Hwc3, leads to hybrid weakness in intrasubspecific japonica rice hybrids.Gene activation caused by long terminal repeat retrotransposon insertion in the promoter of a newly discovered gene,  japonica rice (Oryza sativa ssp. japonica) varieties ‘Aranghyangchalbyeo’ (‘CH7’), ‘Sanghaehyangheolua’ (‘CH8’) and ‘Shinseonchalbyeo’ (‘CH9’), carrying different hybrid weakness genes. The reciprocal progeny obtained from crossing any two varieties displayed characteristic hybrid weakness traits. We mapped and cloned a new locus, Hwc3 (hybrid weakness 3), on chromosome 4. Sequence analysis identified that a long terminal repeat (LTR) retrotransposon was inserted into the promoter region of the Hwc3 gene in ‘CH7’. A 4-kb DNA fragment from ‘CH7’ containing the Hwc3 gene with the inserted LTR retrotransposon was able to induce hybrid weakness in hybrids with ‘CH8’ plants carrying the Hwc1 gene by genetic complementation. We investigated the differential gene expression profile of F1 plants exhibiting hybrid weakness and detected that the genes associated with energy metabolism were significantly down-regulated compared with the parents. Based on our results, we propose that LTR retrotransposons could be a potential cause of hybrid weakness in intrasubspecific hybrids in japonica rice. Understanding the molecular mechanisms underlying intrasubspecific hybrid weakness is important for increasing our knowledge on reproductive isolation and could have significant implications for rice improvement and hybrid breeding.Hybrid weakness is a post-zygotic hybridization barrier frequently observed in plants, including rice. In this study, we describe the genomic variation among three temperate  However, in some cases hybrids between different species show defective development in terms of viability, fertility, and other phenotypic traits. Such defective hybrid characteristics are collectively called hybrid incompatibility and can be further subdivided into hybrid sterility, hybrid inviability, hybrid weakness, and hybrid breakdown, depending on the hybrid phenotype and underlying molecular mechanisms  was first described in the 1950s , ‘Sanghaehyangheolua’ (‘CH8’), and ‘Shinseonchalbyeo’ (‘CH9’) carrying different hybrid weakness genes  varieties ‘Aranghyangchalbyeo’ , ‘Sanghaehyangheolua’  and ‘Shinseonchalbyeo’  carrying different hybrid weakness genes were used in this study. ‘LiyuB’, a wild-type japonica rice, was used as the control plant. Reciprocal crosses among ‘CH7’, ‘CH8’ and ‘CH9’ were performed to generate six F1 hybrid genotypes .Three temperate 1 hybrids. Plant height of seedlings was recorded at 3-d intervals starting from 3 d after transplantation to 60 d after transplantation. Plant height was measured as the distance from the basal nodes to the uppermost tips of the leaf blades and/or panicles. For root length measurements, 10 mature plants from each line at the 3-leaf stage were removed from the soil and the root length was measured (cm) for a period of 7 d. Panicle length was measured as the average value from the panicle neck to the panicle tip based on an evaluation of three panicles from each of 10 mature plants from each line at the maturity stage. To check the significance of results, the data were analysed by employing Prism 6.0 using one-way-ANOVA.In order to characterize the phenotype of hybrid weakness, quantitative analysis of important traits, namely plant height, root length, panicle length, and panicle number per plant, was conducted using 10 mature replicate plants of each of ‘CH7’, ‘CH8’, ‘CH9’, and their eight FHwc3 gene promoter and gene, were amplified using primers Hwc3a F/R and Hwc3b F/R (JXB online). PCR was performed with an initial denaturation step of 3 min at 95 °C, with two PCR cycles  followed by 33 PCR cycles  and a final extension step at 72 °C for 10 min. The amplified products were separated by electrophoresis on a 0.8% (w/v) agarose gel stained with ethidium bromide. The amplified fragments were cloned into the HPE-203 binary vector to generate binary constructs. These constructs were transformed into Agrobacterium tumefaciens strain EHA105 and were used for transformation of ‘CH8’ embryogenic callus, with transgenic plants being generated.To prepare the constructs for complementation tests, a 2-kb fragment, designated AT70 and consisting of the LTR retrotransposon, and a 4-kb fragment, designated AT71 and consisting of the LTR retrotransposon and the Hwc3 candidate gene and to examine its expression pattern, RT-PCR analysis was performed on samples from the parental lines and F1 plants. Fresh leaf samples at tillering stage were taken from the plants and the total RNA was extracted using the TRNzol reagent . The cDNAs were synthesized  from 2 mg total RNA, according to the manufacturer’s protocol. The cDNA primers for the genes were designed using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers for RT-PCR are listed in ACT1) was used as an internal control to normalize the expression of the tested genes. Gel imaging and documentation was performed using the Gel Doc™ XR molecular imager .To confirm the presence of the Hwc3 gene was annotated according to the full-length cDNA sequence on the NCBI database. Sequence similarity was analysed using the BLAST program (http://www.ncbi.nlm.nih.Gov/ BLAST/) provided by the NCBI website. The promoter sequences were analysed through the Plant CARE database (http: bioinformatics. psb.ugent.be/webtools/plant care/html/). Protein homology was performed using BLASTp databases and a phylogenetic tree was constructed with ClustalW software  using the neighbor-joining method.The http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The filtered paired-end reads were mapped to the Oryza sativa ‘Nipponbare’ reference genome using the Burrows–Wheeler alignment (BWA) tool with default parameters (http://broadinstitute.github.io/picard/), and the sorted SAM file was converted to a BAM file (binary version of SAM file) for variant calling using VarScan software (http://varscan.sourceforge.net). Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) (<50 bp) were calculated and identified with snpEff-4.1 software ≤5% combined with standard-fold difference for gene screening with a fold-change≥2 (up-regulation) or ≤−2.0 (down-regulation). Gene description was recorded from NCBI BLAST (https://www.ncbi.nlm.nih.gov/) and Uniprot (http://www.uniprot.org/). The microarray records have been submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119431) and assigned GEO accession numbers as GSE119431.Three biological replicates  for each line extracted at the tillering stage were selected for microarray analysis. Total RNA extraction, cDNA and labelled cRNA synthesis and Affymetrix expression GeneChip hybridization were conducted by CapitalBio Technology. Arrays were scanned on an Affymetrix GeneChip scanner 3000 (Thermo Fisher Scientific). Data were normalized using the Robust Multi-Array Average (RMA) algorithm, which included quantile normalization and background subtraction. Differentially expressed genes were identified using SAM, at the reliability threshold . The genetic variation among the three japonica rice parents, ‘CH7’, ‘CH8’, and ‘CH9’, was individually identified using Samtools. We identified a total of 574 551 polymorphic sites including 487 964 SNPs and 86 587 InDels in ‘CH7’, a total of 1 026 428 polymorphic sites including 874 667 SNPs and 151 761 InDels in ‘CH8’, and a total of 792 465 polymorphic sites including 685 118 SNPs and 10 747 InDels in ‘CH9’. These findings revealed a remarkably high density of molecular polymorphisms among each of the three studied lines. According to their position with respect to genes, SNPs and InDels were divided into different types such as upstream, downstream, intergenic, intron, and exon. The results also indicated that the distribution of SNPs and InDels was mainly in the downstream, exon, intron, intergenic, splice_site_acceptor, splice_site_donor, splice_site_region, transcript, upstream, UTR_3_prime and UTR_5_prime locations  and the detected polymorphism at the Hwc3 locus in inducing hybrid weakness, we analysed the gene expression pattern of the mapped genes at the Hwc3 locus. Results showed that the two genes, Os.69469 and Os.51535, showed expression in ‘CH7’ and ‘CH8’, as well as in the F1 hybrid , which contained the retrotransposon together with the flanking LTR, and a 4-kb fragment (AT71), which contained the LTR retrotransposon, the promoter part and the gene Os.89494  induces the expression of Hwc3, our results preliminarily demonstrate that hybrid weakness in intrasubspecific japonica hybrids may be associated with a two-locus interaction. As the candidate Hwc3 gene is generally not expressed under normal conditions, we speculate that the activation and expression of this gene are responsible for the hybrid weakness phenotype.In order to verify that the gene at the Os.89494 , were amnormally . This inweakness . PreviouHwc3  is 1341 bp. The candidate gene encodes a protein of 446 residues with 10 leucine-rich conserved domains  statistically significant differences in gene expression were detected in both datasets; and (ii) the average of the fold-ratio change in expression level was greater than 2.0 or less than −2.0. The number of significantly differentially expressed genes was 443. Comparing the expression of these genes, our dataset indicated that transcript levels of 184 genes exhibited differential expression in the ‘CH7/CH8’ hybrid compared with either parent. Only those genes whose expression level showed a fold-ratio greater than +3.0 or less than −3.0 are listed in To obtain a global picture of gene expression changes that occurred in the Findica and japonica rice (It is well known that the accumulation of genetic differences ultimately leads to reproductive isolation in plants (ica rice . Strong indica or japonica (1 generation clearly exhibited the hybrid weakness phenotype with its characteristic chlorotic phenotype, short stature, aberrant root formation, and markedly decreased tiller number (japonica rice varieties, in the form of SNPs and InDels (indica rice varieties RGD-7S and Taifeng B, whose F1 progeny exhibited the hybrid weakness phenotype (japonica rice varieties were very different and that they might have originated and evolved under very different conditions. This genome-level polymorphism might not complement one another when the two genomes are hybridized together, which probably leads to the defective development observed in the F1 progeny. The genome-wide polymorphism results obtained in this study will help further in designing InDel markers for further map-based cloning of hybrid weakness genes.Some recent studies, including the current one, have reported that, in addition to interspecific and intersubspecific hybridization in rice, reproductive barriers in the form of hybrid weakness also exist between different lines of the same subspecies, r number , 2. Consr number  among thd InDels . We founcis-activation is caused by the insertion of an LTR retrotransposon in the promoter region of the Hwc3 gene in ‘CH7’, which seems to enhance its expression in the F1 hybrid (Hwc3 gene, could induce hybrid weakness in transgenic ‘CH8’ plants (Drosophila (Hwc3 locus. The candidate Hwc3 gene encoded an LRR protein (Hwc3 gene was found not to be expressed under normal conditions and the plants carrying this gene grew normally, which suggests that the gene has no apparent function and is a silenced gene. However, upon hybridization of ‘CH7’ with ‘CH8’, transcriptional activation occurred and the gene started to be expressed in the hybrid background, a finding that suggests that the ‘CH8’ genome contains some transcriptional factor that activates the gene expression in the F1 hybrid (1 hybrids (Hwc3 by a transcriptional regulator, Hwc1, leads to hybrid weakness in japonica rice.The results of the present study suggest that a 1 hybrid , 4C. Thi’ plants . The wel’ plants . However’ plants . Barbaraosophila . Our stu protein , a type 1 hybrid . Interacjaponica rice, we analysed differential gene expression profiling in the ‘CH7/8’ F1 hybrid at tillering by Affymetrix gene chip microarray. We found that the genes involved in the carbohydrate metabolism pathway had a higher proportion of down-regulated genes, while genes related to cellular defense, calcium signaling and secondary metabolism also tended to be down-regulated, as were genes related to protein biosynthesis and peptide transport (1 progeny observed in our study and the down-regulation of energy-metabolism-related genes might suggest that the observed phenotype could be due to the low energy status of the hybrids. Previously, carbohydrate metabolism-related genes were found to be up-regulated in heterotic rice hybrids (O. sativa (Zea mays (Triticum (Hwc3 might regulate the transcriptional functions of other proteins encoded by the differentially expressed genes in the ‘CH7/CH8’ F1 hybrid.To identify the candidate genes that play key roles in hybrid weakness in the intrasubspecific hybridization of ransport ; Table 1JXB online.Supplementary data are available at japonica rice varieties ‘CH7’, ‘CH8’, and ‘CH9’.Fig. S1. Proportions of InDels and SNPs in different gene regions of the Hwc3 locus in ‘Nipponbare’, ‘CH7’, and ‘CH8’.Fig. S2. Sequence analysis at the Hwc3 gene product and its phylogeny.Fig. S3. The protein sequence of the candidate Hwc3 and XA1 genes using the NCBI database.Fig. S4. Protein homology of the Fig. S5. The differential gene expression patterns related to hybrid weakness.Table S1. Primer sequences used in this study.japonica rice varieties ‘CH7’, ‘CH8’, ‘CH9’, and ‘LiyuB’.Table S2. Coverage of the reads mapping to the ‘Nipponbare’ reference genome from re-sequencing of the Table S3. Annotation of InDels identified in ‘CH7’, ‘CH8’ and ‘CH9’ and ‘LiyuB’ in comparison with ‘Nipponbare’.Table S4. Annotation of SNPs identified in ‘CH7’, ‘CH8’ and ‘CH9’ and ‘LiyuB’ in comparison with ‘Nipponbare’.Hwc3.Table S5. Bioinformatics analysis of the promoter structure of Supplementary Figures S1-S5Click here for additional data file.Supplementary Tables S1-S5Click here for additional data file.


Pseudomonas aeruginosa, strain PA01, was used as a model bacterium. Utilising a simple, cost effective fabrication process, P. aeruginosa was heat-inactivated and formulated into dissolving MNs, fabricated from aqueous blends of 20% w/w poly. The resultant MNs were of sufficient mechanical strength to consistently penetrate a validated skin model Parafilm M®, inserting to a depth of between 254 and 381 µm. MNs were successfully inserted into murine skin and partially dissolved. Analysis of MN dissolution kinetics in murine ears via optical coherence tomography showed almost complete MN dissolution 5 min post-insertion. Mice were vaccinated using these optimised MNs by application of one MN to the dorsal surface of each ear (5 min). Mice were subsequently challenged intranasally (24 h) with a live culture of P. aeruginosa (2 × 106 colony forming units). Bacterial load in the lungs of mice vaccinated with P. aeruginosa MNs was significantly (p = 0.0059) lower than those of their unvaccinated counterparts. This proof of concept work demonstrates the potential of dissolving MNs for intradermal vaccination with heat-inactivated bacteria. MNs may be a cost effective, potentially viable delivery system, which could easily be implemented in developing countries, allowing a rapid and simplified approach to vaccinating against a specific pathogen.This work describes the formulation and evaluation of dissolving microneedle patches (MNs) for intradermal delivery of heat-inactivated bacteria.  The hypodermic needle also results in the creation of biohazardous sharps waste, which pose the risk of infection to both patients and healthcare workers. An estimated 33,800 HIV infections, 1.7 million hepatitis B infections and 315,000 hepatitis C infections annually are caused from unsafe injection practices .The skin represents an attractive route for vaccination, having the advantage of increased patient compliance and increased vaccine effectiveness. The skin is densely populated with professional antigen presenting cells (APCs). Dendritic cells (DCs) present in the dermis and Langerhans cells (LCs) in the epidermis play a pivotal role in induction of immune responses, whereby they capture foreign antigens and present them in draining lymph nodes . The skiSC, and subsequently delivering their payload into viable skin  have been proposed as a novel approach to increase skin permeability and may offer an ideal platform for i.d delivery . MNs areble skin . MNs aresubjects . MNs aresubjects . Furthersubjects .Pseudomonas aeruginosa strain PA01, was chosen as a model bacterium. Utilising a simple, cost effective fabrication process, P. aeruginosa was heat-inactivated and formulated into dissolving MNs. As MNs offer the possibility for self-application, the expected benefit of MN-mediated vaccination with heat-inactivated bacteria would lie in the simplicity and cost effectiveness from the practical perspective. Whole heat-inactivated vaccines are stable, safe for administration to immunocompromised patients and can be easily freeze dried and incorporated into MNs, thus allowing ease of transport and distribution, and a cost-effective approach to vaccinating against a specific pathogen. This approach could potentially allow for mass immunisation within developing countries, helping increase vaccine coverage. To the best of our knowledge, this is the first study that uses dissolving MN arrays for delivery of a heat-inactivated bacterium, intended for vaccination against a bacteria for which no currently licenced vaccine exists.Dissolving MNs have received substantial interest for delivery of currently licenced vaccines. For example, Matsuo et al. demonstrated vaccine efficacy against tetanus and diphtheria, malaria and influenza using dissolving MNs . Moreove22.1Gantrez® S-97 (PMVE/MA), a copolymer of methyl-vinyl-ether and maleic acid , was provided by Ashland . Parafilm M®, 127 µm thick, was obtained from BRAND GMBH . Dulbecco’s phosphate buffered saline (PBS) (endotoxin tested), Luria-Bertani broth and cetrimide agar were obtained from Sigma-Aldrich . All other chemicals were of analytical reagent grade. Rompun™ 100 mg/ml, was purchased from Bayer Healthcare LLC (Bergkamen Germany). Ketalar™ 10 mg/ml ketamine hydrochloric solution was purchased from Pfizer Ltd .2.2P. aeruginosa (strain PAO1), was grown on cetrimide agar at 37 °C overnight. Post incubation a colony was selected and cultured overnight in Luria-Bertani (LB) broth (5 ml) at 37 °C in a rotatory incubator shaker (180 rpm). Post incubation, the culture (5 ml) was transferred into an Erlenmeyer flask containing a further 995 ml of LB broth and incubated as above. After incubation, the culture was transferred to 50 ml Falcon tubes and the bacteria was collected by centrifugation (4000 rpm for 20 min), and the supernatant was subsequently removed. The resultant P. aeruginosa pellets were washed twice by addition of phosphate buffer (PB), followed by centrifugation as described above. Post washing, the pellets were re-suspended in PB buffer and heat-killed by submersion in a water-bath (65 °C) for 1 h. The suspension was inoculated onto LB agar to ensure bacteria were not viable. The remaining suspension was placed in a freezer (−80 °C) for 1 h. Once frozen, each Falcon tube lid was replaced with a KimWipe  and the tubes were transferred to a side arm of a manifold-type freeze dryer (MiVac freeze dryer) under a vacuum for 48 h before being removed and stored until further use.2.3P. aeruginosa diluted in phosphate buffered saline (PBS), pH 7.4, to give a final concentration of 20% Gantrez®S-97. One hundred milligrams of this suspension, containing 200 µg of heat-inactivated P. aeruginosa was poured onto silicon micromoulds. The micromoulds contained 361 (19 × 19) pyramidal shaped needles, perpendicular to the base, 500 µm in height, 300 µm wide and with 50 µm interspacing. The arrays had areas of 0.6 cm2. A pressure of 3 bar was subsequently used to force the MN formulation into the needles of the MN mould. The MNs were dried under controlled temperature conditions (19 °C) for 24 h, before being carefully removed from the MN moulds and stored until further use. A schematic representation showing the method of production of dissolving MNs is shown in Fig. 1.MNs were prepared from aqueous blends of Gantrez® S-97. A stock of Gantrez® S-97 (40%) was diluted with the appropriate volume of a suspension of 2.4When imaging MNs using scanning electron microscopy (SEM), MNs were mounted onto an aluminium stub and observed under a TM3030 microscope . Micrographs of MN structures were obtained and the surface morphology and height/width of individual needles were examined/measured.2.5−1 until the required force of 32 N/array was applied. This has been shown to be the average maximum force a human exerts when applying MNs , a blend of a hydrocarbon wax and a polyolefin, was used as a skin simulant . Inserted MNs were immediately viewed using an EX1301 VivoSight® OCT Microscope with a hand-held probe, the contacting surface of which had an area of 1 cm2. The swept-source Fourier domain OCT system has a laser centre wave-length of 1305 ± 15 nm, facilitating real-time high resolutions imaging of the upper skin layers . The resulting 2D images were analysed using ImageJ®  software. Obtained image files had a scale of 1.0 pixel = 4.2 µm, allowing accurate measurements of MN insertion depth. Five replicates were performed per MN formulation and for each MN inserted, 20 measurements of individual MN insertion depths were taken by random selection of needles, from the 361 penetrating MNs in each array. MN insertion data was presented as (±SD) of 20 replicate measurements. The procedure was repeated at pre-determined time points .The 2.7In vivo studies were performed on C57/BL6 mice, 6–8 weeks old. Mice were randomly divided into 2 groups, those that received blank MNs (bMNs), or P. aeruginosa MNs (p.a MNs), n = 5/group. Briefly, mice were anaesthetised intraperitoneally (i.p) and immunised by manual application of one MN to the dorsal surface of each ear for 5 min, resulting in a corresponding dose of 400 µg of heat killed P. aeruginosa. At 7 days post MN application, both vaccinated and un-vaccinated mice were challenged with 2 × 106 colony forming units (CFU) of P. aeruginosa (PA01), delivered intranasally (i.n). Each mouse received a 20 µl volume of bacterial suspension, diluted in endotoxin-free PBS and administered with a micropipette. Mice were carefully monitored over the course of infection and weights were assessed. Subsequently, 24 h post-challenge, mice were sacrificed and the lungs and spleens were harvested for analysis of CFU counts.2.7.1P. aeruginosa was grown on cetrimide agar at 37 °C overnight. Preparation of bacterial inoculum for challenge study was performed by transferring a colony of P. aeruginosa into LB broth. The culture was grown overnight at 37° C under gentle agitation. Following overnight incubation, the bacteria were harvested by centrifuged for 25 min at 22° C 4000 rpm and the supernatant was carefully removed. The pellet was subsequently washed in PBS. Finally, optical density (OD) measurements were used to dilute the bacterial suspension to the desired concentration (106 CFU) using PBS and this was confirmed by plating serial dilutions of the preparations for enumeration of CFU post-overnight incubation.2.7.2. The agar plates were incubated overnight at 37 °C and the number of CFUs counted for determination of bacterial load.Lung and spleen tissue was homogenized under aseptic conditions in 1 ml of PBS in a sterile bijou using a mechanical homogeniser. Serial dilutions of the homogenized organs (100 µl) were plated onto cetrimide agar  for analysis of bacterial load2.8t-test. In all cases, p < 0.05 was considered acceptable for rejection of the null hypothesis. Statistical analysis was carried out using GraphPad Prism version 5.0 .Data was compared using an unpaired, two-tailed student’s 33.1P. aeruginosa (p.a MNs). The resulting MNs were approximately 500 µm in height, with a width at base of approximately 300 µm, and the fabrication process resulted in MNs with sharp needle tips, essential for insertion into murine skin and successful vaccine delivery. A complete array of needles was formed and MN baseplates were of sufficient strength to be easily removed from the moulds without damage being caused to the array. The addition of P. aeruginosa did not appear to affect MN structure, or the morphology of the individual needles.3.2P. aeruginosa MNs (p.a MNs) by 4.7% (p = 0.805), indicating that the loading of P. aeruginosa within the MNs did not affect their rigidity or compression characteristics. None of the MNs, or the baseplates of the MNs fractured post-application of 32 N, nor did the MNs appear to be compressed  reduced in height by 4.4% C-i and t by 4.7% C-ii, res by 4.7% C-iii , mice underwent bacterial challenge by i.n inoculation with a live culture of e models  and as sP. aeruginosa within the vaccinated group (three out of five spleens were found to be sterile), compared to the unvaccinated group, which received bMNs (one out of five spleens was found to be sterile). These results collectively demonstrate generation of a protective immune response in vivo following application of our dissolving polymeric MNs, resulting in enhanced bacterial clearance from the lungs and decreased likelihood of systemic dissemination of P. aeruginosa.At 24 h post challenge, the number of CFUs in the spleens was overall very low in both groups D, since 4P. aeruginosa (strain PA01) was used as a model bacteria, heat-inactivated and formulated into dissolving MNs. This study was conceived as a proof of concept study for investigating the feasibility of taking a highly pathogenic bacterium, incorporating it into dissolving MNs and successfully delivering the heat-inactivated bacterium in vivo, inducing protective immune responses with a view, in time of working towards more relevant bacteria of the developing world.The emergence of multidrug-resistant bacteria for which no vaccines exist, has resulted in high mortality rates with limited or no therapeutic interventions available . Thus, tMNs were prepared using Gantrez®, previously shown to have been successfully used in the formulation of dissolving MNs for delivery of model protein antigens, importantly, without deterioration of the antigens upon release from the MNs . MNs ladBacterial load post challenge is related to survival status and, therefore, the enumeration of bacteria in vaccinated mice was investigated. MN i.d vaccination resulted in reduced bacterial load in the lungs and reduced bacterial dissemination to the spleens. These results are in line with those of Yang et al. who demonstrated reduced bacterial loads, inflammatory cytokine expression (IL-6) and inflammatory cell infiltration, following intramuscular (i.m) vaccination with POH and adjuvant, in a challenge pneumonia model . Yanyan via dissolving MNs is advantageous for i.d vaccination. Firstly, heat-inactivated vaccines are safe for administration to immunocompromised patients (The use of lyophilised heat-inactivated vaccines delivered patients . Adminispatients . Also, tpatients . At prespatients . Moreovepatients . The Glopatients . Additiopatients  and may patients .via dissolving MNs results in polymer deposition in the skin. Although repeated application of dissolving MNs will not be required for vaccine delivery, regulatory bodies will undoubtedly require assurance of polymer safety. Accordingly, an understanding of the long-term effects of polymer deposition will require further investigation to ensure they do not represent a danger. While dissolving MNs are typically fabricated from biocompatible polymers, they have not been used for i.d delivery. With the MNs utilised in this particular study, each MN deposits approximately 5.4 mg of polymer per array, which is the polymer content in the needles only. As highlighted such polymer deposition may result in effects such as erythema or hepatic/lymphatic accumulation and requires further investigation (The mechanism of vaccine delivery tigation . From a P. aeruginosa. Such experiments should evaluate multiple cellular and humoral facets of the immune response and the cross-protective efficacy among different isolates of P. aeruginosa. Moreover, studies should also take into account the effects of polymer deposition and subsequent impact on immune response. In addition, prime-boost regimes and an investigation into long-term protection are warranted.As this was a proof of concept investigation, studies were not carried out to elucidate the immunological mechanism by which p.a MNs confer protective immunity. This research was designed to develop and evaluate a pre-clinical MN delivery platform for i.d vaccination with heat-inactivated bacteria. Consequently, we describe a simple, cost effective approach to vaccination that could be applicable to a range of pathogens. Future studies are warranted to elucidate the mechanism of immune responses elicited post MN-mediated vaccination against 5P. aeruginosa for i.d vaccination. Notably, MNs were fabricated using pressure only, highlighting the simplicity and cost-effectiveness in the strategy employed. In addition, in vivo murine challenge studies confirmed delivery and the generation of an immune response, resulting in reduced bacterial load in the lungs and spleens of vaccinated mice. Together, these results suggest that dissolving MN-mediated i.d vaccination is a potentially viable delivery system. This technology could simplify vaccine administration and distribution in developing countries, thus improving vaccine coverage. Heat-inactivation of bacteria and its incorporation into MNs offers a novel, simplified approach to vaccination against a specific pathogen. This is the first reported study utilising a dissolving MN platform for successful delivery of a lyophilised heat-inactivated bacterium for vaccination against a pathogen for which no currently licensed vaccine exists.The work presented here reports successful formulation and characterisation of dissolving MNs laden with heat-inactivated This was an initial, but important study highlighting the potential of MNs for delivery of heat-inactivated vaccines. We anticipate that this dissolving MN system, due to its simplicity and ease of use, could potentially have a substantial impact on the prevention of infectious disease. Dissolving MNs offer the prospect of mass vaccination and the incorporation of heat-inactivated bacteria provides a simplified approach, which could easily be implemented in countries of the developing world. This was a proof of concept study and prior to this technology being applied to patients; several additional studies are warranted, including studies to elucidate the mechanisms of induced immune protection and patient usability/acceptability profiles. This technology clearly holds much potential and warrants further investigation.


Perineuronal nets (PNNs) are specialized aggregations of extracellular matrix (ECM) molecules surrounding specific neurons in the central nervous system (CNS). PNNs are supposed to control synaptic transmission and are frequently associated with neurons firing at high rates, including principal neurons of auditory brainstem nuclei. The origin of high-frequency activity of auditory brainstem neurons is the indefatigable sound-driven transmitter release of inner hair cells (IHCs) in the cochlea.Here, we show that synaptic poles of IHCs are ensheathed by basket-like ECM complexes formed by the same molecules that constitute PNNs of neurons in the CNS, including brevican, aggreccan, neurocan, hyaluronan, and proteoglycan link proteins 1 and 4 and tenascin-R. Genetic deletion of brevican, one of the main components, resulted in a massive degradation of ECM baskets at IHCs, a significant impairment in spatial coupling of pre- and postsynaptic elements and mild impairment of hearing.These ECM baskets potentially contribute to control of synaptic transmission at IHCs and might be functionally related to PNNs of neurons in the CNS.The online version of this article (10.1186/s12915-018-0566-8) contains supplementary material, which is available to authorized users. In addition to neurons and glia, the extracellular matrix (ECM) forms a fundamental, non-cellular component of the nervous system. A specialized form of the ECM are the perineuronal nets (PNNs), a pericellular cover that tightly enwraps somata, proximal dendrites, and axon initial segments of specific neurons thereby leaving meshes occupied by synaptic terminals. PNNs are composed of multiple extracellular molecules that interact with each other –5. AmongPNNs are associated with numerous functions, including neuroprotection –11, stabThough many regions within the cochlea such as basilar membrane, spiral limbus, spiral ligament, and stria vascularis are highly enriched in ECM, including collagen, fibronectin, and various unspecified proteoglycans –24, the Here, we aim to clarify whether cochlear hair cells exhibit an ECM coating comparable to PNNs of neurons within the CNS. The present data demonstrate that the main PNN molecules are expressed in the cochlea, including aggrecan, brevican, neurocan, the hyaluronan, and proteoglycan link proteins (HAPLN) 1 and 4, and tenascin-R. These ECM molecules were tightly associated with synaptic contacts, thereby forming basket-shaped complexes surrounding the base of IHCs. Among the proteoglycans brevican was found to be the major molecule constituting this ECM basket. Brevican-deficient mice displayed a remarkable degradation of the ECM basket surrounding IHCs accompanied by an increase in the number of dislocated postsynaptic densities relative to presynaptic calcium channels.In conclusion, the present data demonstrate that the main PNN-constituting molecules are expressed in the cochlea, thereby forming brevican-based ECM baskets that enclose the base of IHCs and their synaptic contacts. Brevican potentially contributes to spatial coupling of pre- and postsynaptic elements and thus may support fast and temporally precise synaptic transmission in the cochlea.Given the sparse information on the expression of the CSPGs brevican, aggrecan, and neurocan, as well as HAPLN1, HAPLN4, and tenascin-R in the cochlea, the initial aim of the present study was to test whether these typical constituents of PNNs can be identified within cochlear tissue and whether these components are associated with cochlear sensory cells. Immunolabeling of those proteins was performed on cochlear cryosections prepared from 3 C57BL/6N mice at the age of P27–P30, complemented by Western blot analyses on 30 isolated cochleae of 15 C57BL/6N mice (P35).All of the tested CSPGs as well as HAPLN1 could be identified in cross-section of the cochlea Fig.  and neurBrevican appeared as a thin layer both (i) underneath the spiral limbus within the osseous spiral lamina and (ii) between the spiral ligament and the bony otic capsule Fig. . These bWestern blot analyses confirmed the presence of brevican (~ 145 kDa full-length protein and ~ 55 kDa fragment), aggrecan (~ 450 kDa), neurocan (~ 150 kDa), HAPLN1 (~ 40 kDa), and HAPLN4 (~ 40–42 kDa) Fig.  in the mIn conclusion, we found that the main PN components are present in the auditory periphery. Despite of the fact that brevican, aggrecan, neurocan, and HAPLN1 exhibit an individual distribution within the cochlea, they conjointly appeared at IHCs, potentially pointing to the presence of an extracellular, PNN-like structure surrounding the sensory cells of the cochlea.n = 15) were prepared, and ECM molecules were labeled using fluorescence immunohistochemistry. The labeling of brevican and HAPLN1 confirmed the prominent presence of these ECM molecules at IHCs along the entire cochlear spiral  also convey afferent information via ribbon synapses and in addition receive three to four efferent fibers, we questioned whether brevican, aggrecan, neurocan, HAPLN1, and HAPLN4 as well as tenascin-R also surround synapses at OHCs. In the majority of cochlear cross-sections see Fig. , b, we oIn conclusion, these data demonstrate that the size and composition of ECM at OHCs differ from that of IHCs if present at all. At IHCs, the proteoglycans aggrecan, brevican, and neurocan as well as HAPLN1, HAPLN4, and tenascin-R form complex ECM baskets surrounding synapses at the entire basal part of the IHCs. At OHCs, only brevican and HAPLN1 seemed to be present and these molecules were found to be spatially restricted to individual ribbon synapses, resulting in a spot-like immunoreactivity.bcan−/−) .Among the CSPGs, brevican immunoreactivity seemed to be the strongest at IHCs. In order to estimate the significance of brevican surrounding IHCs, we analyzed a brevican-deficient mouse , 31. Imm/−) Fig. , b. At fies Fig. , d. Alsobcan−/− mice. The CSPGs aggrecan and neurocan as wells as HAPLN1, HAPLN4, and tenascin-R were labeled in cochlear whole-mount preparations of bcan+/+ and bcan−/− mice, using fluorescence immunohistochemistry. The analysis yielded profound changes in the ECM composition at IHCs in bcan−/− mice  and bcan−/− mice  and corresponding I-V curves  did not indicate any genotype-specific changes in IHC Ba2+ currents. Also, average maximum Ba2+ currents were not altered in bcan−/− mice . Gating parameters of the Ca2+ channels such as the voltage of the half-maximum activation, Vh, and the slope of activation, k, were extracted from fits to the I-V curves  and bcan−/− mice . Likewise, the slope k was not different between genotypes . To summarize, brevican does not affect the function of presynaptic calcium channels.To test for changes in IHC function, whole-cell Casee the “” sectionbcan−/− mice. Click auditory brainstem response (ABR) thresholds were slightly but significantly increased in knockout mice (bcan+/+: 16.7 ± 3.3 dB sound pressure level (SPL), n = 6/12 animals/ears; bcan−/−: 20.0 ± 3.6 dB SPL, n = 6/12 animals/ears, p < 0.01, t test, Fig. bcan−/− mice, covering the best hearing range of mice. The difference was the largest at 11.3 kHz . The results indicate that deficiency of brevican leads to mild impairment of central auditory signal processing.Analysis of the latencies of individual ABR waves revealed timing differences between ice Fig. . Latenciave Fig. . Interesbcan+/+: 23.3 ± 4.5 dB, n = 7/13 animals/ears; bcan−/−: 24.2 ± 2.1 dB, n = 6/12 animals/ears, p = 0.537, t test, Fig. We further tested OHC function by measuring distortion product otoacoustic emissions (DPOAEs), a measure of OHC electromotility Fig. . The 2f1bcan−/− mice might point to impairment of synaptic transmission at the IHC synapse. A characteristic feature of the afferent ribbon synapse is its ability to faithfully transmit signals with high temporal precision and at high rates which requires—among other factors—tight, spatial alignment of presynaptic release sites and postsynaptic receptors. In a recent report by Fell and colleagues . For comparison between genotypes, two-tailed to Sachs   yields a strong immunosignal in the temporal bone and at calbindin-positive IHCs . Maximum intensity projections of confocal stacks of cochlear cross-sections. A, scale 50 μm, B, scale 5 μm. (TIF 3300 kb)Additional file 2:Figure S2. Hearing function assessed by ABR. Individual ABR thresholds in response to pure tones of n = 6/12 ears/animals each genotype . (TIF 3840 kb)Additional file 3:Figure S3. Growth functions of latencies of ABR waves I to IV. Growth functions of the latencies of ABR wave I, II, III, and IV are illustrated for each individual . (TIF 10387 kb)Additional file 4:A detailed list of number of samples, data analyses, and statistics . (XLSX 1


The results revealed differential expression of 14 of these 22 genes in the two varieties. Subsequently, endogenous hormone levels were assessed of the two varieties, along with the number of internodes and internode length. The results suggested that the persimmon could be used as a valuable and powerful natural candidate for providing information on the functional role of dwarfing.The aims of this work were to identify genes related to dwarfing for subsequent dwarfing-related research in persimmon and evaluate the relationship between antioxidant activity, dwarf, and hormones of persimmon trees for analyzing the possible dwarf mechanism oxidation factors. In the present study, a transcriptome analysis of “Nantongxiaofangshi” was used to identify and clone 22 candidate genes related to gibberellin signal transduction pathways and synthetic pathway. The expression of these genes was assessed in two persimmon cultivars, “Dafangshi” and “Nantongxiaofangshi,” by RT-qPCR at different phenological stages and in response to the exogenous application of GA Diospyros kaki Thunb.), characterized by producing performance, is a cultivar suitable for dwarfing and high-density planting due to its weak apical dominance and lack of an obvious trunk. In contrast to “Dafangshi,” “Nantongxiaofangshi” is significantly taller with greater tree height, tree spread, stem diameter, shoot length, and internode length [The persimmon cultivar “Nantongxiaofangshi” (e length . As one Gibberellin (GA) is a plant hormone that plays an important role in the development of stems and leaves. GA is involved in the regulation of many different growth and development processes, including seed germination, stem and leaf elongation, hypocotyl elongation, flower formation, and fruit development . GA metaendo-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20ox, GA3ox, and GA2ox. Among them, GA20ox is rate limiting and thus regulates the synthesis of GA [Arabidopsis have been reported to affect the GA signaling pathway. Mutations in GAI and RGA affect normal GA response resulting in a dwarf phenotype. The deletions in the N-terminal region of RGA alteration were affected by a deletion of 17 amino acids [Arabidopsis, rice, and Brassica [FveRGA1, was suggested to play a role in the formation of strawberry runners [SlDELLA has also been proposed to play a role in the formation of individual leaves in tomato, suggesting that GA may act as a negative regulator of leaflet formation [The GA biosynthesis pathway in higher plants is controlled by three main types of enzymes, which include is of GA –6. The bis of GA , 8. In ris of GA , 10. Theis of GA . Five geno acids , 13. GAIBrassica –16. Receormation . HoweverDiospyros kaki) to increase the environmental stress tolerance and exhibited dwarfing phenotypes [Deguchi et al. have reported that a cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH) of sorbitol biosynthesis from rosaceae was introduced into the Japanese persimmon , the terminal bud senescence stage (2018.5.10), the flowering stage (2018.5.22), the physiological fruit drop stage (2018.6.11), and the fruit color development stage (2018.9.13) [18.4.18, −1 GA3 (GA treatment) or 300 mg·L−1 PP333 (PP333 treatment), with distilled water serving as the control (CK). Leaves of the two persimmon varieties were sprayed and make no precipitation in 24 h after the application. Three plants were used for each treatment. Leaves were collected from both the treated groups and the control group at 0 d, 3 d, 6 d, 12 d, and 24 d after treatment. The samples were immediately immersed in liquid nitrogen and stored at -70°C.The hormone treatments consisted of 100 mg·LNew shoot growth (length) and internode number were recorded beginning on the day of the treatment and at 30 d after the treatment. The measurements were taken on three annual shoots of each of the plants that were similar in the two varieties.3, indole-3-acetic acid (IAA), abscisic acid (ABA), and zeatin nucleoside (ZR) were measured using an enzyme-linked immunosorbent assay (ELISA) method [Endogenous levels of GA) method .Total RNA was extracted from the leaf samples using a Plant Total RNA Isolation Kit Plus  according to the manufacturer's instructions. RNA integrity was checked by 1% agarose gel electrophoresis, and RNA concentration and purity were measured using a Nanodrop ND 1000 Spectrophotometer. Samples with 28S/18S ribosomal RNA between 1.5 and 2.0 and an absorbance ratio OD260/280 between 1.9 and 2.1 were used in the subsequent transcriptomic analysis. Approximately 1,000 ng of total RNA was used for cDNA synthesis using a PrimeScript RT Reagent Kit with gDNA Eraser . The cDNA was diluted with nuclease-free water prior to conducting RT-qPCR.Based on the transcriptome sequencing data obtained from “Nantongxiaofangshi” leaves (unpublished data), 22 candidate genes related to GA biosynthesis and signaling were cloned. Based on the obtained sequences, gene-specific primers for use in the RT-qPCR analysis were designed using Beacon Designer 7.0 software. A list of the gene-specific primers used in this study are presented in μl total volume) included 10 μl of SYBR Premix Ex Taq II , 1.0 μl diluted cDNA (150 ng/μl), 8.6 μl ddH2O, and 0.4 μl of each primer (10 μM). The PCR conditions were as follows: an initial denaturation step of 95°C (30 s), followed by 40 cycles of 95°C (5 s) and 60°C (30 s). Each sample was assayed in quadruplicate, and the DkGAPDH gene was used as an internal control. Relative expression was determined using the 2-△△Ct method.RT-qPCR reactions were performed using a LightCycler 480 II System . The PCR reaction mixture . Significant differences were determined using Duncan's mean separation test .In this work, the expression level of the 22 candidate genes in “Dafangshi” and “Nantongxiaofangshi” persimmon at different phenological stages was assessed in a preliminary study. The results revealed that 14 of the 22 gibberellin-related genes were differentially expressed at a significant level in the five phenological stages. The expression levels of Unigene7595, Unigene18861, Unigene34904, Unigene11769, CL1455.Contig1, and Unigene2304 were significantly higher in “Dafangshi.” In contrast, the expression level of Unigene20143 and CL628.Contig2 was consistently higher in “Nantongxiaofangshi.” Furthermore, the expression level of CL452.Contig2, CL452.Contig3, Unigene25672, CL4153.Contig2, CL4170.Contig4, and CL7393.Contig4 also exhibited a change in expression specifically at the flowering stage, and an opposite trend was exhibited after flowering .ent-kaurene oxidase (KO) activity, which was gibberellin-responsive dwarfing, indicating that KO gene expression was tissue specific, and its expression level was higher in growing tissues, but less in mature leaves and resting stems. It was speculated that the low expression of gibberellin synthase-related gene in the “Nantongxiaofangshi” was one of the reasons for its dwarf character.In detail, the expression levels of these three genes in the whole phenological period were always lower in “Nantongxiaofangshi” than in “Dafangshi,” and the expression levels of these three genes were detected only in “Dafangshi” in the exogenous hormone treatment test. Helliwell et al. pointed out that arabidopsis ga3-1 mutants showed dwarfing characteristics due to lack of 3 was greater in “Nantongxiaofangshi” than in “Dafangshi” and the ability of PAZ to inhibit shoot growth was lower in “Nantongxiaofangshi” than in “Dafangshi.” The number of internodes produced in the two varieties in response to the exogenous application of GA3 was greater in both varieties relative to the untreated control, supporting the premise that an application of a sufficient concentration of exogenous GA3 treatment can promote shoot growth as determined by the number of internodes produced.The length of current-year shoots of “Nantongxiaofangshi” and “Dafangshi” in response to hormone application are presented in Samples were collected from the untreated varieties of persimmon to determine and compare endogenous hormone levels. Results indicated that there was no significant difference between the two varieties in the level of endogenous ZR and IAA. The level of endogenous GA, however, was slightly lower in “Nantongxiaofangshi” than in “Dafangshi,” while the level of endogenous ABA was significantly higher in “Nantongxiaofangshi” than in “Dafangshi” .3, the endogenous level of GA was consistently higher than that of the untreated control ; this then decreases in GA3 but was sustained for PP333 until 24 d, where it decreased to control levels. ZR increased in “Nantongxiaofangshi” on 3 d, though after 24 d, both varieties have basically remained approximately degree as before treatment.Specifically, endogenous hormone can cause different variation of persimmon leaves. The content of ABA increases at 6 d after the application for “Dafangshi” and 12 d for “Nantongxiaofangshi,” but it is not sustained with concentrations dropping below that for the controls in the following days. GA increases for both varieties, and this was sustained. Compared with PP3 and PAZ. Notably, the expression of CL7393.Contig4 was not detected in either variety, so no further analysis was conducted. Three genes, Unigene7595, Unigene18861, and Unigene34904, which are involved in KO synthesis, KAO synthesis, and GA20ox synthesis were only detected in “Dafangshi.” As illustrated in 3, two genes, Unigene 25672, which is homologous to GA2ox, and CL4170.Contig4, which is homologous to DELLA protein, were significantly differentially expressed in the two varieties in response to GA3. In response to the exogenous application of PAZ, the two genes, Unigene 25672 and CL4170.Contig4, were also significantly differentially expressed in the two varieties in response to PAZ. Additionally, the gene CL628.Contig2, which was homologous to DELLA proteins, exhibited significant differences in expression in the two varieties in response to the exogenous application of PAZ  after the exogenous application of GAn of PAZ .In the present study, the analysis identified seven candidate genes in “Nantongxiaofangshi,” including three genes encoding KO, KAO, and GA20ox ; one gene encoding GA2ox (Unigene25672); one gene encoding the GA receptor, GID1 (Unigene 2304); and two genes encoding DELLA proteins (CL628.Contig2 and CL4170.Contig4). Among the seven candidate genes, two genes encoding DELLA protein (CL628.Contig2 and CL4170.Contig4) were found to be associated with dwarfing.3, and endogenous IAA levels also increased slightly. In contrast, increases in endogenous ABA levels were inhibited. In accordance to previous studies, the concentration of endogenous GA and IAA decreased, while the level of endogenous ABA increased subsequent to the exogenous application of PAZ. In previous studies in tobacco [Plant growth and development are affected by plant hormone interactions. Gibberellin plays an important role in breaking dormancy and promoting stem elongation. PAZ, as a growth inhibitor, can inhibit GA biosynthesis and cell elongation that reduces internode length in plant stems. The concentration of endogenous GA increased significantly, relative to untreated control, in response to the exogenous application of GA tobacco  and barl tobacco , the aut tobacco . Based o3 was only detected in “Dafangshi.” Therefore, we speculate that the low expression level of gibberellin synthase-related genes in “Nantongxiaofangshi” is one of the reasons underlying its dwarfing traits. GA2ox plays a key role in gibberellin inactivation [GA20ox is a key rate-limiting enzyme for gibberellin synthesis in plants. Sakamoto et al.  reportedtivation , 30. Sevtivation , 26, 31.3. This indicates that active GA, as a disguised inducer of GID1, may promote the expression of GID1 in “Nantongxiaofangshi” and promote the degradation of DELLA, and this restores GA-induced plant growth responses. DELLA proteins play a negative regulatory role in GA signal transduction and thus repress plant growth and development. Two GAI genes (Unigene20143 and Unigene11769), homologous to DELLA protein, were upregulated in both varieties in response to the exogenous application of GA, while they were both inhibited in both varieties in response to the application of PAZ. However, CL628.Contig2 and CL4170.Contig4 exhibited a completely different expression response in “Dafangshi” and “Nantongxiaofangshi”; this suggests that CL628.Contig2 and CL4170.Contig4 genes which belong to DELLA protein are associated with dwarfing. Branch orientations in “Nantongxiaofangshi” tend to be horizontal. Previous studies have reported that WEEP plays a role in regulating branch orientation and that silencing WEEP in Prunus domestica (plum) resulted in more horizontal shoot growth relative to standard trees where the orientation of branches is more upright [In the current study, the expression of the gene homologous to the GA receptor GID1  was cons upright , 34. TAC upright , 36. The upright . In addi upright .3 and PP333 foliar spraying processing and determine the content of endogenous hormone after processing and the comparison between their annual side length and number of internode, we analyzed 14 or more genes in the two varieties of the expression of exogenous hormone response differences and ultimately determined the 13 differentially expressed genes. These included 3 genes , respectively, from KO, KAO, and GA20ox; 3 genes  from GA2ox; 6 genes (Unigene2304) from GA receptor GID1; and 6 genes (Unigene20143 and Unigene11769) from DELLA protein .Based on the results of the transcriptome and bioinformatics analysis, we randomly selected genes from the “Nantongxiaofangshi” in 22 gibberellin signal transduction pathways and metabolism-related genes and analyzed the difference from “Dafangshi.” In “Nantongxiaofangshi,” different phenological phases of expressing features, so as screening out the differences associated with gibberellin way of two varieties, have 14 genes. At the same time, in order to explore the dwarf varieties of the “Nantongxiaofangshi” and the dwarf phenotype regulation mechanism of the formation in the growing season of “Dafangshi” and “Nantongxiaofangshi” GABased on previous reports in the literature and the results obtained in the present study, we speculate that CL628.Contig2 and CL4170.Contig4, two genes homologous to DELLA proteins, have involved in dwarfing in persimmon through their regulation of GA signal transduction and its complications.


If touch is perceived as pleasant, it can counteract the experience of pain. However, its pain-inhibitory function might be disturbed in chronic pain and this could contribute to pain-related interference. We investigated the perception of pleasant touch and its brain correlates in chronic back pain patients (CBP) compared to subacute back pain patients (SABP) and healthy controls (HC) using soft brush strokes. CBP showed less positive evaluations of touch. We found the highest activation in somatosensory and insular cortices in CBP, ventral striatum (VS) in SABP, and the orbitofrontal cortex in HC. Brain responses were significantly positively correlated with pleasantness ratings in HC and SABP, but not CBP. Further, the insula responses in CBP were positively correlated with pain-related interference and the VS activation in SABP correlated negatively with affective distress. Brain and behavioral changes in the processing of touch and its pleasantness may be a marker of pain chronicity and raise questions about the therapeutic value of pleasant touch in pain prevention and treatment. Pleasant or painful touch representations can drive behavioral adaptations to reach homeostatic balance , which mPleasant touch may act as a positive reinforcer that could have beneficial pain-reducing effects. Touching the body area affected by illness can enhance psychological and physical functioning, induce stress reduction and pain relief, and enhance coping abilities and general health ratings e.g., . This isThe perception and processing of pleasant touch might likewise be altered in the chronic state. Moreover, this might be different already in the early state of the disorder in subacute patients. We examined the brain responses to touch stimulation and ratings of pleasantness during functional magnetic resonance imaging (fMRI) in chronic (CBP) and subacute patients (SABP) with back pain as well as healthy control individuals (HC). Since pain-related interference with different areas of life and pain-related affective distress are core variables in cognitive-behavioral conceptualizations of chronic pain e.g., , we also22.1We investigated CBP , SABP , and HC , matched for education . We used a short stimulation sequence that reached the necessary minimum of stimulation events needed for reliable and valid summation of the touch trials (N = 6), but avoided the development of allodynia symptoms in the pain samples , equipped with a 12-channel head coil. Shimming of the scanner was done to account for maximum magnetic field homogeneity and a standard gradient field map was recorded at the beginning of each measurement. For the functional protocol, 40 contiguous axial slices  were acquired using a T2*-weighted gradient-echo echo-planar imaging (EPI) sequence with GRAPPA technique  = 2350 ms, echo time (TE) = 22 ms, matrix size = 96 × 96, field of view (FoV)= 220 × 220 mm2, flip angle (α)=90°, bandwidth (BW)=1270 Hz/px). For structural reference, we used a T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence  = 240 × 256mm2, flip angle (α) = 9°, bandwidth (BW) = 240 Hz/px) recording 192 sagittal slices.2.4Magnetic resonance imaging The analyses of the fMRI data were performed using Statistical Parametric Mapping software (SPM12 (v6685), Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London, UK), implemented on MATLAB R2016a . The first three scans were excluded from the analyses to account for T1-saturation effects. To reduce geometric distortions and improve spatial accuracy, gradient field map correction was performed on the remaining EPI images using the FieldMap toolbox in SPM. Afterwards the scans were realigned to the fourth image of the session using a rigid body transformation. A mean image was created, and the realigned and unwarped images were corrected for differences in acquisition time. The mean image was then co-registered to the T1 structural image, before the anatomical image was normalized into a standard stereotactic space  using tissue probability maps in SPM12. The nonlinear transformation parameters were then applied to the functional images. Finally, the data were smoothed with a 7 mm3  Gaussian kernel. Event-related blood oxygenation level-dependent (BOLD) responses were convolved with a canonical hemodynamic response function. Each subject’s data set was high-pass filtered  to remove low-frequency drifts and corrected for serial autocorrelations using first-order autoregressive functions AR(1).We modeled the touch stimulation block and included subject-specific regressors with events at the time of touch stimulus onset and of inter-trial interval onset (i.e. the baseline), respectively. As regressors of no interest, we included the six scan-to-scan motion parameters derived from the affine part of the realignment procedure to account for residual movement effects. The individual contrast (stimulation versus baseline) images were subsequently included in a second level random effects analysis using the full factorial model of SPM12. The problem of non-independent data within subjects as well as error variance heterogeneity was addressed by performing a non-sphericity correction. After calculating fixed effect analyses for each subject, second level random effects analyses were performed for the contrasts using analysis of variance (ANOVA) with group as a between factor. T-tests were calculated for significant main effects based on whole brain and small volume correction (SVC). Based on the established literature on pleasant touch representation in healthy individuals e.g., , we emplFor graphical demonstration of the results we plotted the correlation of brain responses with the pain intensity scores and extracted weighted mean beta values of the significant brain regions (masks of the regions were used from the Wake Forest University Pick Atlas v3.0.3). Psychometric and rating data were analyzed with ANOVAs using the Statistical Package for Social Sciences (SPSS) version 15.0 for Windows with pain status  as between subject factor.33.1All groups rated the touch stimulation as pleasant (ratings of 5 and above), however, the CBP group rated the stimulus as significantly less pleasant than the HC (t(2) = 2.49; p = 0.048) and the SABP (t(2) = 2.63; p = 0.049). The SABP group showed the highest, although not statistically significant, variance in pleasantness ratings, see .Fig. 1Pl3.2We found a significant main effect of pain status  for OFC , insula , S1 , and S2 , and VS . Follow-up t-tests between the groups revealed that the HC showed significantly higher responses in the OFC compared to CBP (p = 0.041) and SABP (p = 0.045). The CBP group had a significantly higher response in the insula , the S1  and S2 , and reduced response in the the VS compared to SABP (p = 0.038) , insula (r = 0.376), S1 (r = 0.159), S2 (r = 0.488), VS (r = 0.159); all p < 0.05) and SABP (OFC (r = 0.266), insula (r = 0.465), S1 (r = 0.154), S2 (r = 0.154), VS (r = 0.156); p < 0.05). The correlations were not significantly different between HC, SABP and CBP.3.3We observed a significant positive correlation between pain-related interference and insula responses in CBP  showed touch-related activation in brain regions previously identified as significantly involved in the processing of pleasant touch, including the OFC, insula, VS, ACC, S1 and S2, and also frontal areas. However, except for the ACC and amygdala, the response magnitudes of these regions differed significantly between HC, CBP and SABP. These differences were also apparent in the valence of pleasant touch, with the CBP perceiving the touch stimulation as significantly less pleasant than the HC and SABP. Moreover, we observed a significant association of insula responses and pain-related interference in CBP and VS responses and pain-related affective distress in SABP.4.1In line with CBP showed the lowest VS responsivity to pleasant touch, which was significantly different from SABP, but not HC. While the insula may be involved in the general emotional processing of stimuli e.g., , the VS 4.2The SABP group showed a significantly lower responsivity to pleasant touch in the posterior insula compared to HC and CBP. Previous studies documented an involvement of the insular cortex in the representation of pleasant touch , with th4.3The OFC has been implicated in the processing of affective touch e.g., , and the4.4The present study needs to be considered in the light of some limitations. We did not perform control stimulation (e.g. static touch or vibration) to differentiate the role of the affective component from touch stimulation per se. However, we analyzed the linear relationships between brain responses and affective pleasantness ratings of the touch stimulation within and across groups, which allowed us to directly relate valence and brain activation to the touch stimulation. We also do not know who in the SABP group will develop chronic pain and who will be resilient and how the group differences we found relate to the final outcome on pain chronicity. This will require longitudinal studies. A further limitation of our study is that we covaried out the use of medication. Our sample was too small to include past and current medication use as a separate factor that could explain additional variance in the response to somatosensory and hedonic stimuli. Last, in our experimental design we used 6 stimuli of pleasant touch, applied in the habituation phase at the beginning of a respondent conditioning experiment. Although this short protocol provided significant information, and avoided the occurrence of potential allodynia in the pain samples, a longer protocol with additional somatosensory and hedonic controls might have increased the power and clarified the specificity of the touch effects.4.5.Our data highlight a deficient processing of pleasant touch in CBP and alterations of the response also in the subacute state. These findings suggest that changes in the representation of pleasant touch could be a marker of deficient affective processing in the transition from acute to chronic pain. To what extent the group differences in the processing of pleasant touch extend to other types of somatosensory or hedonic stimuli needs to be determined in future studies. Moreover, our findings can inform longitudinal studies on pleasure and pain and could contribute to the identification of mechanisms predicting or preventing chronicity as well as to the development of therapeutic targets. The neural and perceptual processing of pleasant touch has been a relatively neglected topic in pain research. The possibility to precisely apply brush stimuli in an automated fashion with well defined neural and perceptual responses suggests an inclusion of pleasant touch in quantitative sensory testing as well as brain imaging protocols in pain research


This study investigates the influence of the household registration system on rural–urban disparity in healthcare access , using data from a large-scale nationwide life history survey that covered 150 counties across 28 provinces and municipalities in China. In contrast to the findings of many previous studies that emphasize the disparity in the residence place as the cause of rural–urban disparity in healthcare access, this study finds that the residence place just has a very limited influence on healthcare access in China, and what really matters is the household registration type. Our empirical results show that people with a non-rural household registration type generally have better healthcare access than those with a rural one. For rural residents, changing the registration type of their household  can improve their healthcare access, whereas changing the residence place or migrating from rural to urban areas have no effect. Therefore, mere rural-to-urban migration may not be a valid measure to eliminate the rural–urban disparity in healthcare access, unless the institution of healthcare resource allocation is reformed. Rural–urban disparity in healthcare access is especially reflected in the ease of physician visit, rehabilitative and palliative healthcare services, emergency health service, etc. ,2,3. It In China, a number of rural-to-urban migrants adopt self-medication or refuse to seek healthcare services even though they are in bad health . AlthougFrom this point of view, the institution of healthcare resource allocation can be the culprit of the rural–urban disparity in healthcare access. The separation or asynchronization between the residence place and health insurance coverage further solidifies this disparity and makes it persist among rural-to-urban migrants. The institution that plays an important role in determining the status of basic health insurance is the household registration system . Since 1955, to restrict internal population migration, the government of China has set up a household registration system, the so-called rural–urban dual hukou system ,25,26. TMoreover, with the rapid development of urbanization in the past three decades, China has seen the largest human migration in history and there has been a large influx of migrant workers from the rural areas into the urban areas ,31,32,33This study contributes to the existing literature in several aspects. First, this study advances the research on rural–urban disparity in healthcare access by exploring the institutional factor. Second, although the relation between household registration system and healthcare access has theoretical importance, empirical analyses of this issue are still rare. To fill this research gap, this study investigates the relation between the institutional factor and rural–urban disparity in healthcare access. Third, this study clarifies the limited effect of the residence place on healthcare access. By investigating different groups of people , this study differentiates the respective impacts of the household registration and residence place on healthcare access. In this way, this study also explains why rural-to-urban migrants still have poor healthcare access, even though they have migrated to urban areas.The data used in this study came from a nationwide stratified random sampling survey . This widely used nationwide survey aimed to collect a high-quality and nationally representative sample of Chinese residents over the age of 45 . The surThe dependent variables of this study included several aspects of primary healthcare access, that is, healthcare quality, blood pressure check, blood test, vision test, dental examination, breast exam . Details about dependent variables are shown below.Healthcare quality. Respondents were asked “Can you remember the first time you got to see a doctor? What was the type of the doctor?”. Blood pressure check. Respondents were asked “Did you have your blood pressure checked after you were 16 years old?”.Blood test. Respondents were asked “Did you have your blood tested after you turned 16 years of age, for example, for measurements of cholesterol or blood sugar?”. Vision exam. Respondents are asked “Did you have a vision test after you turned 16 years of age?”.Dental examination. Respondents were asked “Did you have dental examinations after you turned 16 years of age?”. Breast exam . Respondents were asked “Did you have a breast exam after you turned 16 years of age?”. Household registration type. Respondents were asked “What was your first household registration type?”. Registration type change of household. Respondents were asked “Has the registration type changed for your first household change?”. Registered household residence change. Respondents were asked “Has the registered household residence changed for your first household change?”. Residence after migration. Respondents were asked “After your first migration, were you living in a rural or urban area?”. Following previous literature, we also controlled a series of variables that can affect healthcare access. More details are shown below.Gender difference. In light of the noticeable gender inequity in healthcare access, the effect of gender was controlled in this study. One important reason is the social economic disadvantage of females. The other important reason is that traditional values, social culture, or religious beliefs in some countries can hinder females from seeking healthcare services, especially gynecological and reproductive health services which can make them feel embarrassed or humiliated [miliated ,50,51. Educational level. Considering that a lower education level was shown to be associated with lower health literacy, which can prevent people from timely utilizing healthcare services [services ,53,54, wEconomic condition. As a better economic condition can help people access better healthcare services, we controlled the effect of the economic condition. Ethnic minority. We controlled the effect of belonging to an ethnic minority, since previous studies showed that healthcare services can be comparatively restricted for ethnic minority people because of a variety of barriers such as linguistic incompetence, cultural inadaptation, location or settlement disadvantage, “newness” or user ignorance [gnorance ,56,57. Religious belief. We controlled the effect of religious beliefs, since previous studies showed that religious belief can serve as an important barrier to healthcare access. Specifically, Muslim women feel more constraints to accessing healthcare because of the religious obligation to maintain bodily sanctity [sanctity ,59,60. Msanctity ,59,61. Whether or not to have health insurance coverage. We controlled whether or not the respondents had any medical insurance, since previous studies showed that health insurance coverage was closely associated with healthcare access, and uninsured people were more likely to suffer from insufficient healthcare services [services . We matched the data and constructed a dataset that contained the above-mentioned variables with non-missing observations. The overview of variables (grouping) and correlation matrix of core variables are respectively displayed in t-test , t-value = 19.3437; all the above t-values are significant at p-value < 0.01). The results of an independent sample t-test  indicatep-value < 0.01). These results of independent sample t-test and pairwise correlation matrix provide preliminary support for the proposition of this study. The results of pairwise correlation matrix  showed tThis study used the multiple linear regression method to examine the relations between household registration, migration, and healthcare access. Linear regression is a widely used approach for modeling the relationships between a scalar dependent variable y and one or more independent variables denoted x. The least-square method is generally used to fit the linear regression model. To control the heterogeneity of individuals across different communities and villages, the estimated standard errors were clustered on community/village level. The least-square method was used for the estimation, and the statistical software Stata 13.1 was used in the data analysis. More details are shown below.The first set of regressions see  showed tIn the second set of regressions see , this stIn the third set of regressions see , this stIn the fourth set of regressions see , this stHealthcare quality. In total, 19,367 answers to the question “Can you remember the first time you got to see a doctor? What was the type of the doctor?” were obtained; 2191 (11.31%) were “general hospital”, 297 (1.53%) were “specialized hospital”, 2814 (14.53%) were “health clinics in towns”, 4678 (24.15%) were “community health station”, 1414 (7.30%) were “private clinic”, 2460 (12.70%) were “without qualification”, 4445 (22.95%) were “barefoot doctor”, 325 (1.68%) were “others”, 743 (3.84%) were “never see a doctor”.Blood pressure check. Of the total 18,944 answers to the question “Did you have blood pressure checked after you turned 16 years of age?”, 13,533 (71.44%) were “yes”, 5411 (28.56%) were “no”.Blood test. In total, 19,445 answers to the question “Did you have your blood tested after you turned 16 years of age, for example, measurements of cholesterol or blood sugar?” were obtained, of which, 10,863 (55.87%) were “yes”, 8562 (44.13%) were “no”.Vision exam. In total, 199,98 answers to the question “Did you have a vision test after you turned 16 years of age?” were collected, of which, 6844 (34.22%) were “yes”, 13154 (65.78%) were “no”.Dental examination. In total, 20,038 answers to the question “Did you have dental examinations after you turned 16 years of age?” were collected, of which, 5130 (25.60%) were “yes”, 14908 (74.40%) were “no”.Breast exam . In total, 10,512 answers to the question “Did you have a breast exam after you turned 16 years of age?” were collected, of which, 2410 (22.93%) were “yes”, 8102 (77.07%) were “no”.Household registration type. In total, 19,999 answers to the question “What was your first household registration type?” were obtained; 18015 (90.08%) were “rural household registration ”, 1984 (9.82%) were “non-rural household registration  belonging to the non-agricultural type, and 166 (0.83%) belonging to the unified residence type)”.Registration type change of household. In total, 5640 answers to the question “Has the registration type changed for your first household change?” were collected; 2657 (47.11%) were “yes”, 2983 (52.89%) were “no”.Registered household residence change. In total, 5640 answers to the question “Has the registered household residence changed for your first household registration change?” were collected; 3950 (70.04%) were “yes”, 1690 (29.96%) were “no”.Residence after migration. In total, 7108 answers to the question “After your first migration, were you living in rural or urban areas?” were obtained; 5930 (83.43%) were “living in a rural area”, 136 (1.91%) were “mainly living in a rural area”, 19 (0.27%) were “Time living in rural and urban areas was roughly the same”, 64 (0.90%) were “mainly living in an urban area”, 959 (13.49%) were “living in an urban area”.Gender difference. In total, 20,402 answers were collected, of which, 9682 (47.46%) were “male”, 10,720 (52.54%) were “female”.Educational level. In total, 20,393 answers were obtained; 5 (0.02%) were “Ph. D”, 15 (0.07%) were “master”, 185 (0.91%) were “four-year college”, 387 (1.90%) were “three-year college”, 596 (2.92%) were “vocational school”, 1635 (8.02%) were “high school”, 5257 (25.78%) were “middle school”, 12313 (60.38%) were “primary school and below”.Economic condition. In total, 20,248 answers were obtained; 250 (1.23%) were “a lot better off than them”, 1742 (8.60%) were “somewhat better off than them”, 10,305 (50.89%) were “same as them”, 3219 (15.90%) were “somewhat worse off than them”, 4732 (23.37%) were “a lot worse off than them”.Ethnic minority. In total, 20,412 answers were obtained; 18399 (90.14%) were “not ethnic minority”, 2013 (9.86%) were “ethnic minority”. Religious belief. In total, 20,418 answers were collected; 17727 (86.82%) were “no religious belief”, 2691 (13.18%) were “have religious belief”. Whether or not have health insurance coverage. In total, 19,379 answers, of which, 18690 (96.44%) were “have medical insurance”, 689 (3.56%) were “no medical insurance”. p-value < 0.01).p-value < 0.05, healthcare quality; 0.1040, blood pressure check; 0.1451, blood test; 0.2845, vision test; 0.1763, dental examination; 0.1840, breast exam; all with p-value < 0.01). Besides, the relationship between the registered household residence change (from one place to another place) and healthcare access was insignificant . Also, the association between migration and healthcare access was generally insignificant .The regressions of p-value < 0.01, healthcare quality; 0.0877, p-value < 0.05, blood pressure check; 0.1070, p-value < 0.01, blood test; 0.0941, p-value < 0.01, vision test; 0.0950, p-value < 0.01, dental examination; 0.0419, p-value < 0.10, breast exam).The results of p-value > 0.10; and 0.0192, p-value < 0.10, dental examination).Lastly, the results of The empirical findings of this study show that, compared with other countries and regions across the world, the household registration (hukou) system unique to China, rather than the residence place, leads to Chinese rural–urban disparity in healthcare access. This is why the rapid development of urbanization in China has not necessarily solved the rural–urban disparity in healthcare access. This study finds that the change of household registration type from rural to non-rural plays a decisive role in improving healthcare access for rural-to-urban migrants. In this way, they can be eligible to enjoy a broader health insurance coverage. The investigation of different groups  helped further confirm that the mere change of residence place from rural areas to urban areas cannot unconditionally improve healthcare access for migrants, unless they can simultaneously change their household registration type from rural type to non-rural type. Moreover, the investigation of rural residents that never migrated revealed that, although residing in rural areas, they could still get an improvement in healthcare access by changing household registration from rural type to non-rural type, which further confirms the decisive role of household registration type in healthcare access. Meanwhile, this finding also reflects the dilemma of urban villages in China. The savage development of urbanization in China is making the urban areas expand quickly. As a result, those rural areas originally near a city become gradually surrounded by the expanding city and are thus called “urban villages”. In this case, even though the rural residents who live in urban villages already resided inside the city, they still hold the rural-type household registration and thus are legally identified as rural residents. This dilemma leaves quite a large number of rural residents in “urban villages” being denied coverage by the urban basic health insurance, keeping their healthcare access poor. As such, for the great number of rural residents in “urban villages” in China, changing of household registration type from rural type to non-rural means that they can access broader healthcare services that are easily available. Although this is a tricky matter, and a long process is required to reform the current household registration system in China, fortunately, some of the existing literature provide some suggestions to solve the rural–urban disparity in healthcare access. For example, the successful experience of the AiCare platform , a regioIt may be a feasible to build a similar collaborative health portal platform in China. It emphasizes the centrality of the patient, without differentiating rural and urban residents. Also, its affordable care services consider the comparatively lower economic status of Chinese rural residents and rural-to-urban migrants. With the large diffusion of smartphones in China, such online platforms are expected to be an important channel for accessing healthcare and, to some extent, can help narrow the rural–urban disparity in healthcare access. This study is not free of limitations. On the one hand, the nationwide survey used in this study was focused on the traditional and mainstream channels for accessing healthcare and did not consider other emerging and technology-based channels, like online telemedicine service platforms operated by the private sector. Thus, this study could not take into account such information related to healthcare access. Future research can use new data to investigate relevant issues to complement our analysis. On the other hand, because of the type of data available, only a few kinds of primary healthcare access were explored in this study. As more relevant data become available, future research can make more comprehensive explorations. A large influx of rural-to-urban migrant workers has become a striking phenomenon in China as a result of the rapid urbanization that occurred in the past three decades. However, rural-to-urban migration cannot eliminate Chinese rural–urban disparity in healthcare access. The deficiency in healthcare access for this group of people has become an obvious and burning question in China. This study empirically finds that the root cause of Chinese rural–urban disparity in healthcare access lies in the household registration system unique to China. This system causes a separation between the residence place and health insurance coverage, which thus seriously restricts healthcare access for rural-to-urban migrant workers in cities. Only the change of household registration type  can improve their healthcare access, but the channels for changing household registration type from rural to non-rural are very limited for them. Moreover, the phenomenon of “urban villages” is another striking product of rapid urbanization in China. The local residents of “urban villages” face the dilemma that they are still considered as rural residents and continue suffering from the deficiency in healthcare access, although the villages where they reside are now located within cities. Similarly, only the change of household registration type from rural to non-rural can improve their healthcare access. Therefore, with the savage development of urbanization in China, the disadvantages of the current household registration system have become increasingly apparent. There is a need to reform this system to keep pace with the social development and to improve healthcare access for various groups in China.