Eucalyptus blakelyi and yellow box, E. melliodora, occurred in similar proportions irrespective of vegetation density; (2) E. blakelyi had the largest number of stems and basal area, but while E. melliodora had fewer stems, it had a similar basal area to E. blakelyi. E. melliodora also showed fewer signs of dieback than E. blakelyi with between 40–50% trees in good condition compared to 2% for the latter species; (3) woody debris loads were low compared to other studies in woodland, but there were levels of heterogeneity indicating ‘natural’ accumulation was occurring; (4) tree basal area and woody debris loads had a 1:1 relationship across all sites and vegetation densities. Overall, our study indicated that ecosystem recovery was taking place (i.e. with many young trees), but there were fewer large trees that are known to supply most woody debris. Our findings highlight the slow accumulation of this critical resource because the volumes were lower than expected. Based on our results, we recommend: (1) aiming for approximately a 50:50 ratio of yellow box to Blakely's red gum basal area in woodland restoration projects; (2) to accelerate the recovery of woodland structure, addition of woody debris should be added at a minimum ratio of 1:1 to standing basal area  (3) managing for both volume and heterogeneity of woody debris loads; (4) preserving large diameter trees to harness proportionally higher woody debris and litter inputs.Simplification of stand structure of forests and woodlands through human-induced modification is a serious threat to biodiversity. Restoring lost habitat complexity and heterogeneity, such as woody debris, requires an understanding of the relationships between different elements that contribute to stand structure. In this study, we examine the structure and composition of a critically endangered box-gum grassy woodland in south-eastern Australia and relationships with woody debris loads. We found that: (1) despite modification by humans and differing susceptibility to dieback, the two dominant tree species, Blakeley’s red gum,  The structure and composition of vegetation is critically important for maintaining biodiversity, ecological processes and ecosystem services , 2. For Studying the structure and composition of a woodland provides information about its past, and its potential future , 17. ManEucalyptus melliodora) and Blakely’s red gum (E. blakelyi) (ACT Government 2004a). Together with white box , these woodlands once covered a vast area of south-eastern Australia prior to European settlement [The maintenance, restoration and spatial expansion of woodlands through active management is particularly important where an ecosystem type is highly modified and greatly reduced in extent. A good example is the box-gum grassy woodlands in south eastern Australia, which occur in an internationally recognised endangered ecoregion . Box-gumttlement , 20. Howttlement –22. As attlement , with fettlement . Consequttlement , 26.A key effect of the process of modification of woodlands by humans is stand structural ‘simplification’ . This is a result of: (1) removal of woody debris for firewood, ‘tidying up’ and prescribed burns to manage fuel loads, (2) removal and prevention of regeneration of shrubs and trees, (3) clearing and thinning of trees , 27. SimTo restore highly modified or destroyed woodlands requires an understanding of current woodland structure and composition and relationships between key elements. Through better understanding of current, simplified, woodland states we can plan and implement interventions that aim to reverse this process , 29.In this study, we examined relationships between measured vegetation structure and woody debris loads in box-gum grassy woodlands in the northern part of the Australian Capital Territory (ACT) in two adjacent reserves with different management histories. Our aim was to answer three questions:What are the structural and compositional characteristics of woodlands, and how do proportions of dominant species vary?What are the predicted volumes of woody debris across vegetation density classes?What is the relationship between woodland stand basal area and woody debris loads?We make recommendations for future research and conservation in box-gum grassy woodlands specifically, and that also have broader relevance to woodlands generally.The study area was in north-eastern ACT, and comprised two adjacent nature reserves—Mulligans Flat and Goorooyarroo Nature Reserves see . Togethewww.mfgowoodlandexperiment.org.au) [The reserves are the location of a major designed ecological restoration experiment, “The Mulligans Flat–Goorooyarroo Woodland Experiment” (.org.au) , 29. TheOur multi-level experiment consists of 24 polygons, with four 1 hectare sites per polygon High tree cover, low shrub cover (HTLS)Low tree cover, low shrub cover (LTLS)Low tree cover, high shrub cover (LTHS)These classifications describe the vegetation structure (i.e. density) of each polygon, with ‘High Tree’ and ‘High Shrub’ meaning ‘dense’ structure, and ‘Low Tree’ and ‘Low Shrub’ meaning ‘open’ structure.Woody debris and tree inventory data were collected at each of the 96 1 ha sites . SurveysAll woody debris (>2cm diameter) was measured on each transect, always commencing at the same relative side of the site, moving in the same direction. Woody debris was assessed using a continuous line intercept procedure , 32, recWoody debris volume was calculated using the line intercept method , 32 by aWoody vegetation was measured within the 10 m wide ‘bands between the tape transects used for woody debris measurement  being reserve, polygon and site. An appropriate statistical modelling framework for assessing the effects of ‘reserve’, ‘vegetation’ and their interaction, and other relationships, was a general linear mixed model with random effects for polygon and site  and number of stems (between 25:75 and 30:80) for yellow box versus Blakely’s red gum, the proportion did not change significantly with reserve or vegetation classification.3  to 2.28 m3  in Mulligans Flat and from 7.54 m3  to 1.93 m3  in Goorooyarroo.Predicted volume of woody debris (derived by back-transformation) varied between reserves and vegetation class . The preThere was no evidence of any variation in heterogeneity of pattern of woody debris loads within sites (i.e. between bands–2 to 6.05 m2 (a 1% increase), woody debris would be predicted to increase from 3.11 m3 to 3.15 m3 (a 1% increase) tonnes per ha. Although this relationship is strong and statistically significant, prediction of woody debris at specific levels of basal area is imprecise due to the high variability around the relationship between the two variables.Fine woody debris (2–10 cm diameter) and coarse woody debris (>10 cm diameter) were analysed separately, but the result was similar to that for both combined (termed “woody debris”). There was a strong and highly significant  and species associations (e.g. beetles) that each species supports. In this study, the proportions of yellow box basal area and stems to Blakely's red gum was the same in both reserves and across all vegetation classes. This is important information, as each species contributes different ecological values to the woodland environment. For example, McElhinny et al. (2010) found that the amount of litter input from yellow box and Blakely's red gum was not significantly different. However, they did find that Blakely's red gum produced significantly higher loads of leaf and other kinds of litter whereas yellow box had significantly more fine twig litter. These litter differences affect beetle assemblages and functional groups, and a proportion of the beetle assemblages associated with both species are mutually exclusive . Yellow ucophaea . Large Bainsonii  and provainsonii . TherefoBlakely's red gum had a larger basal area and a greater number of stems than any other species. This supports the observation that the species can regenerate vigorously given suitable conditions. Stands of yellow box, the next most abundant tree species in our area, were characterised by considerably fewer stems, but a similar basal area because individuals of the species had a larger mean DBH. Yellow box also was considerably healthier (i.e. less dieback) than Blakely's red gum. These factors, and the consistent proportion of the two species across reserves and vegetation classes  than those published for box-gum grassy woodlands elsewhere [3 per ha (+/- 2.6) for yellow box communities, and Killey et al. (2010) predicted 7.0 m3 per ha . The estimates in this study are also less than those estimated CWD volumes in [3 to 247 m3 per ha, mean 34 m3 for both reserves). The relatively low volumes found in these woodlands reflects the DBH (i.e. age) profile of the trees. Killey et al. (2010) studied yellow box in Goorooyarroo and found that yellow box trees that were 100cm DBH were 10 times more likely to produce CWD than a 50cm DBH tree, and produced 10 times more CWD volume. Most trees measured in this study were under 40 cm DBH, with few over 50 cm DBH. So while this DBH profile indicates a woodland ecosystem in recovery (i.e. many young trees), and the heterogeneity of the woody debris (see below) indicate ‘natural’ falls, the low volumes highlight the slow rate of accumulation of this critical resource. as part of the recovery process. This is an issue that potentially acts as a barrier to persistence of biodiversity and its recovery [This study adds to the growing body of work on woody debris loads in box-gum grassy and similar woodlands , 45, 46.lsewhere , 48. Thelumes in . Results also indicate the level of ‘natural’ variability in woody debris patterns, and indicate that this should be replicated if woody debris is being added to a site.2 basal area per hectare (see above) could constitute 7.6 trees at 100 cm DBH, or 30.5 trees at 50 cm DBH.There was a strong 1:1 relationship between overall basal area of a site and woody debris volumes. This means that managers of areas of box-gum grassy woodlands from which woody debris has been removed can use the basal area of a site to determine what the minimum woody debris volume should be. An example of such a situation could be where a paddock with only scattered trees is to be restored to functional box-gum woodland. Logically, if there are more, larger trees per ha, basal area can be maintained in a woodland but that woodland can be open (i.e. fewer stems) and still produce the same amount of woody debris and litter. For example, 5.99 mPrior to European settlement, there would have been more, large eucalypts in box-gum grassy woodlands . More, lIn light of our findings, management of box-gum grassy woodlands should consider the following:aim for approximately a 50:50 ratio of yellow box to Blakely's red gum basal area in woodland restoration projects;2 requires a minimum volume of 3.11 m3), and higher volumes if possible to enhance biodiversity [add deadwood to accelerate the recovery of woodland structure, and should be, at a minimum, added proportionally 1:1 to standing basal area  harness proportionally higher woody debris and litter inputs, (b) create more open woodland while maintaining these inputs. This could be achieved by encouraging tree regeneration or planting in woodlands with low tree and low shrub cover and/or maintaining woodland structural and compositional heterogeneity i.e. have a range of all four broad vegetation classifications.The field sampling approach and resulting data collected for this study provide the basis for tracking the effects of differing woodland structures and composition on biodiversity and ecological processes. In future, it will be important to model how tree populations change through time, and in relation to existing vegetation structure and experimental treatments, and how this affects biodiversity and ecosystem processes. The relative recruitment and survival of small stems of yellow box and Blakely's red gum, and the associated cascading ecosystem effects on biodiversity will be important information for managers in deciding whether or not to intervene with management actions. Although the characteristics such as woody debris volume and heterogeneity within these reserves suggest a ‘natural’ or undisturbed woodland, the paucity of larger trees reflecting a recovering ecosystem rather than a ‘pristine’ benchmark site. The recovery of key structures in these box-gum grassy woodlands has probably been taking place for a longer period than the formal designation of the reserves. Our results highlight the slow accumulation of woody debris, even after vegetation recovery has been underway for some time. This supports the argument for the addition of woody debris in such woodlands in Australia, and also may have applicability to other woodlands elsewhere.


Ficus pumila L. as a factor that be effective against human T-cell leukemia virus type 1. The significant and novel findings is that symptoms of patients with drinking Ficus pumila L. extracts did not worsen despite a lack of aggressive pharmacotherapy against adult T-cell leukemia, a human T-cell leukemia virus type 1-associated myelopathy, or T-cell leukemia virus type 1 uveitis. Twenty-eight of the 194 inpatients who underwent showed high levels of human T-cell leukemia virus type 1.Human T-cell leukemia virus type 1 was isolated as the retrovirus to be identified in humans. Here, we focused on Ficus pumila L. extracts had no human T-cell leukemia virus type 1-related symptoms, while those who were not administered Ficus pumila L. extracts had human T-cell leukemia virus type 1-related diseases and a significantly poorer prognosis. This suggests that the Ficus pumila L. extracts may show some utility against virus infection.Among human T-cell leukemia virus type 1-infected patients, those who were administered  Human T-cell leukemia virus type 1 (HTLV-1) has been isolated from humans. HTLV-1 is a type C retrovirus that is primarily endemic to Japan, Central and South America, the Middle East, regions of Africa, and the Caribbean. HTLV-1 and related diseases are particularly common in southwestern Japan, especially in rural areas of Kyushu and Okinawa, and Japan is the only developed nation that is an HTLV-1-endemic country.HTLV-1 was first isolated and described by Montagnier and Gallo, and is the first pathogenic retrovirus to be identified in humans. HTLV-1 is a retrovirus that causes malignant tumors of cluster of differentiation 4 (CD4)-positive T lymphocytes, namely adult T-cell leukemia (ATL), along with other diseases such as HTLV-1-associated myelopathy (HAM), an intractable neurological disorder, and HTLV-1 uveitis (HU). ATL tends to be more common in men, and HAM and HU tend to be more common in women, and the virus is mainly transmitted from men to women. Given that women are particularly susceptible to HTLV-1 infection, carrier mothers are forced to wean their infants shortly after childbirth, causing anxiety and mental distress.Ficus pumila L.), inhabits warm areas, such as Okinawa, where is found in rocky areas ) and nine were male (median 88.1 age (82–94 years old)). Of these, 12 inpatients developed symptoms (42.8%); 9 were women and 3 were men. Specifically, 8 were HAM, 2 were ATL, and 2 were HU. HTLV-1-infected cells undergo up-down regulation of microRNA (miRNA) [reatment .Fig. 2TFicus pumila L. extracts decreased soluble interleukin 2 receptor levels, which is a marker of lymphoma, and improved lower limb muscle weakness and gait disorder due to slowly progressive bilateral leg spasm paresis. The ability to walk was restored and urinary tract infections, pollakiuria, and constipation due to bladder and rectal disorders all improved.Although no significant difference was observed between treatments, drinking Ficus pumila L. may inhibit viral transcription and replication, and therapeutic agents for HTLV-1 may be effective against coronavirus 2019-nCoV infection. Although HTLV-1 is zoonoses - the origin of a part of African HTLV-1 was monkeys, it was able to infect human cells due to mutations resulting from repeated RNA rearrangements. RNA is a key component of all multicellular life, and the fact that it is involved in animal and human transmission is thus unsurprising. If Ficus pumila L. can suppress the replication of the HTLV-1 retrovirus, and if HTLV-1 therapeutics work against single-stranded RNA virus infections, then Ficus pumila L. may be an effective therapeutic agent against 2019-nCoV retroviral infection. This suggests that the Ficus pumila L. extracts may show some utility against coronavirus 2019-nCoV infection.Since coronavirus is a single-stranded, plus strand RNA virus and the genome itself acts as mRNA, it is presumed that up-down regulation by miRNA in the early stage of infection is effective for therapeutic miRNA. Ingestion of flavonoid-rich Due to the small number of subjects, this study cannot show a cause–effect relationship strictly. Social acceptance and recall bias were also possible confounding factors.


Viruses are possible pathogenic agents in several autoimmune diseases. Sjögren’s syndrome (SS), which involves exocrine dysfunction and the appearance of autoantibodies, shows salivary gland- and lacrimal gland-oriented clinical features. Epstein-Barr virus (EBV) is the most investigated pathogen as a candidate that directly induces the phenotype found in SS. The reactivation of the virus with various stimuli induced a dysregulated form of EBV that has the potential to infect SS-specific B cells and plasma cells that are closely associated with the function of an ectopic lymphoid structure that contains a germinal center (GC) in the salivary glands of individuals with SS. The involvement of human T-cell leukemia virus type 1 (HTLV-1) in SS has been epidemiologically established, but the disease concept of HTLV-1-associated SS remains unexplained due to limited evidence from basic research. Unlike the cell-to-cell contact between lymphocytes, biofilm-like structures are candidates as the mode of HTLV-1 infection of salivary gland epithelial cells (SGECs). HTLV-1 can infect SGECs with enhanced levels of inflammatory cytokines and chemokines that are secreted from SGECs. Regardless of the different targets that viruses have with respect to affinitive lymphocytes, viruses are involved in the formation of pathological alterations with immunological modifications in SS. The pathogenesis of Sjögren’s syndrome (SS), which affects salivary glands (SGs) as well as lacrimal glands (LGs), involves multiple factors including genetic elements ,2,3 and In addition, viral infections are profoundly associated with the activation of innate immunity, followed by an acquired immune response. Recognition of single-stranded ribonucleic acid (RNA) by toll-like captor 7/9 was a representative sensor of innate immunity in SS ,9. With An acute or chronic viral infection is thought to play a crucial role as a triggering phase of the immunological instability that is required for the formulation of the chronic inflammatory condition in AIDs. As an initial step of innate immunity that involves several functions of cytotoxic T lymphocytes or natural killer T cells, the recognition of mobilized pathogens by receptors of innate immunity is required. Pathogen-associated molecular patterns (PAMPs) are common structures detected in pathogenic microbes that contribute to the initiation of innate immunity . In concIn our previous study, the expression of downstream signal transduction after stimulation with the TLR7 ligand was confirmed in the salivary glands of patients with SS and in an in vitro investigation using cultured epithelial cells obtained from SS patients , and pDCA relationship between typical AIDs and viral infection has been clarified over the past three decades. With respect to outbreaks of type 1 diabetes mellitus, the involvement of coxsackie B virus (an enterovirus) has been suggested, but the epidemiological evidence remains controversial regardless of a systematic review of 26 case-control studies . In contRegarding the relationship between pathogens and the axonal form of Guillain-Barré Syndrome (GBS) with the appearance of antibodies against GM1 or GD1b, the involvement of Campylobacter infection as an exposure to bacteria has been discussed , and theConcerning connective tissue diseases, there are debates with respect to the relationship between systemic lupus erythematosus (SLE) and some viruses , and theHuman T-cell leukemia virus type 1 (HTLV-1) has been vigorously investigated because HTLV-1 transgenic mice showed chronic erosive arthritis that resembled the synovitis observed in RA ,26. HoweEBV is a member of the family of herpes viruses. It is a double-stranded DNA virus, and the nomenclature of EBV is human herpes virus 4 (HHV-4). EBV was originally isolated from culture medium of Burkitt tumor cells , and EBVIn the field of rheumatology, the involvement of EBV in methotrexate (MTX)-associated lymphoproliferative disorder (MTX-LPD) is a considerable problem in the era of molecular-targeted therapy for RA . In addiWith respect to the relationship between SS and EBV infection, our PubMed search identified 149 reports, with many conflicting results and opinions regarding SS per se and EBV-associated hematological tumors. Representative studies about the detection of EBV in SS are listed in A first definitive study  regardinIt was reported that, compared to control subjects, the titer of anti-ENBA antibodies but not that of anti-cytomegalovirus (CMV) antibodies was higher in sera from SS patients, and anti-EBNA antibodies were dominantly detected in anti-La/SS-B antibody-positive patients with SS . VenableBecause benign lymphoepithelial lesions (BLELs) are unique pathological features in SS, Di Giuseppe et al. examined the involvement of EBV in BLELs : they den = 10) were diagnosed with EBV-associated follicular lymphoma, and 8 of these 10 patients showed a positive expression of LMP1 [Regarding the involvement of EBV in the pathogenesis of lymphomas, Royer et al. examined the occurrence of non-Hodgkin’s lymphoma (NHL) and they identified cases with low-grade marginal zone lymphoma (MZL) and mucosa-associated lymphoid tissue (MALT) lymphoma; however, no obvious association between these SS and EBV infection was observed based on the presence of LMP proteins and EBER RNA . In addi of LMP1 .The word ‘reactivation’ in SS was first used in the 1980s by researchers examining the reactivity of monoclonal antibodies against EBV in salivary glands of individuals with SS ,62. SincISH was later used to detect EBV-specific DNA in patients with secondary SS in which epithelial cells positive for EBV DNA were observed around areas with salivary gland destruction or lymphoepithelial lesions . By usinRegarding a link between tumorigenesis and EBV reactivation, increased reactivity was demonstrated toward EBV EA proteins such as BHRF1  . A clearImmunological and virological considerations have shaped the perspective on the involvement of EBV in the pathogenesis of Sjögren’s syndrome. Yamaoka et al. reported an increase in the proportion of polyclonal B cells in accord with the elevation of antiviral capsid antigen in SS . DiffereRegarding the hypothesis of the direct involvement of EBV in SS, positive reactivity to EBV-related nucleo-cytoplasmic antigen was reported in 24 patients with SS, although the reactivity data differed between immunofluorescence and immunoblot analyses . CorrelaAlpha-fodrin was indicated as a possible autoantigen for SS , and Ino+ B cells and CD138+ plasma cells around ELSs expressed EBER. (3) The expression of epithelial LMP was observed in both SS and nonspecific sialadenitis. (4) An antigen expressed during the lytic cycle, BFRF-positive CD138+ plasma cells that EBV lytically infected around ELSs, displayed Ro52. (5) SCID mice that received transplants of SS salivary gland tissue containing ELSs exhibited the ability to produce anti-Ro52/SS-A and anti-La/SS-B antibodies. (6) EBV that had increased affinity to ELSs in SS salivary glands was closely associated with impaired CD8+ T-cell cytotoxicity. These observations clearly demonstrated that the lytic phase of EBV has affinity to both ELSs and plasma cells with the capability to produce SS-specific autoantibodies.In the most recent development regarding the pathogenesis of SS, Croia et al. demonstrated SS-specific B-cell reactivation by EBV in their examination of ectopic lymphoid structures (ELSs) . AlthougRepresentative studies on the involvement of retroviruses in SS are listed in The possibility of retrovirus infections other than HTLV-1 infection in individuals with SS was a research focus in the early 1990s. In 1989 it was reported that patients with HIV-associated salivary gland disease (HIV-SGD) showed a high frequency of parotid enlargement, sicca symptoms, and a low CD4/8 ratio in LSG biopsy specimens . Interes+ T cell-dominant infiltration was observed in salivary gland biopsy specimens without anti-Ro/SS-A, La/SS-B antibody [The diagnostic criteria of DILS are as follows: (1) HIV infection with positive serology, (2) bilateral salivary gland enlargement or xerostomia, (3) persistence of signs or symptoms for ≥6 months, and (4) histologic confirmation of salivary or lacrimal gland lymphocytic infiltration without granulomatosis or neoplastic involvement. All four of these criteria must be met for the diagnosis of DILS. A difference in the T-cell receptor (TCR) beta-chains was also revealed between SS patients and subjects with DILS . In a coantibody . An asseantibody . In theiantibody . Their fThe presence of human retrovirus-5 (HRV-5) in salivary glands was examined by nested PCR in 55 SS patients and 37 non-SS subjects, and no association of HRV-5 with SS was observed . The estIn parallel with investigations of involvement of the HIV in Sjögren’s syndrome, the engagement of HTLV-1 in SS has been researched. In this section, we summarize the clinical and epidemiological studies of the association between HTLV-1 infection and SS. HTLV-1, a member of the type C delta-retrovirus family , is a caAlthough several case reports regarding SS associated with HTLV-1 infection are available, it was initially reported that HTLV-1 carriers with SS showed high frequencies of extra-glandular manifestations including uveitis and myopathy . It was The high prevalence of SS in HAM patients was also observed in a later investigation in which chronic sialadenitis and exocrine dysfunction were detected by Saxon test and Schirmer’s test, respectively . MagnetiWe also re-evaluated the complications of SS experienced by patients with HAM 18 years after we reported the association , becauseVale et al.  examinedtax gene  was detected by both ISH and PCR in labial salivary glands of two of nine patients with SS. They also observed the localization of tax in the nuclei of both SGECs and lymphocytes. Sumida et al. [tax gene in labial salivary glands from Japanese patients with SS, and they reported that the nucleotide sequence of the pX region in labial salivary glands was completely identical to that of the MT-2 cell line, suggesting that the findings of these two studies [tax gene might be involved in the pathogenesis of SS.Similar to the involvement of EBV in SS, the infection of salivary glands by HTLV-1 has also been debated. In 1992, it was reported that the expression of HTLV-1 p19 (a member of the family of gag antigens) was observed in SGECs from 31% of SS patients, 24% of RA patients with SS, and 21% of patients with sicca , suggesta et al.  later de studies ,107 supptax gene and the local synthesis of IgA class anti-HTLV-1 antibody in saliva was discussed. In contrast, a study of salivary glands from 49 patients with SS in Great Britain demonstrated no tax expression unlike that of ERV-3, an endogenous human retrovirus [In an aforementioned study conducted in Nagasaki , the sertrovirus , suggesttax gene in 18% of the salivary glands of HTLV-1-seronegative SS patients by nested PCR [tax gene was expressed in LSGs from these patients [With respect to HTLV-1-seropositive patients with SS, an investigation using an in situ PCR technique detected HTLV-1 proviral DNA in infiltrating MNCs but not salivary gland epithelial cells of these patients . In contsted PCR , suggestpatients . Sasaki patients .tax gene in LSGs from SS patients and patients with chronic inflammatory diseases [tax gene in 30% of the SS patients and 28% of the non-SS patients. These results suggested that HTLV-1 may contribute to the development of chronic inflammation regardless of the presence/absence of SS. Although the number of publications concerning HTLV-1-related genes in the salivary glands of individuals with SS has decreased since 2000, a 2012 report from Korea noted that tax gene was detected in 3.8% of a series of SS patients, and HTLV-1 p19 or Tax protein was revealed by IHC in LSGs from >40% of the SS patients [HBZ) and tax gene by ISH, which showed that both genes expressed in infiltrating MNCs and SGECs from HAM-SS patients and a patient with adult T-cell leukemia (ATL), although the expression of tax gene was dominant in MNCs of the HAM-SS patients [As for other inflammatory conditions, Mariette et al. examined the expression of diseases . They depatients . With repatients   mice [tax Tg mice showed human SS-like pathology including cellular infiltration as well as a proliferation of ductal epithelial cells. It was also demonstrated that the introduction of env-pX gene to Wistar-King-Aptekman-Hokudai (WKAH) rats induced human SS-like characteristics such as chronic sialadenitis and dacryoadenitis as well as arthritis, necrotizing arteritis, polymyositis, myocarditis and dermatitis with elevated RF [env-pX rats showed a direct involvement of HTLV-1 infection in the induction of autoimmunity.The association between the pathogenesis of SS and HTLV-1 infection has been addressed with experimental approaches including in vitro studies, animal studies, and studies with human tissue samples including salivary gland tissue. As a catalyst of experimental studies, Green et al. developed HTLV-1 Tg) mice . Interesvated RF . These cConcerning the involvement of HTLV-1 infection in WKAH rats, Yoshiki et al. showed that one of their WKAH rats that possessed the RT1k haplotype presented neurological symptoms resembling HAM , suggestIn our study of the involvement of anti-apoptotic molecules in HTLV-1-seropositive SS patients, we investigated the expression of Bcl-2 family proteins in PBMCs and LSGs from SS patients , becauseWe subsequently detected members of the mitogen-activated protein kinase (MAPK) superfamily  includin+ T cells [+ T cells in the salivary glands of HAM-SS patients with hypergammaglobulinemia resembles that observed in DILS. In addition, we treated an ATL patient with bilateral parotid gland enlargement, xerostomia, and xerophthalmia [+ T cell-dominant lymphocytes, antibodies against Ro/SS-A, La/SS-B were negative. There are differences in the phenotypes of infiltrating T cells, but the existence of DILS in HIV-positive patients and a patient with ATL suggests the possibility that a massive infiltration of T cells with sicca symptoms would fulfill the classification criteria of SS [Concerning chronic lymphocytic infiltration in salivary glands without specific autoantibodies in SS, the DILS observed in salivary glands of individuals affected by HIV with the infiltration of CD8 T cells  is a remhthalmia , and altia of SS ,150.+ T cells [Lastly, we discuss the immunological alterations observed in that area after the integration of HTLV-1 into host non-lymphoid cells including SGECs. HTLV-1 infection confirmed by semiquantitative PCR or ELISA was observed in 2.6% of retinal glial cells along with the production of inflammatory cytokines including IL-6 and TNF-α . A simil T cells .Regarding the HTLV-1 infection of SGECs from SS patients, we confirmed HTLV-1 proviral DNA and proteins by in situ PCR and immunofluorescence, respectively after co-culture with an HTLV-1-infected T-cell line ; in addiWe have outlined the involvement of viruses  in the pathogenesis of SS . These ttax or env-pX Tg animal models and in vitro studies. However, unlike EBV infection, in HTLV-1-infected subjects there is no direct evidence indicating molecular biological effects on the production of autoantibodies in SS patients.EBV showed robust affinity to the ELS, whereas HTLV-1 infection might inhibit the formation of the ELS in accord with its proviral load (PVL). In addition, EBV has high affinity to B cells or plasma cells (PCs) in accord with the alteration of its phase from the latent mode to the lytic mode, which is suggested to be stimulated by environmental substances. However, SS patients with HAM who show higher PVLs than HTLV-1 carriers have a low frequency of the autoantibodies that are found in typical SS patients. Regarding the HTLV-1-mediated induction of SS, various theories have attempted to explain the pathogenesis of SS. A definite epidemiological relationship between HTLV-1 infection and SS has been established by the findings obtained in + T cells mobilized by HTLV-1 infection simply form an aggregation of DILS-like lymphocytes as observed in HIV infection or the sialadenitis observed in ATL. Unlike HIV infection with sialadenitis, a great mass of HTLV-1 infection occurs by maternal infection; in contrast, SS usually develops in middle age. Clinicians should thus be concerned about the effects and or the immunological modulation of HTLV-1 infection during the 40–50 years before the development of SS.To address this scarcity of evidence, it is necessary to determine whether HTLV-1 infection has immunological influences on specific autoantibody production systems. In other words, it should be clarified whether or not HTLV-1 simply modulates the pathogenesis of existing typical SS. For example, HTLV-1 might influence immune cells including B cells, PCs, and/or T follicular helper cells in the ELS to reduce autoantibody production. It is also possible that CD4+ T cells for several decades. Investigations that may determine the triggers of the formation of SS-like sialadenitis in salivary glands over a prolonged period are further research tasks that will also elucidate the differences in engagement between EBV and HTLV-1.In cases of EBV infection, it is suspected that reactivation in the lytic phase promotes immunological dysfunction. However, when HTLV-1 infection occurs by maternal infection, HTLV-1 that shows no change of infective form continues to infect CD4Molecular biological approaches have enhanced our understanding of the contribution of Epstein-Barr virus to the pathogenesis of Sjögren’s syndrome. The involvement of HTLV-1 has been thought to vary among endemic areas, whereas EBV infection is ubiquitously observed regardless of regional characteristics. In addition, the aging of HTLV-1-infected populations might influence the prevalence of HTLV-1-seropositive SS. To determine the direct contribution of both EBV and HTLV-1 in the pathogenesis of SS, the integration of epidemiological and experimental studies including virological and molecular immunological approaches is strongly desired.


SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil  Hh signaling is initiated by Hh ligands, including Sonic hedgehog (Shh), Indian hedgehog (Ihh), or Desert hedgehog (Dhh), followed by activation of its downstream cascade that comprises Patched homolog (PTCH), Smoothened homolog (SMO), Suppressor of Fused homolog (SuFu), and the family of Gli transcriptional factors. Among the three Gli homologs, Gli2 and Gli3 can undergo partial proteolysis to generate a suppressive form in the absence of Hh ligands, while Gli1 lacks this suppressive domain3.Deregulation of Hedgehog (Hh) signaling, which is essential for cell proliferation and differentiation in embryonic development, often results in multiple developmental defects4. Additionally, Hh signaling can be self-controlled by activating the expression of PTCH, an upstream suppressor in the pathway5. While the Hh pathway normally regulates cell proliferation and differentiation, its dysregulation promotes tumor formation and progression7. A number of oncogenes, such as Sox2, c-Myc, Bcl2, FoxM1, etc.8, have been identified as Hh targets. Overexpression of these oncogenes due to deregulation of Hh signaling plays critical roles in the initiation and maintenance of multiple types of tumors, such as melanoma10, glioblastoma11, and colorectal cancer (CRC)14. Dysregulation of the Hedgehog signaling pathway plays a critical role in colorectal cancer (CRC) growth, but its molecular mechanism remains unclear.Because of its key role in cell fate determination, Hh signaling is tightly regulated by both positive and negative feedback mechanisms. Specifically, Gli1 upregulates the expression of its target genes in response to Hh ligands in a positive feedback fashion15. Conditional SuFu knockout leads to abnormal cardiac looping17 and other developmental defects19. Additionally, SuFu can function as a tumor suppressor, as SuFu depletion accelerates tumorigenesis in TP53−/− mice21. In humans, SuFu mutations are associated with Gorlin’s syndrome22, a hereditary disease with an increase incidence of tumors, such as basal cell carcinoma and medulloblastoma. Germline SuFu mutations have also been identified in these tumors26.SuFu, a PEST-domain protein, serves as a key negative regulator of Hh signaling by preventing the nuclear accumulation of Gli transcriptional factors. It plays an essential role in embryonic development, as knocking out the SuFu gene results in early embryonic lethality at gestation with neural tube defects27. Two E3 ligase complexes, SCFFbxl17 28 and β-arrestin2-Itch29, have been reported to mediate the ubiquitination and turnover of SuFu. Its ubiquitination is further controlled by multiple post-translational modifications, such as phosphorylation by PKA and GSK3β30. However, it remains incompletely understood whether there are additional factors that regulate SuFu stability in response to Hh activation.SuFu stability is tightly regulated. Hh activation induces SuFu degradation via the ubiquitination-proteasome pathway. Mono- and polyubiquitination of SuFu have been reportedC18orf56 (GenBank: NM_001012716) as a novel Hh target gene. It is located at chromosome 18p11.32 and encodes a protein of 121 amino acid residues without any reported functions, although its transcription and translation have been verified via high-throughput screening31. In this study, we showed this protein as a SuFu suppressor and thus named it “SuFu negating protein 1” (SNEP1). We showed that SNEP1 can promote SuFu degradation by interacting with an E3 ubiquitin ligase called ligand of numb-protein X1 (LNX1) and enhancing its activity toward SuFu in response to Hh activation. Additionally, SNEP1 is highly expressed in human CRCs, and this high expression is associated with poor prognosis. Thus, our study uncovers SNEP1 as a positive feedback regulator of the Hh signaling pathway, a crucial oncogenic player in colorectal cancer development and progression, and a potential drug target for the future development of anti-CRC therapy.In this study, we identified 33, were treated with the small molecule Gli inhibitor GANT61 or subjected to ectopic expression of Gli2, and the gene expression profiles were determined by next-generation sequencing. Among 157 genes whose expression was dramatically regulated by both GANT61 and Gli2, 32 had no annotated function in the gene ontology (GO) database  overexpression construct, which increased SNEP1 messenger RNA (mRNA) Fig.  and prot35 from Genomatics (http://www.genomatix.de/) revealed three potential Gli-binding sites  in the promoter region of SNEP1. In HT-29 cells, Gli2 was able to bind to all of these Gli-responsive DNA elements, as shown by chromatin immunoprecipitation (ChIP) analysis  injection and dextran sulfate sodium (DSS) administration were used to induce colitis and adenoma in both LSL-SNEP1/+ and LSL-SNEP1/+ ;villin-cre/+ mice   assays. As a result, both endogenous and exogenous SNEP1 and SuFu bound to each other in reciprocal Co-IP analyses in HT-29 or HEK-293T cells Fig. . This inP1) Fig. . Domain P1) Fig. . AdditioP1) Fig. , the truP1) Fig. . FurtherP1) Fig. . Togethe37 but has never been reported to regulate Hh signaling. We confirmed the interaction between SNEP1 and LNX1 by Co-IP assay and GST pull-down assay . However, an E3 ligase LNX1 was found in the SNEP1-interacting proteins. LNX1 is a RING-finger E3 ubiquitin-protein ligasesay Fig. . Among tsay Fig. .Fig. 4SNNext, we wanted to test whether LNX1 is required for the regulation of SuFu stability. As shown in Fig. 39. Therefore, we assessed the nuclear distribution of Gli2 when LNX1 or SNEP1 was overexpressed in HCT-116 cells. As shown in Fig. SuFu attenuates Hh signaling by downregulating full-length Gli transcription factors and preventing their nuclear importWe tested whether LNX1 is required for SuFu ubiquitination. Indeed, LNX1 mediated the ubiquitination of SuFu Fig. , and LNXDrosophila to vertebrates  and disease-free survival (DFS) rates by Kaplan–Meier analysis and log-rank tests. We found that patients with higher SNEP1 protein levels exhibited much lower OS rates and DFS rates Figs.  and S6F.40. Activation of Smo-independent Hh signaling may induces vismodegib-resistance. We found SNEP1 activates the Hh signaling pathway by regulating SuFu, which is Smo downstream component. We reasoned that elevated expression of SNEP1 may induce resistance to vismodegib in CRC. To evaluate this conjecture, clone formation and cell viability assay were used to analyze the effects of vismodegib in control and SNEP1-overexpressing colorectal cancer cells. Overexpression of SNEP1 suppressed the inhibitory effects of vismodegib on clone formation , a Smo-targeting inhibitor, was recently approved by FDA for the treatment of basal cell carcinoma. Vismodegib has preclinical activity in colorectal cancer (CRC) models, but it failed to show benefit in Phase II clinical of CRCion Fig. . Moreoveion Fig. . Taken t41. Among the three Gli homologs, Gli2 plays a more critical role in development and tumorigenesis43, and deficiency of Gli2, but not Gli1, in mice leads to embryonic lethality at later embryonic stages44. Here, we report SNEP1 as a novel target of the Gli transcription factors in response to Hh signaling. Our data indicate that SNEP1 acts as a positive feedback regulator of Hh signaling. As an Hh target, SNEP1 binds to LNX1 and enhances its E3 ubiquitin ligase activity toward SuFu, thereby mediating SuFu proteasomal degradation in response to Hh signaling. As a result, Gli2 becomes activated, enters the nucleus and induces the expression of its target genes to promote cell proliferation and survival. It is through this positive feedback regulation of Hh signaling that SNEP1 acts as an oncoprotein to promote CRC development. Interestingly, although Hh signaling is a conserved signaling pathway from Drosophila to humans, SNEP1 is a novel gene found in humans, with no homologs even in many mammals, including mice. This indicates that this unique Hh regulator might play certain roles in human development and tumors, but these roles remain to be discovered in the future.Hh signaling promotes the expression of a series of oncogenes by activating the Gli transcriptional factors that bind to the consensus sequence GACCACCCA through their zinc finger domainsFbxl17-mediated ubiquitination facilitates SuFu degradation28, whereas β-arrestin2-Itch–mediated K63 ubiquitination protects SuFu from proteolysis29. We identified LNX1 as another E3 ligase that promotes SuFu ubiquitination and degradation. LNX1 and SCFFbxl17 may utilize different mechanisms to target SuFu for degradation. Indeed, they target different lysine residues in the SuFu protein, as LNX1 ubiquitinates SuFu at K59 and K470, and SCFFbxl17 acts at K257, while Itch ubiquitinates at K321 and K457. It would be interesting to explore whether there is any interplay between SCFFbxl17 and the SNEP1-LNX1 complex in the regulation of SuFu stability in response to Hh signaling or other signals and whether β-arrestin2-Itch competes with the SNEP1-LNX1 complex to regulate SuFu stability.Two E3 ligase complexes have been previously reported to mediate SuFu ubiquitination in mammals. Intriguingly, these two E3 ligases have opposite effects on SuFu stability: SCFDrosophila, Hh-induced BTB protein expression downregulates SuFu levels by repressing its translation without affecting its stability46. It has also been shown that dual phosphorylation of SuFu by PKA and GSK3β leads to SuFu stabilization30, while dephosphorylated SuFu is recognized by Fbxl17 for Skp1-mediated proteolysis28. Bcl2, an Hh target, was also reported to directly bind to SuFu and facilitate its turnover, consequently suppressing SuFu interaction with Gli proteins47. Our studies, as presented here, uncover SNEP1 as novel Hh target was also directly bind to SuFu and facilitate its degradation, consequently activating Hh signaling pathway.The reduction in SuFu protein levels in response to Hh signaling has been known for more than a decade, and many regulators of this process, including protein kinases, interacting adaptors, and E3 ligases, have been reported. In 48. Three principal models of aberrant Hh signaling have been proposed in Hh pathway-dependent cancer: (i) ligand-independent signaling, (ii) ligand-dependent autocrine signaling, and (iii) ligand-dependent paracrine signaling. Ligand-independent Hh signaling is associated with a subset of human tumors; this type includes PTCH1 inactivating mutations, Smo activating mutations, SuFu loss-of-function mutations, and gene amplifications of Gli149. As SNEP1 expression in the intestinal epithelium activated Hh signaling by facilitating SuFu degradation and consequently promoted CRC cell proliferation and growth in vitro and vivo, we speculated that SNEP1 might have a positive effect on CRC progression by activating Hh signaling in a ligand-independent manner. Indeed, ectopic expression of SNEP1 in mice increased the number and size of tumors in AOM-DSS-induced CRC. Moreover, our analyses of a cohort of 395 pairs of primary CRC tissues and patients suggested that SNEP1 may be used as an independent prognostic factor for CRC.It has been shown that aberrant Hh signaling plays a key role in the initiation and/or maintenance of gastrointestinal tumors, including CRC40. In this study, we demonstrates that SNEP1 activates Hh signaling by regulating downstream of the Smo, and thereby induces resistance to Smo inhibitors in CRC cells. Moreover, we found that Gli inhibitor GANT61 attenuates SNEP1-induced Hh signaling activation and CRC cell proliferation. These findings suggest that SNEP1 may cause the failure of clinical treatment of Smo inhibitor in CRC.Smoothened (Smo), a vital receptor of Hh signaling pathway, is an important therapeutic target in Hh signaling pathway-related cancer therapy. Vismodegib (GDC-0449) is the most widely used drugs for targeting the Hh pathway, the first drug approved by the FDA to treat basal cell carcinoma (BCC). However, Vismodegib appear to be largely ineffective in the treatment of solid tumors other than BCCIn summary, our comprehensive basic and clinical studies, as described here, unravel two new players, SNEP1 and LNX1, in the Hh pathway. Biochemically, SNEP1 can partner with LNX1, a Ring E3 ligase, to ubiquitinate and degrade SuFu, a suppressor of the Hh responsive Gli2 transcriptional factor. Since SNEP1 is also an authentic transcriptional target of Gli2, this LNX1 coworker acts as a positive feedback regulator of Hh signaling. Biologically, highly expressed SNEP1, such as in xenograft or primary human CRC tumors, can promote CRC cell proliferation and survival as well tumorigenesis by inhibiting SuFu and consequently activating the Hh pathway. Clinically, our analysis of a large cohort of primary CRC tissues and patients suggests that SNEP1 could serve as a prognostic biomarker for CRC and might be used as a target for future anti-CRC therapy development.Transformed human embryonic kidney cell line HEK-293T and human CRC cell lines, including HT-29, HCT-116, Caco2, and SW620, were purchased from ATCC. All cell lines were authenticated using short tandem repeat profiling and were negative for mycoplasma contamination detected via PCR-based assay. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS (Gibco) and antibiotics . Cells were transiently transfected with Lipofectamine 3000  in accordance with the manufacturer’s instructions.SNEP1 polyclonal antibody was purified from rabbit by Beijing Boer Mai Biotechnology Company, China. Other antibodies were purchased from Abcam , Cell Signaling Technology , Sigma , Santa Cruz , LSBio , Millipore , Genetex  and Thermo Fisher Scientific .Reagents were purchased from Selleck , Invitrogen , Solarbio  and Sigma , Wako . The other analytical-grade chemicals were purchased from Sigma-Aldrich .Gli2 (Cat. RC217291) were purchased from OriGene Technologies . The human full-length SNEP1 (NM_001012716) construct was subcloned into pcDNA3.1-Flag (Invitrogen). The shRNAi-Gli2 expression vectors were purchased from GeneChem . The shRNAi-SNEP1 and shRNAi-LNX1 expression vectors were generated using the BLOCK-iT™ Pol II miRNAi Expression Vector Kit  in accordance with the manufacturer’s protocol. The target sequences of the aforementioned shRNAi expression constructs are listed in Appendix Table Expression plasmids of human full-length p-values < 0.05 were considered differentially expressed genes (DEGs). All raw data are available at the sequence read archive (SRA) with accession no. PRJNA623247. To screen the novel target genes with unknown functions, the Gene Ontology database was used to annotate the DEGs, and these genes that had no annotation except that inferred from physical interaction (IPI) were identified as function unknown.HT-29 cells infected by lentivirus expressing Gli2 or empty vector or treated with GANT61 or DMSO were harvested for RNA extraction with TRIzol reagent (Invitrogen). Gene expression profiles were determined via next-generation sequencing (NGS) with Illumina NovaSeq by Novogene Co., Ltd. , and genes with expression changes over 2-fold and adjusted Cells were harvested and lysed in lysis buffer . Cell lysates were cleared of debris via centrifugation at 4 °C and 12,000 rpm for 15 min and subjected to IB with the indicated antibodies, as described previously 50. Total RNA was harvested using TRIzol Reagent (Life Technologies™) and evaluated via qPCR. Briefly, total RNA (1 μg) was employed to prepare cDNA via reverse transcription using a PrimeScript® RT reagent Kit with gDNA Eraser . qPCR was carried out in an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems) using SYBR® Premix Ex Taq™ Tli RnaseH Plus . The primers used are shown in Appendix Table SNEP1 was subcloned into the pGBKT7 vector as bait, resulting in a fusion with the Gal4 DNA-binding domain. The resulting plasmid and Saccharomyces cerevisiae strain Y187 containing the cDNA library were simultaneously transformed into the yeast Y2HGold strain, as described previously 48.E. coli and conjugated to glutathione-Sepharose 4B beads (Sigma-Aldrich). Protein–protein interaction assays were processed using cell lysates with mammalian-expressed HA-SuFu or Flag-LNX1. The cell lysates were incubated with the indicated GST fusion proteins before being immobilized on glutathione-Sepharose beads. Bound proteins were eluted from the beads and analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis/Coomassie Blue staining and/or subjected to immunoblotting.HEK-293T cells were transfected with GFP-SNEP1 and Flag-SuFu or Flag-LNX1 for 48 h and harvested for immunoprecipitation analysis, as described previously 48. For the GST pull-down assay, GST-tagged SNEP1 was expressed in HT-29 cells at 90% confluence were cross-linked in 1% formaldehyde. Their DNA was sonicated into fragments of 100 to 400 bp using a Bioruptor Sonicator (Diagenode) for 30 cycles of 1 s on with 2 s off. The lysates were precleared in BSA-blocked protein A/G beads and incubated with specific antibodies or IgG control overnight. After washing, DNA was eluted and reverse cross-linked at 65 °C overnight. Eluted DNA was used as a template for semiquantitative PCR. The input control was obtained from the supernatant before precipitation. The predictive binding sequences and the primers used for SNEP1 promoters are listed in Appendix Table http://www.genomatix.de/). To construct the reporter vector for the luciferase assay, the 5ʹ-fragment of human SNEP1 containing Gli2-binding sites was amplified via PCR and cloned into the firefly luciferase reporter plasmid pGL3-Enhancer . Cloned promoter sequences were validated via Sanger DNA sequencing. The primers used for the luciferase reporter constructs are listed in Appendix Table In silico analysis of transcription factor Gli2-binding sites in the human SNEP1 5ʹ-upstream region (−1062 to +971) was performed by using Genomatix MatInspector software . The number of colonies was enumerated microscopically and determined via ImageJ software .5, were incubated with 1 × 108 IU of virus and 8 μg/ml of polybrene  for 12 h. Protein expression was induced in 1 μg/ml doxycycline  for 48 h.For lentiviral infection, HT-29, HCT-116, SW620, and CaCo2 cells, at a density of 4 × 107 stably infected HT-29 (LV-sh-control and LV-sh-SNEP1) and HCT-116 (LV-control and LV-SNEP1) cells were resuspended in sterilized PBS (200 μl) and hypodermically injected into bilateral inguinal parts of 5-week-old female BALB/c-nu mice . Three days after injection, mice were administered 2 mg/ml doxycycline in drinking water, which was replenished every 2 days. Tumor sizes on both sides of mice were monitored using a Vernier caliper every alternate day. After 2 weeks, xenografts were harvested for IHC and IB analysis. Eight female nude mice were used for each group.For ex vivo experiments, 2 × 10em1(CAG-LSL-SNEP1-IRES-eGFP)/NCU (LSL-SNEP1) mice, B6-Tg(Vil1-cre)997Gum/J (vil-cre) mice were purchased from Shanghai Model Organisms Center. LSL-SNEP1/+ mice were crossed with vil-cre/+ mice to generate LSL-SNEP1/+; vil-cre/+ mice and mice lacking vil-cre were used as controls.CAG-loxP-Stop-loxP-SNEP1 cds-IRES-EGFP-WPRE-pA was knocked in the Rosa26 site to generate B6-Gt(ROSA)26SorFor the inflammation-dependent colorectal cancer model, mice were injected with AOM  at 10 mg per kg bodyweight before oral administration of 2.5% DSS solution (5 ml/day) for 5 days. DSS cycles were repeated three times within 14 days of normal drinking water Fig. . The DSSA cohort of 395 CRC patients undergoing surgery was reviewed at the First Affiliated Hospital of Nanchang University between January 2009 and August 2014. All patients had primary tumors that were untreated before surgery. Biopsy specimens were reviewed by a pathologist to ensure that all the specimens contained tumors and adjacent normal tissues. We defined tumor size as the maximum tumor diameter measured intraoperatively in the tumor specimens. Histological types of the 395 samples were defined in accordance with the WHO classification criteria as grade I (64 cases), grade II (243 cases), and grade III (88 cases). Clinical stage was defined in accordance with the 7th edition of the AJCC. This study was approved by the Ethics Committee of the Frist Affiliated Hospital of Nanchang University .Paraffin sections (3 μm thick) of formalin-fixed CRC tissue and adjacent tissue were dewaxed, rehydrated, and incubated in 3% hydrogen peroxide solution for 10 min. Antigen retrieval was performed by heating them in EDTA buffer (pH 9.0) for 24 min and cooling them down naturally. Normal goat serum (10%) was used to block nonspecific staining, and then the tissue sections were exposed to the indicated antibodies. The stained sections were observed by at least two independent investigators blinded to the histopathological features of the samples. The German semiquantitative scoring system was employed for assessing the staining intensity and stained area. Each specimen was assigned a score in accordance with the staining intensity of the nucleus or cytoplasm  and the extent of staining in cells . The final immunoreactive score was determined by multiplying the intensity score with the score of the extent of staining, ranging from 0 (the minimum score) to 12 (the maximum score).t-tests. IHC scores between two or three independent groups were compared using the Mann–Whitney U-test or the Kruskal–Wallis H-test. The χ² test was used to analyze the correlation between gene expression and clinicopathological characteristics. The Kaplan–Meier method and the log-rank test were performed for survival analysis. The Cox proportional hazards model was used to determine independent factors influencing survival and recurrence based on the variables selected from the univariate analysis. The sample size were calculated by G Power 3.1. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software version 21.0 .Differences in quantitative data between two groups were analyzed using two-sided paired or unpaired Student’s Supplementary Figure LegendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Table


MAPK-MK2 signaling axis in mediating synaptic plasticity through control of AMPA receptor trafficking and the morphology of dendritic spines. These processes are mediated through control of cytoskeletal dynamics via the activation of cofilin-1 and possibly control of the expression of Arc/Arg3.1. There is evidence that MK2 is necessary for group I metabotropic glutamate receptors long-term depression (mGluR-LTD). Disruption of this signaling may play an important role in mediating cognitive dysfunction in neurological disorders such as fragile X syndrome and Alzheimer’s disease. To date, the role of neuronal MK2 mediating synaptic plasticity in response to inflammatory stimuli has not yet been investigated. In immune cells, it is clear that MK2 is phosphorylated following activation of a broad range of cell surface receptors for cytokines and other inflammatory mediators. We propose that neuronal MK2 may be an important player in the link between inflammatory states and dysregulation of synaptic plasticity underlying cognitive functions. Finally, we discuss the potential of the p38MAPK-MK2 signaling axis as target for therapeutic intervention in a number of neurological disorders.p38 is a mitogen-activated protein kinase (MAPK), that responds primarily to stress stimuli. p38 has a number of targets for phosphorylation, including MAPK-activated protein kinase 2 (MK2). MK2 primarily functions as a master regulator of RNA-binding proteins, indirectly controlling gene expression at the level of translation. The role of MK2 in regulating the synthesis of pro-inflammatory cytokines downstream of inflammation and cellular stress is well-described. A significant amount of evidence, however, now points to a role for the p38 MAPK), phosphorylates MAPK-activated protein kinase 2 (MK2). The role of this p38MAPK-MK2 signaling axis in cellular responses to stress and inflammatory signals, including control of the synthesis and release of inflammatory signaling molecules, has been delineated in detail, largely from immune cells  on dendritic spines. Expressed in neurons throughout the brain, activated in response to inflammatory cues and with a demonstrated role in mediating synaptic plasticity, the p38MAPK-MK2 signaling axis is an attractive candidate for mediating crosstalk between inflammatory and neuronal signaling. Here, we will discuss what is known about activation of the p38MAPK-MK2 axis in response to inflammatory stimuli, the role it plays in mediating synaptic plasticity, discuss the potential of this signaling pathway for mediating cross-talk between inflammatory signals and neurotransmission and discuss settings within which targeting this signaling system may prove efficacious.Over recent years, the importance of bidirectional cross-talk between the immune system and central nervous system has become increasingly clear. Immune responses are subject to neuroendocrine modulation , while ine cells . In neurMAPK-signaling cascades are highly conserved intracellular signaling pathways that convert external stimuli, usually through cell-surface receptors, into a range of cellular responses . These sMAPK is a MAPK, that in mammalian cells is expressed in four splice variants: p38α, p38β, p38γ, and p38δ, with differential phosphorylation of molecular targets  and TIR-domain-containing adaptor-inducing interferon-β (TRIF) (Toll-like receptors (TLRs) are a family of archetypal PRRs, which detect molecular patterns associated with pathogens . Lipopoltivation , 1994. Ttivation  where ittivation   . MK2, thMAPK-MK2 signaling axis  domain . This gring axis . Evidencing axis , IL-33  (Transforming growth factor-β (TGF-β) is a multifunctional cytokine, released in latent form and activated by proteolysis by matrix metalloproteinases on the cell surface . Upon ac1 (TAK1) .Tumor necrosis factor-α (TNFα)  is a proMAPK-MK2 axis is broader than can be described within the scope of this review. Other receptors for inflammatory stimuli which have been demonstrated to activate the p38MAPK-MK2 signaling axis include (but are not limited to): monocyte chemoattractant protein 1/chemotactic cytokine ligand 2 (MCP-1/CCL2) activation of chemotactic cytokine receptor 2 (CCR2)  , and int2 (CCR2) .MAPK isoforms are expressed in the mouse brain, with the p38α and β isoforms particularly prominent in the cerebral cortex and hippocampus, with p38α most abundant in neurons , MKK3/6, p38MAPK and MK2 , which is phosphorylated at Ser-323  and regurphology . These sAMPA-R trafficking mediating mGluR-LTD is largely coordinated by the expression of the activity-dependent immediate early gene, Arc/Arg3.1 . TranscrMAPK-MK2 signaling axis mediate changes in synaptic strength that impact at cognitive and behavioral levels. Intact mGluR-LTD is necessary for maintaining cognitive flexibility  (As described in section “Activation of p38luR-LTD) , we suggMAPK-MK2 signaling axis has been identified as an effector of cellular responses to inflammatory signals and stimuli. The same signaling axis has also been shown to be a key link in the processes mediating mGluR-LTD. Neuroinflammation is a ubiquitous or near ubiquitous feature of neurological disorders and we identify the p38MAPK-MK2 signaling axis as a potentially important mechanistic link between neuroinflammation and synaptic dysregulation underlying cognitive impairments in neurological disorders, such as Alzheimer’s disease. MK2-mediated mGluR-LTD is necessary for maintaining cognitive flexibility, the loss of which is an early clinical sign of Alzheimer’s disease. Inhibitors of p38MAPK have been trialed in a number of contexts where inflammation makes an important contribution to disease etiology (MAPK mediated activation of MK2 or target the activity of MK2 itself may offer more promise, with a narrower focus. The p38MAPK-MK2 signaling axis is a promising target for therapeutic intervention, where inflammation contributes to dysregulation of neuronal network activity. More evidence, however, is necessary to clarifying the contribution of this signaling axis.A key role for the p38etiology . These ietiology . InhibitEB and SALC conceived the review and shared in writing the manuscript. Both 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.


Macrophages play a central role in dictating the tissue response to infection and orchestrating subsequent repair of the damage. In this context, macrophages residing in the lungs continuously sense and discriminate among a wide range of insults to initiate the immune responses important to host-defense. Inflammatory tissue injury also leads to activation of proteases, and thereby the coagulation pathway, to optimize injury and repair post-infection. However, long-lasting inflammatory triggers from macrophages can impair the lung's ability to recover from severe injury, leading to increased lung vascular permeability and neutrophilic injury, hallmarks of Acute Lung Injury (ALI). In this review, we discuss the roles of toll-like receptor 4 (TLR4) and protease activating receptor 2 (PAR2) expressed on the macrophage cell-surface in regulating lung vascular inflammatory signaling. Macrophages (MΦ), initially classified as phagocytes by Metchnikoff in 1893 , constitAlveolar macrophages (AMΦ) and interstitial macrophages (IMΦ) constitute the two key resident MΦ populations in the lungs. AMΦ, as the name suggests, are located within the airspace of the alveoli, juxtaposed to epithelial cells . InterstInflammation is also known to activate the coagulation cascade, which in turn affects inflammatory processes by generating a further suite of proteases such as trypsin, thrombin, elastases, FVIIa and FXa , 27. Pro+ embryonic MΦ from the yolk sac migrate into the lung bud around E10.5. These MΦ persist in the adult lung as “primitive interstitial MΦ” and localize peripherally and perivascularly. The second wave is initiated by Mac2+ embryonic monocytes at E12, most likely from the fetal liver  (2)   or the g (2) (2) . Because (2) (2) –40. On a (2) (2) . In huma (2) (2) . These A (2) (2) . However (2) (2) . It is r (2) (2) , 45. ButIMΦ, initially referred to as septal cells , comprishi and MHCIIlo IMΦ residing near blood vessels and Lyve1lo MHCIIhi IMΦ residing near nerve fibers or endings. One study has suggested that Lyve1hi IMΦ are responsible for exacerbated fibrosis and that both IMΦ populations are slowly replaced by Ly6Chi monocytes over time  along with MΦ-peroxisome proliferator-activated receptor γ (PPARγ) is required for differentiation and maintenance of the AMΦ phenotype from embryonic precursors , 69. ThiPattern or pathogen recognition receptors (PRRs) are a class of receptors that recognize pathogen-associated molecular patterns, PAMPs, of pathogenic organisms or endogenous signals from damaged cells, referred to as damage-associated molecular pattern or DAMPs. Upon binding with PAMPs or DAMPs, PRRs activate signaling cascades that lead to the production of pro-inflammatory cytokines and interferons, an important step in the initiation of adaptive immunity –86. EndoTLRs contain 22–29 residue long leucine-rich repeats- (LRR-) N-terminal ectodomains and intracellular toll-interleukin-1 receptor (TIR) signaling domains. The LRR motif of TLRs play a key role in the protein-protein interactions involved in downstream signaling . MΦ haveTLR4 is unique among the various TLRs due to its ability to activate signaling from the cell-surface as well as intracellularly. Cell-surface TLR4 propagates signaling through both a MyD88-dependent and independent pathway, resulting in generation of proinflammatory cytokines and type I interferons, respectively , 96. UpoEndocytosed TLR4 can sense cytosolic LPS to induce NFκB and IRF3 mediated transcription, which is critical to full regulation of innate immunity during pathogenic insult , 102. St+ across the plasma membrane is a key factor regulating the activation of NLRP3. Di et al. recently showed that NLRP3 activation of K+ efflux by two-pore domain weak inwardly rectifying K+ channel 2 (TWIK2) played a critical role in regulating inflammasome formation in AMΦ  and caspase-1 –105. Infn in AMΦ .Recent studies show that, in mice, caspase-11 (caspases-4 and 5 in humans) can bind cytosolic LPS and induces the NLRP3 pathway as well as gesdermin D to stimulate pyroptosis , 107 andCell death leads to the generation of reactive species and activation of Z-DNA binding protein 1 (ZBP1). ZBP1 results in the release of mtDNA and/or dsDNA. Cyclic GMP-AMP synthase (cGAS) catalyzes generation of cyclic GMP-AMP (cGAMP) upon binding to dsDNA, which leads to the activation of STING/IFN-β signaling and lung injury , 110–112Inflammation-induced injury releases a mélange of proteases, complements, chemokines, prostaglandins, and other inflammatory molecules, which activate several receptors, including G-protein coupled receptors (GPCRs) . Thus, iProtease activated receptors (PARs), PAR1, PAR2, and PAR3 encoded by the genes F2R, F2RL1, F2RL2, and F2RL3, respectively are also expressed on the MΦ cell-surface. As the name indicates, PARs are activated by several different proteases, including those involved in the coagulation pathway , 28, 120αs, Gαq, Gαi, and Gα12/13 and triggers several signaling cascades to mediate its diverse cellular functions  and SLIGKV (human), in turn binds to initiate PAR2 signaling. Other proteases, including thrombin, neutrophil elastase, cathepsin G, cathepsin S, proteinase-3, gingipain-R and kallikrein-14, cleave PAR2 at sites other than the tethered ligand site, leading to bias signaling  Ca2+ stores, followed by Ca2+ entry through plasmalemmal channels (2+ levels via the phospholipase C-inositol trisphosphate (PLC-IP3) axis  axis –138. Ca2 Gαi/Gαo , 140, in2+-permeable non-selective cation channels involved in MΦ activation. Studies showed that TRPM2 and TRPV4 but not STIM-mediated store-operated calcium channels play an important role in mediating Ca2+ entry in MΦ  are a group of Cary in MΦ , 141. Hoy in AMΦ . TRPV4 iy in AMΦ . It is ay in AMΦ –146. TRPy in AMΦ –149. Alsy in AMΦ –152.2+ entry in PAR2-null BMDM more than in wild-type BMDM  compared to wild-type BMDM, indicating that PAR2 suppresses TRPV4 activity . Also, dype BMDM . FurtherCyclic AMP (cAMP) is a ubiquitous second messenger involved in numerous physiological processes in all domains of life. Adenylyl cyclases (AC) generate cAMP from ATP . AC have2+ entry in PAR2-null BMDM  and elastase in leukocytes and monocytes, which facilitates activation of the coagulation pathway in part through the production of thrombin , 177. TFin vivo –181. Ralin vivo . Thus, wLiang et al. followed up on the TLR4 and PAR2 receptor co-operativity concept discussed above and showed that TLR4 transactivated PAR2, which then enhanced TLR4 signaling  Figure . In thisper se failed to increase IL-12p40 production in MΦ without PAR2 expression . Nh. Nh2+ enurned on . These rurned on . Furtherin vitro vs. in vivo. We also described recent advances in TLR4 signaling, such as the role of inflammasomes and STING in regulating AMΦ functions. We noted herein that generation of cAMP through PAR2 activity is critical in suppressing NFAT activity, thereby dampening AMΦ inflammatory signaling. Intriguingly, data also show the key role of cAMP in blocking TRPV4 activity in MΦ. However, several questions remain to be addressed: as for exampleThis review describes recent mechanistic developments in lung MΦ regulation of tissue-fluid homeostasis with an emphasis on PAR2-mediated signaling in AMΦ and its intersection with TLR4 signaling to modulate inflammation and lung vascular injury. We highlighted the subsets of lung resident MΦ and their dichotomous phenotypes, as regulated Where does this cAMP comes from to bind TRPV4 in AMΦ and how does cAMP alter the affinity of TRPV4 for its agonists?Does PAR1 expression in AMΦ augment TLR4 activity in the absence of PAR2 expression?How does PAR2, or PAR1 expression, for that matter in IMΦ or monocyte-derived MΦ, which are known to be recruited to the lung during injury, regulate AMΦ inflammatory signaling?in vivo along with genetic mouse models will likely advance our understanding of lung MΦ subsets generation and function under normal conditions and during inflammation.Are cAMP-induced transcription factors, such as CREB, involved in dictating AMΦ function? Further studies employing state of the art techniques such as macrophage imaging SR and DM: conceptualized the manuscript. IR and DM: edited the manuscript. SR, JJ, IR, BJ, SB, and DM wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Public pension insurance has become a major form of social protection around the world. However, little is known about the association between public pension expansion and individuals’ fertility in developing economies. In this paper, we examine the effects of the New Rural Pension Scheme (NRPS) on the fertility of married women in rural China. Using data from the China Family Panel Studies (CFPS), the difference-in-differences approach is employed to estimate the impact of NRPS expansion on fertility outcomes. The robustness of results is checked through additional estimations, including difference-in-differences with propensity score matching, fixed-effects model, and instrumental variable approach. Results show that the NRPS expansion has a significantly negative effect on the number of children, and it reduces the likelihood of having a second child. The fertility-reducing effect of the NRPS is larger for the younger, well-educated women and those in high-income families. Considerations of the fertility effects and their population differences are needed in the impact evaluations of relevant public pension reform. Public pension insurance is considered as one of the major social security programs to improve social welfare and reduce inequalities. The expansion in pension coverage of young adults affects the lifetime budget constraint. This, in turn, may lead to changes in individual behavior, such as fertility. While numerous studies of the association between the old-age pension and fertility have been conducted for industrialized countries –6 the feChina is the world’s largest developing economy and has achieved rapid industrial and economic growth during the past few decades. According to the World Bank Open Data (WBOD), however, it has seen the percentage of population aged 65 and above increase sharply over the last years, from about 5.63% in 1990 to 10.35% in 2017. Meanwhile, the total fertility rate decreased from 2.3 in 1990 to 1.6 in 2015, which is lower than the replacement-level fertility. The shifts of population structure in China reflect a demographic transition from a population pattern previously characterized by demographic dividend and large working-age population to low fertility rates and aging population, raising considerable concerns about the pension arrangement and sustainability. Yet, little is known about the Chinese people’s fertility responses to their country’s public pension system.Researches in economics have assumed that the parents are altruistic toward their children, which are considered as a consumption good to produce utility for their parents –8. BasedSeveral studies have empirically examined the impacts of social security or public pension on fertility, and most of them revealed a negative association. For instance, Cigno 1992, 1993) found that the expansion of social security coverage has a significant negative impact on fertility in European countries 992, 1993. Using dSince the public pension system plays an important role in protecting against the future earnings uncertainty, young adults may adjust their saving for old-age consumption to maximize utility throughout their lifetime 14]. On . On 14].This paper attempts to fill the gap of existing research concerning the link between public pension and fertility. First, different from the previous studies, we offer a new insight into the fertility preferences among the rural women of a rapidly growing country, China. This study extends existing analysis on the fertility effect of pension, which has largely focused on advanced countries. Second, we use individual-level microdata from a nationally representative survey to empirically test the hypotheses derived from the theoretical model, revealing the fertility differences between pension and non-pension scenarios. We also employ a series of identification strategies to examine the causal effect of public pension insurance on individuals’ fertility, and investigate whether the effect varies by population groups.Since 2009, the Chinese government has expanded public pension availability for rural residents. Using data from 2010 and 2014 waves of the China Family Panel Studies, we employ a difference-in-differences strategy to examine the fertility effects of New Rural Pension Scheme on targeted populations. The actual number of children and whether to have a second child are the two indicators used to measure the fertility outcomes among rural married women aged between 20 and 45. Moreover, we implement difference-in-differences with propensity score matching, fixed-effects model, and instrumental variable approach to check the validity of our results and analyze the heterogeneity of treatment effects by age, education, and household income. Our empirical findings, which are robust to several specification checks, indicate that the implementation of rural pension scheme has a significantly negative effect on the number of children, and it reduces the likelihood of having a second child. The fertility-reducing effect of the public pension is larger for the younger, well-educated women and those in high-income families. By contrast, the rural pension expansion does not significantly reduce women’s fertility for the elderly, people with less education, and low-income families.The remainder of this paper is organized as follows. Section 2 describes the institutional background; Section 3 provides a conceptual framework. Section 4 presents the data source and the main empirical strategy. Section 5 describes the main results including descriptive statistics, the effect of the NRPS on fertility and subgroups analyses, robustness checks (sample selection and endogeneity). Lastly, discussion and concluding remarks are in Section 6.China’s public pension system is comprised of three main types of pension scheme: the Urban Employees’ Pension Plan (UEPP), the New Rural Pension Scheme (NRPS), and the Urban Resident Pension Scheme (URPS). Among them, the NRPS is designed for covering rural residents aged 16 years or above who are not students and not covered by the basic urban pension scheme. Before the NRPS, the Chinese government experimented with pilot programs to extend a rural pension scheme starting in 1986. The program placed most of the financial responsibility on individuals, was supplemented by local collectives, and covered both the collective and private sectors in rural China. However, this financial arrangement led to a very low take-up rate with a lack of sustainability and effectiveness of public financial support, and the pilot scheme expansion was halted in the late 1990s.To improve the economic well-being of the rural elderly, the Chinese government launched the NRPS in 2009. Since its inception the NRPS has rapidly expanded, where participating counties have increased over time, from 838 in 2010 to nearly all 2,853 counties by 2012 and 326.4 million rural residents were participating in this scheme in 2011 . CompareThe pension returns of NRPS consist of the individual account benefits and a non-contributory basic pension. Individual account benefits are calculated based on the participant’s accumulated contributions from the individual account and accrued investment returns . A minimTo control the growing population, the Chinese government introduced a birth planning program (the one-child policy) in 1979. The one-child rule was strictly enforced for urban residents. In rural areas, however, it was virtually unenforceable and from 1984 rural couples in most provinces were allowed to have a second child if their first was a girl. Particularly, rural couples from the six northwestern provinces were allowed a second child, irrespective of the sex of the first child . In ordeWe extend the overlapping generations (OLG) model, by incorporating public pension and saving, to investigate the fertility response of young adult to the change in pension coverage using comparative statics. In this model, the economy is populated by individuals who live through three periods: childhood, adult and old age. When the individual is a child, she lives with her parents who provide necessary inputs for child-rearing and development. In the period of old age, the individual is retired and her consumption consists of saving and pension income. Young adults, the main labor force and wage earner, acts as a sole role in making family decisions such as fertility and consumption.t obtain utility (Ut) from the number of children (nt) in the family as well as the consumption in young  represents the utility weight of raising children relative to consumption. We first consider the scenario that the person does not participate in any pension scheme, and thus the individual in generation t has to face following the budget constraint:Here, St denotes the savings for consumption in the retirement period, p is the cost of each child's human capital inputs, ϕ (ϕ>0) is the rate of the expenditure to support the elderly, and wt is an adult’s wage income. The budget constraint in the old age period is written as:Where Rt+1 = 1+rt+1, and rt+1 is the interest rate in period t+1. Except for saving, consumption in old age also includes the pecuniary return to having children, represented by ntϕwt+1. Maximizing one’s lifetime utility function as described in (1), while it subjects to budget constraints (2) and (3). We obtain the optimal solution of fertility level Where θ, then the budget constraint for young adults at generation t can be written as:We next examine the fertility decisions for the individual who had pension coverage. Assuming that the contribution rate of social security involving the public pension is denoted by θ)wt is an individual’s net wage, which is distributed into consumption, saving, money spent on child-rearing, and support for the elderly. In the retirement period, the individual’s consumption (StRt+1), pension income from government (G), and pecuniary return to children (ntϕwt+1). Hence, the budget constraint is:Where (1−Maximizing the utility function (1) subjects to the budget constraints (5) and (6), the optimal fertility level In order to compare the fertility differences between pension and non-pension, we subtract the Eq  from Eq θ and the interest rate Rt+1 are assumed to be constant. Since the parameters p,β,γ, and Rt+1 are greater than zero, and 0<θ<1, the fertility response to pension plan depends on the sign of (pRt+1−ϕwt+1) and (G−θRt+1wt). The former shows the comparison between cost and pecuniary return of a child, while the latter reflects a tradeoff between future pension benefits and the current earnings. Particularly, if people are provided a high transfer income from public pension  and the economic return is more important for people to have children , having pension would generate large negative impacts on fertility. In the empirical analyses below, we will use survey data and employ identification strategies to examine the causal relationship between pension and fertility, and investigate whether the effect varies by population groups.To simplify the analysis, the contribution rate The data used in the present study comes from the China Family Panel Studies (CFPS), a nationally representative and longitudinal survey conducted by the Institute of Social Science Survey (ISSS) of Peking University starting in 2010. A multi-sage probability proportional to size (PPS) strategy with implicit stratification was performed in the sampling process that comprises three stages: county level as the primary sampling unit, a community or village for the second-sage sampling unit and the final sampling unit was household . The CFPGiven that the NRPS coverage rapidly extended during 2011 and 2012, we construct a two-wave balanced panel data by merging the baseline in 2010 with a follow-up survey in 2014 from the adult and family dataset of CFPS, so as to compare the fertility outcomes of individuals with NRPS and non-NRPS before and after program implementation. To meet the requirements of participation in NRPS, the sample is restricted to tracked individuals having an agricultural Hukou, the eligibility for participation in the NRPS. Aside from NRPS, those who participated in another pension scheme, such as an urban and private pension, are excluded from the sample of our study. This is because other types of pension may also alter individual reproductive behaviors. Introducing these pension programs might cause a bias on the results for estimating the impact of NRPS on fertility. For the fertility analysis, we restricted the sample to married women aged between 20 and 45 because 20 is the legal minimum marriage age in China and the age range represents the majority of the childbearing population. The final study sample consists of 6,930 women observations.The CFPS asks respondents to report their pension status, based on the question “What type of pension plan(s) do you have?” in each wave of the survey. According to respondents’ answers, we define the treatment group as those respondents who reported no pension in 2010 but having an NRPS in the wave of 2014. The control group consists of those individuals who were not covered by any pension scheme for the survey in both waves of 2010 and 2014. As a result, the number of cases in the treatment and control group is 4,374 (63.1%) and 2,556 (36.9%), respectively.The first dependent variable for this study, the number of children, measures the number of children among rural married women. In the CFPS data, however, there is no specific question to reflect the number of children born to a woman directly. Alternatively, we create this outcome variable by using the codes from the list of family members to locate the respondent’s each child and to calculate the total number of children the respondent has. We also create an additional outcome variable, having a second child, to investigate the effect of public pension on the likelihood of a second child for rural women. It is measured by a dichotomous variable that equals 1 if the respondent has two or more children and 0 otherwise.All regression specifications adjust for several covariates that may confound estimates of the effect of public pension on fertility. Age is a continuous variable that may reflect attitudes toward childbearing at different stages of life. The women’s age at first marriage, a strong correlate of fertility, is also included. To capture the nonlinear effect of education on fertility, the individual education is coded into one of 4-level categorical variable: primary school or less, middle school, high school, and college or more. Since China is a large and diverse country, we control for respondent nationality by a dummy variable, 1 if from an ethnic minority group and 0 for Han nationality. Religion is associated with fertility preferences 23] and  and 23] To empirically examine the effect of NRPS on fertility, the average treatment effect on the treated (ATT), a causal impact parameter for comparing the difference between expected fertility outcomes for women with NRPS coverage  before and after a policy change that might have a potential impact on fertility outcomes can be differenced out by a DD estimator. To augment the robustness of the estimated ATT for NRPS on fertility, a standard DD linear regression model allows for several controls for the differences in observed characteristics between treatment and control group:i, refers to the individual, and t, to the period. Yit is the dependent variable related to the outcomes of female fertility. For the main independent variables, the interaction term, NRPSi×Postt, is the most important regressor and its coefficient, β1, is the DD estimator that captures the ATT estimator of the NRPS on individual fertility outcomes. NRPSi is a dummy variable, equaling one for the NRPS treatment group and zero for the non-NRPS control group. Postt is a dummy variable coded one for the post-treatment period, 2014, and zero for the pre-treatment period in the baseline, 2010. Vector Xit is a group of covariates including individual and family characteristics that may affect the fertility outcomes, and which might have differed between the treatment and comparison groups. Pj is a full set of province dummies accounting for heterogeneity in pension effects by region. Moreover, as the pension program is implemented by county, standard errors in all regressions are adjusted to cluster at the county level.Here, the subscript, In a difference-in-differences model, the parallel trend assumption that treatment and control group would not trend differentially during this time period in the absence of a policy change should be satisfied. A common approach to test this assumption is to compare the time trend of fertility outcomes between the treatment and control group before the treatment  takes place 28]. How. How28].Mean of the study variables are presented in The DD regression results are presented in Apart from the pension status, several sociodemographic variables affect fertility as well. We find that older women tend to have more children, but the age at first marriage is negatively associated with the number of children and the probability of a second child to be born. As expected, due to the opportunity cost of childbearing and rearing among educated persons, people with a higher level of education report a lower desire for fertility. Those who come from an ethnic minority group are more likely to have more children, showing a stronger motive for fertility. Women in poor health have fewer children and are less likely to have a second child. However, health insurance has no significant effect on fertility, probably because most of women are insured in the study sample. The fertility-reducing effect varies by employment status. We find a significant reduction in fertility for wage-employed women, but not for the self-employed. Because self-employed jobs are more autonomous and flexible in work arrangements, which make married women have a low opportunity cost during childbearing and they may prefer more children. Finally, the association between household saving and fertility outcomes is found to be significantly negative in both cases of the number of children and the probability of second child, supporting the research findings of Lugauer et al (2019) .In this section, we address the concern about sample selection that might bias our main results. That is, the estimated effect might be biased if treatment (NRPS) and control (non-NRPS) observations have heterogeneity in initial conditions as treatment is not randomly assigned to women. Following the study of Van den Broeck and Maertens (2015) , we combResults from the PSMDD estimation are still significantly negative but the magnitudes of effects become smaller, as shown in One more threat to the main results may come from the potential unobserved heterogeneity, such as unobserved norm and attitude, which may affect both pension participation and fertility preferences. These unobserved factors would cause our estimates of NRPS and fertility effects to be correlated with the error term, resulting in a bias. Following Rokicki et al (2014) and Cheng et al (2016) 31], we , we 31],Pensionit is an indicator variable for whether the individual participated in the NRPS, and its coefficient β1 is our main interest, revealing the effect of NRPS participation on women’s fertility. Xit is the same vector of time-varying covariates from the previous analyses, including age, health status, health insurance, employment status, and household saving per capita (log). Wt is a full set of year dummies, while ci is the individual fixed effect, which accounts for the unobserved characteristics that are fixed across time.Where The first two columns in We also apply the instrumental variables (IV) approach to further reduce the unobserved heterogeneity bias. Several scholars have pointed out that as the NRPS operates at the county level, the implementation status of county pension program has a strong association with the individual participation in NRPS but appears to be the exogenous nature for other individual characteristics that would potentially affect one’s behaviors since the pilot areas of the scheme were decided by central government of China 31]. The. The31].The last two columns in As the impact of the public pension on fertility is likely to have differential effects in terms of individual characteristics, the sample is stratified by age, education, and household income. Panel A of The effects of NRPS on fertility vary by different education levels. As shown in the Panel B of Panel C of This paper contributes to research on the link between public pension and fertility. We first develop a theoretical model to compare the fertility differences between pension and non-pension scenarios, and then, using Chinese individual-level microdata, we present an empirical analysis for the impacts of NRPS expansion on rural married women’s fertility outcomes. Results from DD estimation show that the implementation of NRPS has a significantly negative effect on women’s demand for the number of children and having a second child. This is in line with the findings of the previous studies 5], reve, reve5],Previous research has indicated that the NRPS expansion in rural China significantly improves the health status of children up to 15 years of age, and the health effect is stronger for boys and left-behind children . CombingOur results in this paper are specific for the case in rural China. The effect of public pension on fertility is likely impinged on by economic conditions and cultural background, which calls for caution in generalizing our results to other countries. One limitation in the present study is that, due to the data limitations, we are not able to identify the parallel trend on the fertility outcomes of the treatment and control group, which is a crucial assumption for DD analyses. If this specific assumption is violated, the estimates of the fertility effects could be biased. Although we can conclude that the public pension is associated with decreased fertility in rural China and several robustness checks, including PSMDD strategy, fixed-effects model, and IV approach, support our findings, these estimates are still subject to some limitations. Additional studies with other data sources and approaches will help to further strengthen our findings.In spite of the limitations, this study has important implications for social protection in China and other developing countries. First, when evaluating the effect of the pension policy, most previous studies only emphasized the well-being of the elderly while ignored the effect on other family members, for instance, younger women and children. Considerations of such population groups and intergenerational spillovers are needed in the impact evaluations of relevant public pension reform. Second, while implementing a universal coverage pension system is considered to be one major policy to improve social welfare and reduce inequalities, it is worth to pay more attention to the adverse effect of pension on fertility, particularly for the countries facing low fertility problem. In this context, relevant supportive measures are also needed to maintain the number in the labor force and enhance the feasibility and sustainability of public pension systems.S1 Appendix(DOCX)Click here for additional data file.