-azabutadieneis being reported tentatively.28 However,to the best of our knowledge, astronomical searches of 2-azabutadienehave never been reported. This is probably due to the fact that inaddition to the limited spectral data reported in the literature,no entry for 2-azabutadiene is available in the widely used CDMS database30 or in the JPL catalog.31Exhaustive characterization of the high-resolutionrotational spectrafor these butadiene-like species is also crucial for a different aspect:the study of the chemistry occurring in the interstellar medium (ISM).Despite the harsh conditions of molecular clouds, i.e., very low temperaturesand number densities, unsaturated carbon chains are often abundant, straightforwardly derived from the equilibrium structure, andthe vibrational corrections (ΔBvib)i denotesthe inertial axis , and the sum runs over all vibrationalnormal modes. Within vibrational perturbation theory to second order(VPT2), the vibrational corrections require computation of the vibration–rotationinteraction constants (αri).32 Their evaluationrequires an anharmonic force field,33 whichhas been calculated at the MP2/cc-pVTZ34 level, within the frozen-core (fc) approximation. The MP2 acronymstands for Møller–Plesset perturbation theory to the secondorder.35The leadingterms in rotational spectroscopy are the rotationalconstants, which can be computationally predicted by summing two contributions:the equilibrium rotational constants  method (coupled-cluster (CC) singles and doubleswith perturbative treatment of triples39) and requires minimization of the following energy gradientEHF–SCF∞/dx and dΔECCSD(T)∞/dx are gradientsfor the extrapolation to the complete basis set (CBS) limit of theHF-SCF energy  and of the CCSD(T) electron correlation contribution(n–3 extrapolation expression41), respectively. Since the extrapolation to theCBS limit is performed within the frozen core approximation, core–valence(CV) correlation effects are incorporated by adding the correspondingcorrection, dΔECV/dx. This involves the difference of all-electron (ae) and frozen-coreCCSD(T) calculations using the same basis set. In the CBS+CV scheme,for the extrapolation to the CBS limit, we used the cc-pVnZ basis sets,34 with n = T – 5 for HF-SCF and n = T, Q for CCSD(T).For the CV correction, we resorted to the cc-pCVTZ basis set.42Despite being importantfor quantitative predictions,33The semirigidity of the molecules alsorequires one to accountfor the effect of centrifugal distortion. Different orders of centrifugaldistortion terms are possible according to the power of the angularmomentum operator considered in the effective rotational Hamiltonian.However, from a computational point of view, only the quartic andsextic centrifugal-distortion constants can be obtained: the formeras a byproduct of harmonic force-field computations, and the latterfrom the anharmonic force field. Therefore, the sextic terms havebeen computed at the MP2/cc-pVTZ level, while the quartic centrifugal-distortionconstants have been purposely derived from a fc-CCSD(T)/cc-pVTZ harmonicforce field. This latter calculation also allowed for determiningthe electric dipole moment (μ) components as well as the electricfield gradients at the nitrogen nucleus, which straightforwardly providethe nitrogen quadrupole coupling constants.44All quantum-chemical computations have been carried out withtheCFOUR package.The sample of 2-azabutadiene was synthesized in a two-step procedure,and its rotational spectrum was recorded with a frequency-modulationspectrometer.46 has been slightly revised, andthe experimental conditions were optimized with the aim of maximizingthe signal-to-noise ratio (S/N) of the rotational spectrum and minimizingthe number of possible interfering lines. The production of 2-azabutadieneproceeds in two steps, detailed in the following: synthesis of theprecursor (step I) and its transformation into the title molecule(step II).The synthetic reaction reported in the literaturefor obtaining2-azabutadiene46 Distillation under vacuum (0.1 mbar) gave the pure product in a43% yield (5.1 g).The precursor is2-[(2-chloroethyl)amino]acetonitrile, which has been synthesized ina Strecker reaction starting from 2-chloroethylamine hydrochloride and sodium cyanide  in methanol (30mL) and water (20 mL). The reaction mixture was cooled to 0 °C,and formaldehyde  was added slowly. The solutionwas allowed to warm up to room temperature and stirred for 1 h. Organiccompounds were then extracted with dichloromethane (3 × 50 mL).The organic phase was dried over magnesium sulfate, and the solventswere removed in vacuo. The crude product obtained in a 90% yield (10.6g) was sufficiently pure to be used directly in the next step.2-Chloroethylamine hydrochloride, formaldehydein water, and sodium cyanide were purchased from Sigma-Aldrich andused without further purification.tert-butylate  introduced in a horizontal tube and filling its lowerhalf. In this reaction, tert-butanol was also producedand selectively trapped in a U-tube immersed in a cooling bath, whilea gaseous flow of almost pure 2-azabutadiene was directly introducedinto the spectrometer cell.2-Azabutadienewas synthesized by vaporizing 2-[(2-chloroethyl)amino]acetonitrile(2 g) on potassium JKa, Kc = 270,27 ←261,26 transition of trans-2-azabutadiene,predicted on the basis of literature data.20t-BuOK, and (iii) the cooling bath.Ultimately, the maximum yield of 2-azabutadiene (and consequentlythe best S/N in the spectrum) was achieved by maintaining the precursorcompound between 65 and 90 °C, the potassium tert-butylate sample at room temperature, and the cooling bath at −80°C. The cooling bath was obtained by mixing acetone with liquidnitrogen and allowed for complete removal of the interfering linesbelonging to the t-BuOH formed during the reaction.The best conditions were optimizedby monitoring the spectral S/N for the 48 Briefly, the primary radiation source—whosefrequency and phase stability are ensured by a phase-lock loop (PLL)—isa Gunn diode emitting in the W band (75–110 GHz). The Gunndiode is coupled to a passive multiplier  to produce higher frequencies. The modulated radiationsource is fed into a 3 m glass absorption cell where the vapors of2-azabutadiene are injected at a pressure between 5 and 10 μbar.The output signal is detected by a Schottky barrier diode and demodulatedby a lock-in amplifier set at twice the modulation frequency (2f). The uncertainties of the present experiment are around30 kHz.The rotationalspectrum of 2-azabutadiene has been recorded between 225 and 330 GHzby means of a frequency-modulation millimeter-wave spectrometer inBologna; a detailed description of the instrument is given in previousworks.trans form (ϕ = 180°) and the more energetic gauche isomer (ϕ = 54°). The optimized equilibrium structuresare shown in Supporting Information). Except for the dihedral angle, most of the bond lengths and anglesare similar in the two forms, i.e., they agree within few milliAngstromsfor the former and ∼1° for the latter. The largest differencesare observed for the H–C=N angle (3.7°) and thetwo involved bonds (7 mÅ for C=N and 9 mÅ for C–H).Understanding the reasons at the basis of these differences is beyondthe scope of our paper; however, they can be simply rationalized byperforming natural bond orbital (NBO) analysis. The results of suchanalysis are collected in the SI and canbe used to understand how the conjugation effects stabilize the trans species more than the gauche form.2-Azabutadiene exists intwo conformations, depending on the C=N—C=Cdihedral angle (ϕ): the most stable planar ΔE[gauche – trans])betweenthe two conformers is 6.5 kJ/mol. By incorporating the harmonic zero-pointenergy (ZPE) correction, computed at the fc-CCSD(T)/cc-pVTZ levelof theory, ΔE[gauche – trans] increases to 7.2 kJ/mol. On the basis of this energy difference,the population of gauche-2-azabutadiene is about7% of that of the trans form at room temperature.Since the computed (μb(gauche)/μb(trans))2 ratio is ∼0.7 .At the CBS+CV level, the equilibrium energy difference. In its principal inertia system,the molecule lies on the ab plane with the c axis perpendicular to it. The computed dipole moment componentsare μa = 0.47 D and μb = 1.46 D (see c being null for symmetry reasons (the point group symmetry is Cs).Moving to the fully characterized 46 D see 1, with μa and of b type,were recorded between 225 and 330 GHz  with respect to the much smaller B and C values (∼4.4 GHz). Indeed,the population of the rotational energy levels decreases quickly byincreasing Ka and slowlyby increasing J. Our newly observed rotational transitionswere combined with previous low-frequency data20 and analyzed in a weighted least-squares procedure usingthe SPFIT program.49 The optimized energylevel positions were calculated employing a Watson S-reduced Hamiltonian with centrifugal distortion terms up to thesixth power of the angular momentum operator.50 Despite the relatively high J values targeted inthis work, the hyperfine structure arising from the quadrupolar 14N nucleus has been partially resolved for some low Ka, b-typetransitions. Therefore, we were able to determine the (χbb – χcc)/4 parameter, whereas the value of 1.5 × χaa has been kept fixed to its computed value.The derived spectroscopic parameters are collected in Exploiting the bestexperimental conditions, almost 600 new rotationaltransitions, both of 20 The semirigid model is appropriate for the descriptionof the rotational energy levels, as demonstrated by the standard deviationof the fit (σ), which is 1.09. The rotational constants A, B, and C have beendetermined with remarkably good accuracy, the associated errors beingsmaller than 1 kHz. Notably, all quartic centrifugal distortion constantshave been obtained with an uncertainty smaller than 0.01%, and mostof the sextic terms could be derived with the exception of two off-diagonalconstants (h2 and h3) that have been kept fixed at the corresponding computedvalues. The agreement between the experimental and the theoreticalparameters is very good. Minor discrepancies are only found for thesextic constants and the value of the b componentof the dipole moment, experimentally determined by means of Starkeffect measurements.20 The latter discrepancyis unexpected based on the literature available for dipole momentcomputations.51 Indeed, even though theuse of augmented basis sets is recommended to ensure a flexible descriptionof the outer valence region, the accuracy of the fc-CCSD(T)/cc-pVTZvalues is expected to be on the order of the experimental uncertainty.51 In this respect, the limitations of the experimentaldetermination deserve to be mentioned; in fact, for both μa and μb, this relied on the observation of only two transitions.From inspection of 20 isevident  representsthe generic parameters of the X isomer. The scaled parameters arecollected in the last column of Concerning the trans and cis/gauche species, the results being reportedin trans species are concerned, the similarity is evident. The order of magnitudeand the sign of each constant listed in Given the similar heavy-atom skeleton and theconjugated πsystem shared by 2-azabutadiene, butadiene, acrolein, and 1-azabutadiene,it is quite interesting to qualitatively compare the values of therotational and centrifugal distortion constants available for eachspecies. This comparison is performed separately for the gauche/cis speciesis slightlydifferent. Here, the similarity is less pronounced due to two facts.First, the higher energy conformer of acrolein displays a planar cisgeometry, while both butadiene and 2-azabutadiene possess nonplanargauche forms. Second, gauche-butadiene exhibits tunnelingbetween two equivalent minima, whereas no large amplitude motion occursin the heterodienes. That being said, it is likely that the differencesobserved for the two inversion states of butadiene are due to an unsatisfactorytreatment of the Coriolis interaction occurring between the 0+ and the 0– states. In this framework, thecentrifugal distortion constants of acrolein and 2-azabutadiene mightbe used as a reference to guide the analysis toward a better modeling.The case of the trans formnow can be predicted with high accuracy in a wide range of frequencies.This represents a key prerequisite for enabling astronomical searchesof this species in dense spectral line surveys of molecular-rich objects,where an unambiguous spectroscopic identification typically requiresa match within a few tens of kilohertz.Centrifugal analysis of 2-azabutadiene has another important outcome.The rotational spectrum of the most stable 55 while another isomer, i.e., 1-azabutadiene, is reported as tentativelydetected in G+0.693.28 These discoveriestell us that molecules showing the same degree of complexity as 2-azabutadienecan be formed efficiently in interstellar conditions, thus makingthe title heterodiene a good candidate for future detection. Besidesgiving information about the kinetics and thermodynamics of the C3H5N isomeric family, 2-azabutadiene can be used—togetherwith the other heterodienes—to proxy the abundance of interstellarbutadiene or even ethene. While we do not have yet any evidence thatthis suggestion might work, indirect support to this idea is providedby the relative abundance of CCH over CN cyclic-hydrocarbon derivativesin TMC-1, which are highly correlated.56 In work by Cernicharo et al.,56 it wasalso suggested that their relative abundance probably reflected theabundance of the CCH and CN radicals. In this respect, we note that2-azabutadiene can be seen as substituted ethene, where the H2CN radical is linked to the latter through the N atom.One isomer of 2-azabutadiene,namely, ethyl cyanide, has been observedin high-mass star-forming regions with remarkable high abundance,Finally, Titan’s chemistry deserves a note. Organic nitrilesand, more generally, N-substituted hydrocarbons are largely presentin the nitrogen-rich atmosphere Titan. In this view, spectroscopiccharacterization of 2-azabutadiene might be useful to gain furtherinformation on the chemistry occurring in what is considered to resemblethe primitive Earth.


The impact of benign gynecological conditions on life of women and on costs for the society is high. The purpose of this study is to gain knowledge and understanding of costs of the treatment of these disorders in order to be able to improve the clinical care processes, gain insight into feasible savings opportunities and to allocate funds wisely.The healthcare processes of 311 women attending university or community hospitals in the Helsinki and Uusimaa Hospital District between June 2012 and August 2013 due to a benign gynecological condition were followed up for two years and treatment costs analysed.Total direct hospital costs averaged 689€ at six months and 2194€ at two years. The most expensive treatment was that of uterine fibroids in the short term and that of endometriosis and fibroids later on. Costs did not depend on hospital size. Surgical operations caused nearly half of hospital costs. Productivity loss caused biggest expenses outside of the hospital. LNG-IUD (levonorgestrel-releasing intrauterine device) accounted for the largest pharmaceutical costs for patients. Hospital treatment was associated with a reduced need for outpatient services during follow-up.A majority of direct hospital costs arise over time. This stresses the need for prolonged healthcare management. To control costs, the need for repetitive doctors’ appointments, monitoring tests, and ward treatments should be carefully evaluated. Procedures not needing an operation theatre (for example hysteroscopy for polypectomy), should be done ambulatorily. The impact of common benign gynecological conditions on the life of women  and on tLimited healthcare resources mandate that more focus is set on optimizing healthcare processes and on the cost-effectiveness and opportunity costs of treatment. As the ageing of the population and the desire for improved quality-of-life are associated with high healthcare costs , it is iThis study provides new, original, short- and long-term data on the healthcare processes and costs of treatment of common benign gynecological conditions in a publicly funded healthcare setting. Our study also reports the indirect costs of treatment and the more seldom reported other out-of-hospital costs. Furthermore, the study is set to answer whether the distribution of costs differs depending on the treated condition or the hospital size and whether hospital treatment diminishes the need to seek care elsewhere and thus decreases costs and indirect societal costs. This knowledge enables better recognition of patient groups whose treatment protocols need further development.st, 2012 and August 31st, 2013 were invited to participate. Patients with uterine fibroids or some other benign uterine neoplasms, endometriosis, adenomyosis, uterine prolapse, endometrial or cervical polyps, gynecological bleeding disorder, dysmenorrhea or lower abdominal/pelvic pain or some other benign uterine finding were included in the study. The patients were grouped according to the ICD-10 classification codes after clinician’s routine assessment. If a specific condition/pathology was found behind the presenting symptoms , the patient was categorized according to this primary diagnosis. If no apparent reason for the condition was found, the patient was grouped according to this idiopathic symptom (for example idiopathic menorrhagia in the bleeding disorder group).A prospective observational cost study conducted in the obstetrics and gynecology departments of the hospitals of the Helsinki and Uusimaa Hospital District: the Helsinki University Hospital  and four smaller community hospitals. All women treated for a benign gynecological condition in these hospitals between June 1Patients were treated according to typical clinical practice in the Finnish publicly funded (taxation based) healthcare system. The clinical care process was tracked regarding doctor’s appointments, laboratory examinations, imaging, pathological examinations, outpatient clinic procedures, surgical operations, and ward/inpatient treatment in the different patient groups. The direct hospital costs were acquired from the clinical patient administration database and are reported at the current cost level of the follow-up time (from June 2012 –August 2013 (study intake) until June 2014 –August 2015 (last follow-up survey). These direct hospital costs were analysed both in short-term (six-month follow-up) and in long-term (two-year follow-up). The average exchange rate during the study period varied between 1EUR = 1.11–1.33USD.To take out-of-hospital costs into account, the patients were asked to fill in a questionnaire concerning the use of services outside of the hospital before treatment , and at the six-month and the two-year follow-up. Patients reported nurses’ and doctors’ appointments at primary health care centres or private clinics, laboratory examinations, imaging, treatment for the same condition in other hospitals or clinics, drug purchases (including pain killers (for example NSAIDs), blood loss diminishing medicines (for example tranexamic acid), progesterone derivates  and separately LNG-IUD (levonorgestrel-releasing intrauterine device)) and absence from work during a three-month time period before each follow-up. Healthcare unit costs, productivity costs in Finland , 16 and Paired samples t-test was used to test the statistical significance of the change in costs during follow-up. The statistical significance of the differences in the mean costs between two unrelated groups was tested by independent samples t-test. Two-sided p-values < 0.05 were considered statistically significant. The data were analysed using SPSS for Windows statistical software version 22.0 .The Ethics Committee of the Helsinki University Central Hospital approved the study protocol (registration number 538/E0/02). The study was conducted in accordance with the 1964 Helsinki declaration and its later amendments. Informed written consent was obtained from all participants.Of the 1173 women invited to participate, 543 (46.3%) completed the baseline questionnaire. Of these, 425 (78.3%) answered also at six months and 397 (73.1%) at two years. The inclusion criteria  were met by 389 patients at six months and 311 patients at two years. Of them, 178 (57.2%) were treated in the university hospital and 133 (42.8%) in community hospitals. Patients were treated because of bleeding disorders (n = 94), uterine fibroids (n = 79), polyps (n = 68), dysmenorrhea/pelvic pain (n = 24), endometriosis (n = 23), uterine prolapse (n = 7) or some other benign uterine disorder (n = 16). The mean duration of the long-term follow-up was 24 months 11 days (SD±12days). The mean age of the patients at baseline was 49.6 years .Of all patients, 167 (46.3%) underwent surgery in an operation theatre. Of university hospital patients 44.4% (n = 79) and of community hospital patients 48.9% (n = 65) were treated operatively . Patients with uterine fibroids or a prolapse were most often treated operatively  and patients with bleeding disorders, pelvic pain or some other benign gynecological condition least often . The only statistically significant difference in operative treatment between the hospitals was that polyp patients were more often treated in the operation theatre in the community hospitals compared to the university hospital (66.7% versus 36.8%) .The total direct costs averaged 688.6€ (SD±1098.8 €) at six months and 2193.8€ (SD±2158.6€) at two years. Treatment of uterine fibroids was the most expensive intervention at six months. At two years, the costs were highest in the endometriosis and uterine fibroids groups. The treatment of other benign gynecological conditions was less expensive during the whole follow-up . There wNearly half of total direct costs (48.4%) was caused by surgery. Doctor’s appointments, inpatient/ward treatment, and outpatient procedures all accounted for similar proportions (13–17%) . Doctor’Mean direct costs outside of the hospitals  decreased in the whole study group significantly compared to baseline (450.2€ ±339.7) at both the six-month (309.0€ ±204.9) and two-year follow-ups (281.3€ ±421.9) (p<0.001). This decrease was seen in patients treated in university hospitals , but not in those treated in community hospitals . These direct costs were similar for patients treated conservatively or operatively. However, the productivity costs were temporarily, at six months, higher for patients treated operatively than for those treated conservatively , but returned to low levels by two years (58.3€ vs. 117.7€).The highest mean direct costs outside of the hospital were at baseline in the endometriosis group (735.0€ vs. 225.4–570.2€ in the other groups) and at six months in the pelvic pain group (598.3€ vs. 221.8€ - 370.5€). At the two-year follow-up, patients with a bleeding disorder, prolapse or some other benign gynecological condition reported significantly lower direct out-of-hospital costs than at baseline (p<0.05) .At baseline, endometriosis and pelvic pain patients  and at six months patients with fibroids, pelvic pain or prolapse  had the highest mean productivity costs compared to the other patient groups. In the long-term, however, marked productivity costs were observed only in endometriosis patients (803.5€ vs. 0–89.3€ of patients in the other groups). A significant decline in these costs during follow-up was noted in patients with a bleeding disorder or pelvic pain .Absence from work and ultrasound examinations performed by private gynecologists generated the highest costs outside of the hospital at baseline followed by appointments with general practitioners at primary health care centres or private clinics, LNG-IUD purchases, and appointments with private gynecologists. The burden of sick leave declined from the six-month high back to baseline levels by two years, however, still accounting for the highest proportion of costs outside of the hospital. Already by six months, costs due to private gynecologists’ appointments had, instead, diminished. Appointments with general practitioners at primary health care centres decreased throughout the whole follow-up, whereas costs due to appointments with general practitioners at private clinics and purchases of LNG-IUD persisted throughout the follow-up. Purchases of painkillers, progesterone derivatives, and pharmaceuticals diminishing blood loss decreased from baseline to two years. Cost distribution was otherwise similar in patients of the university hospital and community hospitals throughout the study but only the costs of blood loss diminishing medicines declined significantly in the community hospitals .At baseline, patients with pelvic pain used private gynecologists’ services least frequently but visited general practitioners most regularly. At six months, the difference in general practitioners’ visits at primary health care centres prevailed, but there were no longer differences in costs caused by private clinic general practitioner visits or private gynecological services between the diagnosis groups. At two years, also the use of general practitioners’ services at primary health care centres had evened out. At two years, endometriosis patients used private gynecological services significantly more than patients with a bleeding disorder or pelvic pain .At baseline, patients with endometriosis or pelvic pain purchased more analgesics than the other diagnosis groups. This tendency persisted throughout the follow-up. At six months, endometriosis patients used significantly more oral progesterone derivatives but fewer bought LNG-IUDs compared to patients with a bleeding disorder. Patients with fibroids used blood loss decreasing medicines significantly more often at the short-term follow-up than patients with polyps. At the long-term follow-up, endometriosis patients purchased more pharmaceuticals to diminish blood loss than patients with a bleeding disorder or a polyp .Benign gynecological conditions are often thought to be minor inconveniences rather than actual diseases. However, these conditions greatly affect the quality-of-life of women , 8, 9 anPrior studies have shown annual direct hospital costs of gynecological patients to vary from $5400  to $9500  , 11. Thest century [Very few studies have analyzed how money is spent in the actual day-to-day environment of gynecological clinics. One previous study with a six-month follow-up from the same area  in Finland was published ten years ago . Only pa century  and bleeIn endometriosis patients, 29% of direct costs have previously been reported to be caused by surgery, 19% by monitoring tests, 18% by hospitalization, and 16% by doctors’ appointments . In our Our study showed the treatment of fibroids to be the most expensive in the short-term. By two years, costs of endometriosis and fibroids were the highest. This is in line with two previous studies reporting similar annual costs in these groups , 11. As Less money, on the other hand, was used on the treatment of polyps in the university hospital. In accordance with previous reports , 19, ourOur study showed that the economic burden outside of the hospital declines after treatment initiation. However, indirect costs due to sick leave do not decrease in the long-term. This finding may indicate a relatively minor need for absence from work in the study group which is reflected in stable productivity costs. The reason why treatment reduces out-of-hospital costs more in patients treated in the university hospital than when treated in community hospitals remains unclear. Since baseline expenses do not differ, the explanation cannot be in different healthcare behaviour in the more rural areas.Cost data of common benign gynecological conditions in a typical real-world setting, and based on actually realized costs, are rare. Thus, as a strength of the study, our results are valuable for future cost-effectiveness analyses. A small uncertainty is associated with the out-of-hospital costs since these data were gathered by asking patients to recall their use of healthcare services, purchases of pharmaceuticals and days absent from work. In order to minimize bias caused by uncertain memory the surveys were limited to a three-month time period, which also enabled the comparison of equal time periods. Non-medical out-of-pocket costs such as menstrual pads and incontinence pads were unfortunately not gathered from study patients which could also increase the cost burden of some of the study patient groups. The patient material of the study is that of a referral hospital out-patient clinic. The patients were grouped according to clinical assessment using the ICD-10 classification codes based on primary disease. The authors feel that this kind of classification serves clinicians and healthcare assessment better than a symptom based classification as patients most likely present with more than one lead symptom and different diseases present with partly the same symptoms. The authors also feel that this approach may facilitate comparison to data obtained elsewhere as the ICD-10 classification codes are global and often used both in clinical work and scientific studies. Clinical data of women who chose not to participate in the study is unfortunately out of reach of this study. Generally, as clinical practice and resources vary in different countries, cost data should be interpreted by taking into account factors such as local purchasing power parity. As relatively few studies have been published in this field, the possibility of international cost comparison is limited.A majority of direct costs does not arise during the first months of treatment but accumulate with time showing the need for prolonged healthcare management and cost follow-up. Lower costs could be achieved by fewer repeated doctors’ appointments, fewer monitoring tests and by re-evaluating the need for hospitalization and length of ward treatment. Procedures that do not need to be done in the operation theatre should be done ambulatorily. Hospital treatment reduces the need to seek medical attention outside of the hospital and decreases out-of-hospital expenses.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


Candidatus Liberibacter spp., which is transmitted by psyllids. The bacteria is responsible for the synthesis of callose at the phloem sieve plate, leading to the obstruction of the pores that provide connections between adjacent sieve elements, thus limiting the symplastic transport of the sugars and starches synthesized in leaves to the other plant organs. The Persian triploid lime (Citrus latifolia) is one of the most HLB-tolerant citrus varieties, but the determinants associated with the tolerance are still unknown. HLB-infected diploid Mexican lime (Citrus aurantiifolia) and Persian lime were investigated. The leaf petiole was analyzed using scanning electron microscopy (SEM) to observe callose deposition at the phloem sieve plate. Leaf starch contents and detoxification enzyme activities were investigated. In the field, Persian lime leaves present more limited symptoms due to HLB than the Mexican lime leaves do. Photosynthesis, stomatal conductance, and transpiration decreased compared with control plants, but values remained greater in the Persian than in the Mexican lime. Analysis of the petiole sieve plate in control petiole samples showed that pores were approximately 1.8-fold larger in the Persian than in the Mexican lime. SEM analyses of petiole samples of symptomatic leaves showed the important deposition of callose into pores of Mexican and Persian limes, whereas biochemical analyses revealed better detoxification in Persian limes than in Mexican limes. Moreover, SEM analyses of infected petiole samples of asymptomatic leaves showed much larger callose depositions into the Mexican lime pores than in the Persian lime pores, whereas biochemical traits revealed much better behavior in Persian limes than in Mexican limes. Our results reveal that polyploids present specific behaviors associated with important physiological and biochemical determinants that may explain the better tolerance of the Persian lime against HLB compared with the Mexican lime.Huanglongbing (HLB) is presently a major threat to the citrus industry. Because of this disease, millions of trees are currently dying worldwide. The putative causal agent is a motile bacteria belonging to  Candidatus Liberibacter spp.; Candidatus Liberibacter asiaticus (Las) (Candidatus Liberibacter africanus (Candidatus Liberibacter americanus (Diaphorina citri), which efficiently spreads the disease, is the vector of Las. HLB affects tree development, fruit quality (Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide and is caused by a phloem-restricted gram-negative α-proteobacteria (us (Las) , Candidafricanus , and Canericanus . In the ericanus . The Asi quality , and yie quality  and can  quality . Dieback quality  follows  quality , thus li quality . Sugars  quality  because  quality . Such mo quality  have con quality . When in quality .Citrus  is one of the least susceptible to HLB (Citrus x aurantiifolia (Christm.) Swing and a haploid female gamete of Citrus x limon L.  as the male parent and C. x aurantium L. and Citrus medica x micrantha Wester for C. x lemon and C. x aurantiifolia, respectively  is an important, relatively stable, non-radical ROS. At low concentrations, H2O2 acts as a signal molecule involved in the regulation of specific biological and physiological processes . Oxidative stress and eventual cell death in plants can be caused by excess H2O2 accumulation. As stress factors provoke an enhanced production of H2O2 in plants, severe damage can be induced at the cellular level. Plants are endowed with several H2O2-metabolizing enzymes and, primarily, with catalases (CAT) or ascorbate peroxidases (APX). CAT is a peroxisome-localized enzyme which do not require any reductant for catalyzing a dismutation reaction. APXs have a high affinity for H2O2 and utilizes ascorbate (AsA) as a specific electron donor (Polyploidy is recognized as a major force driving plant evolution . Polyploon donor .C. x. aurantiifolia) and the Persian triploid lime, which are known for their contrasted tolerance to HLB, respectively. We performed investigations in asymptomatic (Las+AS) and symptomatic (Las+S) leaves of infected trees compared to the control to understand the disease development process better. Control (Las−) and asymptomatic samples were collected on trees grown in distinct insect-proof greenhouses. Infection was obtained after grafting-infected budwoods to determine the precise moment of the infection. Thus, it was possible to limit the impact of other environmental factors. Symptomatic samples were collected on trees naturally infected by psyllids in the field because, in the greenhouse, we rarely acquire symptomatic leaves. The physiological leaf behavior was evaluated by measuring leaf gas exchanges. In this article, we performed anatomical measurements using scanning electron microscopy (SEM) and fluorescent microscopy, which were associated with the investigation quantification of antioxidant molecules enzymes involved in detoxification processes. Taken together, our findings provide new knowledge on the determinant of the phenotypic differentiation between Mexican limes and Persian limes, which highlighted the importance of ploidy for HLB disease tolerance within the genus Citrus.In the present work, we compared the behavior under the HLB constraint and the control condition of the Mexican diploid lime (Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) seeds from the collection of the “CRB Citrus” biological resource center (The INRAE-CIRAD of San Giuliano in Corsica (France) provided diploid Citrumelo ] and the 3x Persian lime . The budwoods of asymptomatic HLB leaves were collected from a single tree initially infected in the greenhouse by using psyllids collected in the field. Budwoods of that tree tested molecularly positive for HLB. Inverted t-budding was performed 30 cm above the ground. Six control and infected trees of each genotype were grown in 40-L pots in close, but distinct, greenhouses for 18 months with day and night temperatures of 25–32°C and 22–28°C, respectively, and a relative humidity varying between 70 and 98%. These trees were used for the physiological, microscopic, and biochemical investigations.Thirty genetically identical and uniform 2× seedlings were selected for further investigation. To compare the HLB tolerance of genotypes, controlling the development of the disease and transmitting the disease by grafting was necessary. Ten 10-month-old rootstock seedlings were grafted using budwoods collected near asymptomatic HLB leaves and control leaves of the 2× Mexican lime  and the “LB8-9” Sugar Belle® mandarin hybrid  Mexican limes. The higher callose depositions in Las+AS petioles of Mexican limes compared to Persian limes, as observed by SEM, were confirmed by the presence of a strong white ring targeting the callose deposit when using aniline blue. The level of callose deposition is associated with a higher fluorescence labeling of Las in the Mexican lime and correlates with the lower Ct values measured in that genotype compared with the Persian lime, suggesting the faster development of the bacterium and a stronger impact at the physiological level. Starch accumulation is a classical symptom of a HLB-infected tree. A large number of starch grains were visible only in the Mexican lime, emphasizing the stronger impact of HLB in that genotype, as previously observed in different aerial organs , H2O2 can give rise to high deleterious radical species: hydroxyl radicals (2+ + H2O2 would favor the production of OH. Maintaining a relatively low level of H2O2 relates to the reduction of H2O2 through APX and its use in the Fenton reaction.The Fe and Cu increase that we observed in Las+AS and Las+S was probably transient and would cause the plant to limit HLB-induced stress through the translocation of these important micronutrients from the root to the leaf. Indeed, a foliar application of Feus trees . Also, tradicals . Indeed,In the field, the triploid Persian lime is one of the most HLB-tolerant cultivars. We have shown that this variety behaves better at a physiological and a biochemical level than its diploid Mexican lime parent does. Anatomical, physiological, and biochemical differentiations due to ploidy variation explain a large part of the behavior of the Persian lime under an HLB constraint. Using SEM analysis, we observed that the Persian lime’s better tolerance to HLB was associated with the larger pore size in the sieve plate of the phloem cells of the leaf petiole compared with the Mexican lime. In addition, the investigation of infected petiole samples of asymptomatic leaves showed much larger callose depositions onto the Mexican lime versus the Persian lime, whereas symptomatic leaves showed important depositions onto Mexican lime and Persian lime pores. Our results provide insights into specific traits associated with polyploidy, such as the size of the pores of the phloem, as well as the Persian lime’s detoxification processes, which can help maintain the phloemic flow in the plant and, thus, result in a better HLB-tolerance . Thus, iThe original contributions presented in the study are included in the article/GS, LC, SB, and RB-N performed the experiments and collected the physiological data. GS, PB, PO, and RM performed the statistical analyses, interpreted the results, and drafted the manuscript. GS and OG performed the analysis by SEM. LC and OG helped to draft 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.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.


Viruses are key population regulators, but we have limited knowledge of the diversity and ecology of viruses. This is even the case in wild host populations that provide ecosystem services, where small fitness effects may have major ecological impacts in aggregate. One such group of hosts are the bumblebees, which have a major role in the pollination of food crops and have suffered population declines and range contractions in recent decades. In this study, we investigate the diversity of four recently discovered bumblebee viruses , and two previously known viruses that infect both wild bumblebees and managed honeybees  from isolates in Scotland. We investigate the ecological and environmental factors that determine viral presence and absence. We show that the recently discovered bumblebee viruses were more genetically diverse than the viruses shared with honeybees. Coinfection is potentially important in shaping prevalence: we found a strong positive association between River Liunaeg virus and Loch Morlich virus presence after controlling for host species, location and other relevant ecological variables. We tested for a relationship between environmental variables , but as we had few sampling sites, and thus low power for site-level analyses, we could not conclude anything regarding these variables. We also describe the relationship between the bumblebee communities at our sampling sites. This study represents a first step in the description of predictors of bumblebee infection in the wild. Viruses are among the most abundant and diverse groups of organisms on Earth and have a key role in regulating natural populations ; whereveApis mellifera, thus the knowledge of the ecology and evolution of viruses of bumblebees is more limited. Some of this work is transferable to bumblebees; for instance, viruses known from honeybees have pathogenic effects in the buff-tailed bumblebee, Bombus terrestris , Mayfield virus 2 (MV2), River Liunaeg virus (RLV), and Loch Morlich virus (LMV), where fitness effects have not yet been tested, and contrast this with two viruses known from honeybees, Acute bee paralysis virus (ABPV), and Slow bee paralysis virus (SBPV). We also explore whether or not there are facilitative or suppressive interactions between these viruses and explore the effect of differences in temperature, UV radiation, wind speed and precipitation on their prevalences. We show that the viruses described only in bumblebees are universally more diverse than ABPV and SBPV and that there is a strong positive association between LMV and RLV infection.Bombus muscorum were caught, but did not go forward to the extraction stage and were instead used in Samples were derived from the field collections described in n = 544) and Acute bee paralysis virus (ABPV) (n = 385); these were the only common honeybee viruses detected by next generation sequencing analyses , Mayfield virus 2 (MV2), River Luinaeg virus (RLV), Loch Morlich virus (LMV) at the individual level by RTPCR . We testanalyses .B. terrestris , Bombus pascuorum  and Bombus lucorum , each sequenced twice by BGI Genomics, once using duplex specific normalization and once using poly-A selection both of which reduced the contribution of ribosomal RNAs to the final sequencing data, and a pool of mixed Bombus species , sequenced only with poly-A selection. MV1, MV2, RLV, LMV, SBPV Rothamsted (EU035616.1) and ABPV (AF486072.2) sequences were taken from GenBank and aligned on the TranslatorX server , ABPV (median depth: 189 reads) and MV1 (median depth: 5373.5 reads). B. terrestris was analyzed for SBPV (median depth: 400 reads), MV1 (median depth: 5786.5 reads) and MV2 (median depth: 35 reads). B. pascuorum was analyzed for ABPV , SBPV , and MV2 (median depth: 5433.5 reads). The mixed Bombus pool was analyzed for all six viruses .To analyse sequence diversity, we used the raw reads from the RNA sequencing described in X server , using iX server . Post-alX server . OverlapX server . Reads wX server . Both meX server . There wX server . Varianton 1.2.1 . Base quon 1.2.1 . VariantThe number of polymorphic sites was calculated for each virus. Variants with allele frequencies of 1 were removed as these represent fixed differences from the underlying reference sequence. To measure genetic diversity, we used these counts to approximate Watterson’s estimator  for eachapprox is the approximation to Watterson’s estimator, n is the number of variants, l is the length of the sequence and p is the number of putative positives. This method makes two strong assumptions: (1) there is no mixed infection of viral variants in individuals  and (2) all variants present are detectable. The impact of deviation from the first assumption is likely to be small. The marginal change in the partial sum in the denominator decreases with every extra count, so a few missed counts will result in little change to the resultant estimate. The second assumption is more influential, given the larger impact that a missing variant has on the generated number. Given this, we acknowledge that our presented estimates may be conservative. We tested for an association between the approximation to Watterson’s estimator and the median read depth over called variants using Kendall’s tau, a method of rank-based correlation that does not make assumptions about the normality of the two variables. A 95% confidence interval was generated for the correlation using a naïve percentile bootstrap.where θClimatic data for each of the nine sites at which bees were collected was taken from the WorldClim database at 1 km resolution . PredictWe tested associations between individual prevalence and climate data using Stan version 2.18.2  via the Bombus muscorum samples that were excluded from the other analyses, so as not to bias the sampling results. We use a Dirichlet prior with these 24 categories and a concentration parameter of 1 for each category, implying complete uncertainty about the underlying probability. This has the advantage that the posterior has a known analytical form. Probabilities were estimated independently for each site. Ten thousand simulations were taken from the posterior distributions generated for each site to generate possible values of the underlying sampling probabilities of each bumblebee species at each site, which we assume to be roughly equivalent to the frequency of that bumblebee species at that site. For each of the 10,000 simulations from the posteriors at the sites, we generated estimates of the community dissimilarity using the Morisita-Horn index  appears to exhibit much higher species specificity with 13/16 detections being in B. jonellus. It was also strongly associated with RLV, with 13/16 detections being coinfections. No species other than B. jonellus were detected with LMV infection in the absence of RLV coinfection. Mayfield virus 1 (MV1) appears to be a generalist, with frequent infections across bumblebee species. Its prevalence data showed large differences between the degree of infection of different bumblebee species between sites. Edinburgh and Gorebridge, two sites around 15 km apart with large sample sizes, have dramatically different MV1 prevalences in B. terrestris, B. pratorum, and B. pascuorum, being between 30 and 60% in Edinburgh, and below 15% in all species in Gorebridge. Mayfield virus 2 (MV2) shows a similar pattern but without obvious differences in infection levels between sites. The prevalence of MV2 is generally lower than that of MV1, but beyond that, the range of species infected is largely similar. Acute bee paralysis virus (ABPV) was found at intermediate modal prevalences of above 10% in all species apart from B. terrestris and B. lucorum. The prevalence of SBPV was universally high.River Luinaeg virus (RLV) was detected in We also tested for excess co-infection beyond random between viruses using multivariate probit models that allowed us to calculate the correlation in the error terms of the multivariate normal latent variable. This measures the degree to which, after accounting for the predictors, there is still shared error, as caused by unobserved factors affecting infection risk. In this case, these measure the extent to which there is excess coinfection after accounting for the location of sampling, the species of bee and the various location-level environmental variables. Some viruses exhibited excess coinfection . RLV andViral prevalence was related to environmental covariates in some cases . Higher B. terrestris, B. pascuorum, and B. lucorum dominated communities, whereas those further north had Bombus jonellus and Bombus hortorum dominated communities  and Slow bee paralysis virus (SBPV) show considerably less diversity than Mayfield virus 1 (MV1), Mayfield virus 2 (MV2), River Luinaeg virus (RLV) and Loch Morlich virus (MLV) within the study region. ABPV and SBPV were initially described in honeybees , while tIt is likely that much of the observed variation in the “bumblebee viruses” LMV, MV1, MV2, and RLV is neutral, as most variation is at 3rd codon positions and codes for either identical or similarly charged amino acids, which are unlikely to have large fitness effects in either direction on the virus. Given the frequent bottlenecking that occurs during transmission , even muV. destructor, a parasite that can vector viruses and is associated with the prevalence of ABPV  can be found below: Sequencing data can be found at Sequence Read Archive under the following BioProject ID: LW and DO designed the sampling regime, acquired funding for the work, and provided the funding. LW, DO, and MT performed the sample collection. LW and DP performed the wet lab work. DP performed the data analysis and bioinformatics. DP and BC created the figures. DP wrote the first draft of the manuscript. All authors contributed to subsequent drafts and editing.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.


The clinical assessment of hip rotation range-of-motion (ROM) is important for managing hip and groin injuries in footballers. Previously published reliability studies on hip ROM have employed protocols that are difficult to replicate under everyday clinical conditions. This single trial, intra- and inter-rater reliability study included 41 male academy football (soccer) players, aged 14–15 years, from one European football academy. Passive hip internal rotation (IR) and external rotation (ER) ROM were measured in supine with hip and knee flexed to 90°. The ROM was determined using a smartphone application, with the smartphone attached to the lower leg. The tests were performed on two separate occasions, one week apart, by two different physiotherapists and on both sides (left and right hips). Reliability was evaluated using Intra-Class Correlation Coefficients (ICCs) and Minimal Detectable Change (MDC). Hip IR and ER ROM displayed moderate to good intra-rater agreement (ICCs 0.54–0.75), with MDCs ranging from 10.9° to 16.4°. Inter-rater reliability displayed poor to moderate reliability (ICCs 0.33–0.75), with MDCs ranging from 11.7° to 16.5°. A hip rotation ROM test using a smartphone application and a protocol closely reflecting everyday clinical conditions displayed moderate to good intra-rater reliability and poor to moderate inter-rater reliability. Due to the high MDCs, the practical applicability of this test procedure is limited and further refinement is necessary. Hip and groin injuries are common in football, accounting for 14% of all injuries in professional teams . These iHip rotation ROM is commonly assessed using a goniometer ,10,11,12Technological advances in optical and inertial motion capture have presented new alternatives for measuring joint ROM. Three-dimensional motion capture is commonly recognized as the gold standard, but its cost and the required expertise limit its clinical application.  WearableRecently, a number of studies focusing on the reliability of goniometric smartphone applications have emerged . AlthougFor clinicians applying the findings of these published reliability studies to everyday clinical practice, it is important to carefully consider the specific study population, sample size, and test procedure employed in studies . Most reIn professional sports settings, clinical tests will often be performed under time pressure, before training sessions (to decide if the player can participate), and in the absence of standardized warm-ups ,30. Hip To address these limitations, our study aimed to evaluate the reliability of a hip rotation ROM test, using a smartphone application and a test procedure closely reflecting everyday practice in the football academy soccer setting . We hypothesized that excellent reliability (ICC > 0.90) and low Minimal Detectable Change (MDC) values (<10°) could be achieved. The findings of this study can directly inform clinicians employing these tests under real-world clinical settings for the diagnosis, rehabilitation, and prevention of hip and groin injuries.The participants were male football players from two football teams (Under-15 and Under-16) in one European football academy. The teams competed in the highest national youth competition and typically played one game per week, in addition to 6–7 training sessions. The players routinely took part in a bi-annual musculoskeletal screening test battery, including the hip rotation ROM test investigated in this study. All players presenting to the routine screening test in December 2020 were eligible for participation. Current injuries, including hip/groin pain, were not exclusion criteria unless the team’s medical staff judged the injury to be a contraindication for hip ROM testing. A-priori sample size calculations, based on the methods of Walter et al.  were conPassive hip IR and ER ROM were measured in two separate testing sessions, separated by seven days. On each occasion, the players were tested on both the left and right hips by two different testers (both physiotherapists with four years of experience). The testers were in separate rooms, and the order in which players were tested in each session (physiotherapist 1 and physiotherapist 2) was randomized, using block randomization due to the different school timetables of the players. Additionally, both the testers and participants were blinded to the hip rotation ROM results, with an assistant recording the values. In order to replicate normal conditions for testing hip rotation ROM in the academy, the sessions were conducted with a single measure prior to training with no warm-up.TM document. The same procedure was used to test hip ER  for analysis. The normal distribution (Shapiro-Wilk test) and the variance homogeneity (Levene’s test) were calculated. For all data satisfying statistical assumptions (variation and homogeneity), Intraclass Correlation Coefficients (ICC), Standard Error of Measurement (SEM), and Minimal Detectable Change (MDC) were calculated. For ICC, two-way mixed models with absolute agreement  were used. Descriptive statistics and ICC values were calculated in SPSS, while SEM and MDC were calculated in Excel, using the equations of Weir (SEM = SD × √ (1 − ICC) and MDC = SEM × 1.96 × √2) [The raw data were entered into Microsoft Excel96 × √2) . The Coe96 × √2) . Spearma96 × √2) .p > 0.05) for both sessions and testers. For the left hip, all data with the exception of ER from one tester in the first session were normally distributed (p > 0.05). The TR of the left hip by tester one was normally distributed in both sessions, as was the TR for the right hip of tester two in the first session (p > 0.05). There was no significant difference in the variance homogeneity for all normally distributed data (p > 0.05). Descriptive statistics for hip rotation measurements are presented in A total of 41 players (82 hips) were tested in the first session. For the second session, there were 11 dropouts: three due to injury, one due to illness, and seven due to COVID quarantine measures. Accordingly, 30 players (60 hips) were included in the final analysis. The data for IR and ER of the right hip were normally distributed (s) indicated moderate agreement (0.47–0.67). The ICCs, SEMs, MDCs, and CVs for inter-rater reliability of hip rotation measurements are displayed in The mean IR hip ROM of the participants ranged from 40.4° to 43.5° (SD 7.2° to 7.9°), while the mean ER hip ROM ranged from 58.4° to 60.7° (SD 7.0° to 8.8°) and the mean TR hip ROM ranged from 98.9° to 104° (10.6° to 12.9°). The ICCs, SEMs, MDCs, and CVs for intra-rater reliability of hip rotation measurements are displayed in Hip and groin injuries are a major issue in elite football ,2, and aDirect comparison of the study findings to previously reported studies is difficult due to differences in study populations and protocols. Charlton et al.  found moTaken together, the above research findings suggest that hip rotation ROM can be measured with a high level of reliability on healthy subjects under controlled conditions ,26,28. TThe MDC values can assist clinicians in interpreting hip rotation ROM measurements, with values beyond the MDC indicating, with 95% probability, an actual change as opposed to measurement error. For intra-rater reliability, MDCs in our study ranged from 10.9° and 13.4° for hip IR, slightly lower than the values (range 12.6° to 19.1°) reported by St. Pierre et al. . In our A secondary outcome of this study is the normative data for hip rotation ROM in male academy football players. The mean IR values (40.4° to 43.5° across testers and left/right hips) and ER values (58.7° to 60.7°) align with the findings of one previous study in ten academy athletes, 15.3 ± 1.6 years old, four of whom were football players . AnotherA strength of this study was the large sample size with 41 participants compared to 20–24 participants in previous studies ,26,28. AAs two smartphones were used, angular position differences between the two inertial sensors may have been a source of error . To our A supine hip rotation ROM measurement, using a smartphone application, demonstrated moderate to good intra-rater reliability and poor to moderate inter-rater reliability. The MDCs for IR and ER ROM ranged from 10.9°–16.4° for measurements by the same tester and 11.7°–16.5° for measurements between testers. The test protocol in this study closely simulated normal clinical conditions, but the high MDC values limit its practical application. Employing a standardized warm-up, extensive training of testers, and improvement of the smartphone fixation to the leg might improve reliability values, but this remains to be proven.


Flaviviridae have a wide global and economic impact due to high morbidity and mortality. Flaviviridae infection usually leads to severe, acute or chronic diseases, such as liver injury and liver cancer resulting from hepatitis C virus (HCV) infection, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) caused by dengue virus (DENV). Given the highly complex pathogenesis of Flaviviridae infections, they are still not fully understood at present. Accumulating evidence suggests that host autophagy is disrupted to regulate the life cycle of Flaviviridae. Organelle-specific autophagy is able to selectively target different organelles for quality control, which is essential for regulating cellular homeostasis. As an important sub process of autophagy, lipophagy regulates lipid metabolism by targeting lipid droplets (LDs) and is also closely related to the infection of a variety of pathogenic microorganisms. In this review, we briefly understand the LDs interaction relationship with Flaviviridae infection, outline the molecular events of how lipophagy occurs and the related research progress on the regulatory mechanisms of lipophagy in Flaviviridae infection. Exploring the crosstalk between viral infection and lipophagy induced molecular events may provide new avenues for antiviral therapy.Diseases caused by  Flaviviridae consists of a group of enveloped viruses with positive single strand RNA genomes, including hepatitis C virus (HCV) of the genus Hepacivirus, dengue virus (DENV) and Zika virus (ZIKV) of the genus Flavivirus, bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) of the genus Pestivirus  disease, placing nearly 3.9 billion people, mainly in poor countries, at constant risk of infection due to the lack of completely effective vaccines and antivirals against DENV. Recently, an increasing number of studies have shown that Flaviviridae extensively regulate host lipid metabolism, underscoring the importance of LDs in viral infection , as an intracellular degradation system that maintains cellular metabolic homeostasis and cell survival when cells face various stresses such as nutrient deprivation and accumulation of damaged organelles, is an essential defensive cellular program. Autophagy for the degradation of various organelles has been well-known in the last century, but its function in the degradation of LDs has been intensively studied only in recent years  catalyze the last step in the TG biosynthesis pathway, while acyl-CoA cholesterol acyltransferases 1 and 2 (ACAT1 and ACAT2) determine CE synthesis   for subsequent delivery to the autolysosome in which the LDs are degraded, the molecular basis of which is the attachment of tagged cargo to the autophagosome via adaptor proteins  can exploit host resources, evade host immunity mechanisms and hijack autophagy to facilitate its own replication, which may also be a potential mechanism by which they utilize LDs   was reduced and inversely correlated with lipophagy induction in DENV infected-cells. Although AMPK normally stimulates tuberous sclerosis complex 2 (TSC2)-dependent inactivation of mTORC1 signaling , and recently it was found to play a relevant role in lipophagy induced by DENV and other flaviviruses , decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication  RNA viruses  has the potential to be new observational approach of metabolic disorders as well as infectious diseases.Over the past decade, our knowledge of the importance of viral remodeling of cellular metabolism has increased dramatically. Although many examples have been well-documented, there is still relatively little understanding of the detailed mechanisms of how these metabolic alterations are promoted. LDs are the crux of replication assembly of flavivirus control  and Forkhead box protein O1 (FoxO1), calcium signaling transduction, small molecules including ethanol, etc. How these cues relate to each other, and what are the triggering molecular mechanisms specific to lipophagy are unclear. To this end, the dynamic relationship of lipophagy and viral infection remains to be further explored.KW: writing—original draft preparation. SF, LZ, and FZ: writing—review and editing. MZ, HY, and JC: conceptualization, supervision, and project administration. All authors contributed to the conception and design of the work. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.