Artificial rearing system, commonly used in prolific sheep breeds, is associated to increased mortality and morbidity rates before weaning, which might be linked to perturbations in digestive tract maturation, including microbiota colonization. This study evaluated the effect of rearing mode  on the establishment of the rumen and intestinal microbiome of lambs from birth to weaning. We also measured immunological and zootechnical parameters to assess lambs’ growth and health. GIT anatomy as well as rumen and intestinal epithelium gene expression were also analysed on weaned animals to assess possible long-term effects of the rearing practice.Fibrobacter succinogenes and rumen protozoa did not establish correctly before weaning in artificially-reared lambs. Enterobacteriaceae and Escherichia coli were dominant in the fecal microbiota of mothered lambs, but main E. coli virulence genes were not found differential between the two groups, suggesting they are commensal bacteria which could exert a protective effect against pathogens. The fecal microbiota of artificially-reared lambs had a high proportion of lactic acid bacteria taxa. No difference was observed in mucosa gene expression in the two lamb groups after weaning.Total VFA concentrations were higher in mothered lambs at 2 months of age, while artificially-reared lambs had lower average daily gain, a more degraded sanitary status and lower serum IgG concentration in the early growth phase. Metataxonomic analysis revealed higher richness of bacterial and eukaryote populations in mothered vs. artificially-reared lambs in both Rumen and Feces. Beta diversity analysis indicated an evolution of rumen and fecal bacterial communities in mothered lambs with age, not observed in artificially-reared lambs. Important functional microorganisms such as the cellulolytic bacterium The rearing mode influences gastrointestinal microbiota and health-associated parameters in offspring in early life: rumen maturation was impaired in artificially-reared lambs which also presented altered sanitary status and higher risk of gut dysbiosis. The first month of age is thus a critical period where the gastrointestinal tract environment and microbiota are particularly unstable and special care should be taken in the management of artificially fed newborn ruminants.The online version contains supplementary material available at 10.1186/s42523-023-00255-7. The microbial colonization of the digestive tract during early life is a dynamic process in mammals which plays a key role in host health and development , 2. IndeFibrobacter succinogenes and of protozoa in their rumen [The rumen microbiota is inoculated at birth and in early life from various sources, including the mother vaginal canal, udder skin, colostrum but also environment, fecal material on the litter and saliva from the dam . It has ir rumen . Furtherir rumen  in additAlthough the presence of the dam is of importance for the newborn gut microbiota inoculation and its health and welfare, artificial rearing systems, i.e., separating newborns from their mothers and feeding them with milk replacer, have been developed in ovine. This is due to the selection of prolific breeds such as Romane breed with ewes giving birth up to 3 or even 4 lambs, because of the important economic impact of reproductive efficiency in sheep, particularly for meat production . HoweverRumen development could also be negatively affected by artificial rearing, and kids isolated and fed with milk replacer could also show a significant growth impairment . Howeverp < 0.05). In addition, the MOT group showed higher variability in ADG after weaning.Lamb growth was monitored all along the experiment. On the whole experiment, no significant difference was observed in average daily gain (ADG) of MOT and ART groups, although the ADG was significantly higher for the ART group in the post-weaning period Fig. A   of microbiota to compare the rumen microbial colonization of mothered and artificially-reared lambs from birth up to two months of life and monitored the evolution of the fecal microbiota of the two groups during the same period of time.Ruminococcus albus and Fibrobacter succinogenes were quantified in the rumen of all animals from the two groups using qPCR , but no significant difference was found between ART and MOT lambs, and a high variability was observed, especially during the first 7 days of life. No difference could be detected on R. albus population levels in the two groups, but F. succinogenes established more rapidly at high levels in the MOT lambs compared to the ART lambs .A group effect was observed for ASV numbers and Shannon index with higher values in the rumen microbiota of mothered lambs compared with artificially-reared lambs Fig. A.Beta diversity analysis of the rumen microbiota showed an evolution of bacterial communities with age in MOT lambs not observed in ART lambs Fig. B. A signAt the phylum level, MOT lamb rumens had much more relative proportions of Proteobacteria at day 7 than ART lambs . Twenty-four differential taxa were identified at day 7, 120 differential taxa at day 14 and 51 at day 28 . At day 7, only 5 taxa were more abundant in ART rumens while 19 other taxa were more abundant in MOT rumens. Differential taxa at day 7 included Lactobacillaceae, Enterobacteriaceae, Streptococcaceae and Veillonellaceae which were more abundant in ART rumens (Log2FC > 2.5) and taxa such as Acidobacteriaceae (subgroup 1), Bradyrhizobiaceae, Anaeroplasmataceae or Neisseriaceae more abundant in MOT rumens (Log2FC<-3.5). Fibrobacteraceae were also much more abundant in MOT rumens (Log2FC<-2).The differential analysis also identified differential taxa in relation to age (p < 0.05), these differential taxa being much more numerous in the MOT group compared with the ART group. The number of differential families between day 7 and 14 of age were 10 and 44 for ART and MOT rumens, respectively, and between 14 and 28 days, the differential families were 23 for ART and 114 for MOT.Lactobacillus  and E.coli (5.2%) were characterized as indicative of Art group at d7 (p < 0.05). Some Streptococcus species were significantly associated with Art group (d7 and d14) but at low relative abundance (~ 0.05%). Anaeroplasma varium (1.5%) was indicative of Mot group at d28 while several Acidobacteria species were identified as significantly indicative of Mot group at d14 but at low relative abundances (ranging from 0.003 to 0.09%).The observations made through DESeq2 analysis were confirmed by the indicspecies analysis  in the rumen microbiota of mothered lambs compared with artificially-reared lambs Fig. A, indicaEntodinium, Eudiplodinium and Polyplastron genera, which were also the dominant taxa in these animals, while in ART lambs the dominant genera were Isotricha and Dasytricha  and day 28 , except Debaryomyces sequences which were more abundant in MOT lamb rumens at day 14  were identified as significantly indicative of Art group at d14, while Mot group was associated with E. coli , Faecalibacterium prausnitzii (0.044%) and Prevotella HUN102 (0.4%).Deseq2 analysis  identified 10 differential taxa between MOT and ART feces (p < 0.05) at day 14: Lactobacillaceae, Alcaligenaceae, Streptococcaceae, Gemmatimonadaceae and Erysipelotrichaceae families were more abundant in ART feces, while Bacteroidaceae, Coriobacteriaceae, Enterobacteriaceae, Moraxellaceae and Pseudomonadaceae were more abundant in MOT feces. At day 28, 19 differential taxa were identified between ART and MOT fecal microbiota, with high value of Log2FC. These included Bifidobacteriaceae and Lactobacillaceae much more abundant in ART feces. Deseq2 analysis also identified 27 taxa differentially distributed according to age in MOT fecal samples (p < 0.05). Two species of lactic acid bacteria  was more frequently detected in ART feces at d14  while gene encoding Eagg EC heat stable enterotoxin (East1) was more prevalent in MOT feces at d14 . The heat labile enterotoxin (LT), East1 and Shiga toxin 2 (Stx2) genes were present in some samples at d14 but not detected anymore at d28 in any sample, while Stx1 was below detection threshold in all fecal samples tested at both sampling times  in the fecal microbiota of mothered lambs compared with artificially-reared lambs at day 28   , and Chytridiomycota were more abundant in MOT feces at day 28 (p < 0.001)  on rumen and lower gut microbiota colonization in the first month after birth as well as possible post-weaning effects on GIT development.Cryptosporidium taxa. Cryptosporidium is an ubiquitous protozoan parasite that infects a broad range of vertebrate hosts, including sheep [Eimeria, a well-known coccidian parasite in lambs [No difference in lambs’ weight and ADG was observed between groups before weaning or on the whole experiment. Several previous works reported impaired early growth rate in lambs submitted to artificial rearing compared with mothering , 22. On ng sheep . Note thin lambs , were alin lambs . In addiin lambs . Delayinin lambs . In agrein lambs . In addiin lambs . LimitedF. succinogenes population level).To investigate possible long-term effects of the newborn rearing mode on the lamb physiology post-weaning, the GIT anatomy and cellular activity were evaluated in the two groups. No impact of the rearing mode was observed at day 64 on the GIT anatomy as assessed by measuring the relative weight of organs, rumen wall thickness and papillae dimensions. We also quantified the expression of a selection of genes coding for proteins involved in epithelium integrity, metabolism or immune response in the rumen and colon mucosa, but no significant difference could be observed between the ART and MOT lambs. This suggests that after 64 days, the epithelium metabolism and activity is similar whatever the rearing system, even though it could have been different earlier in life. Indeed, the rumen metabolism of pre-weaning lambs has been shown to be mainly stimulated by SCFA , 32 and Claudin1, HMGSC2, HMGSL and MCT1 were more expressed in the rumen wall while IGF-1, IL-10 and TLR4, participating to intestinal homeostasis, epithelial regeneration, and immunity, appeared more expressed in the colon epithelium. Expression of genes involved in innate immune functions has demonstrated variations according to GIT segment in bovines [TLR4  was previously found higher in the colon epithelium than in the rumen mucosa both in calves and cows [HMGSC2 and HMGSL encode the 3-hydroxy-3-methylglutaryl-CoA synthase isoform 2 and a 3-hydroxy-3-methylglutaryl-CoA lyase, respectively, these two proteins being involved in the ketogenesis pathway. It is known that, while the neonate rumen epithelium is not ketogenic, the ketogenesis pathway is expressed in the sheep rumen mucosa from 42 days of age [Finally, our data showed differential expression of certain genes in the rumen vs. the colon epithelium of lambs irrespective of the rearing mode and bring additional information to the few papers comparing gene expression in these two GIT segments in sheep. Among the differential genes identified here,  bovines . The expand cows , 35, accand cows . HMGSC2 s of age . MCT1 prs of age . More prs of age , and itss of age . Our resThe present study highlights huge modifications in the rumen and lower gut microbiota colonization depending on the mode of rearing before weaning. Artificial rearing led to lower richness and slower maturation of the rumen microbiota, with delayed establishment of functional fibrolytic populations and protozoa, leading to lower rumen fermentative activities, particularly after weaning.Bifidobacterium, Enterobacteriaceae, Streptococcus and Lactobacillus taxa decreased while strictly anaerobic bacteria increased and fibrolytic bacteria such as Ruminococcus flavefaciens and Fibrobacter sp. become detectable only around weaning [The rumen and distal gut microbiota of MOT animals diversified and matured with age, according to known stages of GIT colonization in sheep and bovines: Proteobacteria, including facultative anaerobic bacteria, constitute the major phylum during the first days of life, and they consume the oxygen present allowing the gradual colonization of strictly anaerobic bacteria and fungi in the first two weeks , 40–43.  weaning . Such su weaning , 20, 22.Bifidobacterium in the feces of pre-weaned ART lambs. These families/genera are generally associated with primarily milk feeding diet, as it has been shown in humans that milk oligosaccharides and glycans can act as prebiotics stimulating the growth of Bifidobacterium and Lactobacillus spp [R. albus and F. succinogenes were already well established at day 7 in the MOT lamb rumens, but, although R. albus was also quantified at high levels in ART rumens, F. succinogenes establishment was clearly delayed in ART animals. This species was found in lower proportions in the artificially reared lambs, even after weaning. Strong delay in establishment of F. succinogenes was previously detected in calves or lambs separated from their dam [Fibrobacter may be quite sensitive to changes in the ecological conditions of the rumen. F. succinogenes is known as an important fibrolytic microorganism, particularly efficient in degrading recalcitrant cellulosic substrates such as straw [The ART rumen and feces microbiota revealed a lactic profile with high proportions of Streptococcaceae and Lactobacillaceae, respectively, compared with MOT microbial communities. This was confirmed by qPCR quantifications which showed higher proportions of lactic acid bacteria and llus spp . Streptoheir dam , 10, 46.heir dam , indicatas straw , 49 and Escherichia as dominant genus in the feces. Neisseriaceae are known to colonize the rumen mucosal surface and belong to the core rumen epithelial bacteria of several ruminant species [Another remarkable finding is the much lower relative proportion of Proteobacteria in ART newborn lambs compared with MOT animals, both in rumen (at day 7) and feces (at day 14), the major phylum in ART samples at these ages being Firmicutes. In MOT lambs, the Proteobacteria sequences are in majority affiliated to Neisseriaceae in the rumen and to Enterobacteriaceae including  species , 51. The species . Thus, m species ); howeveEscherichia was a dominant genus in the feces of MOT lambs, we sought the presence of E. coli virulence genes in these samples. No difference between MOT and ART lambs’ feces was detected, except for 2 genes with opposite detection frequencies. Despite a strong increase of Enterobacteriaceae relative abundance observed in MOT lambs’ feces at d14, we could not find any strong association with virulence genes detection frequencies of the colibiota, in accordance with the good sanitary status of these animals. Previous studies in piglets have shown that E. coli identified during the pre-weaning period were non-pathogenic microorganisms that conferred health benefits to the host [E. coli might be mostly commensals and that they could fill an ecological niche no more available for pathogenic E. coli colonization, potentially providing a protective effect to the mothered lambs.Because the host . We coulAmong other differential bacterial taxa in the rumen of the two lamb groups, Anaeroplasmataceae and Acidobacteriaceae were much more abundant in MOT rumens. These two families have been associated to the succinate to propionate pathway in goat . They maEntodinium, Eudiplodinium and Polyplastron as dominant genera. It is known that the establishment of ciliate protozoa in the rumen requires the preliminary establishment of a diverse bacteria community [Candida spp. have been found in the sheep farm environment and animal feed, as well as in ewe’s milk and teat surface or ruminant digestive tract [Regarding the Eukaryota community, artificially reared lambs had no rumen ciliate protozoa at weaning, while natural rearing promoted an early colonization of the rumen by a diverse protozoal population. These results confirm previous findings on lambs and goat kids , 22, 23.ommunity , 23. Theommunity . Howeverve tract , 59, butFinally, the dysbiosis observed in the ART rumen and hindgut microbiota reveals impaired establishments of functionally important microbes, which may be due to several factors. Nutritional factors such as limited colostrum feeding or quality of milk replacer may be important in the artificial rearing management, but other stresses such as the separation with the dam and other adult ruminants may also affect the health and wellbeing of the lamb .E. coli populations in the intestine of MOT lambs could exert a protective effect against pathogenic E. coli. The first month of age is thus a critical period where the GIT environment and microbiota are particularly unstable and special care should be taken in the management of artificially reared newborn ruminants.The presence of the mother and rearing mode influence GIT microbiota and health associated parameters in offspring in early life. Although ART and MOT lambs presented similar GIT development and performances, rumen maturation was delayed in ART lambs with very late establishment of functional fibrolytic populations, linked to lower rumen fermentative activities. ART lambs presented also altered sanitary status, probably indicative of higher risk of gut dysbiosis, as reflected by the measured higher LAB fecal concentrations. Higher proportions of commensal http://www2.vet-lyon.fr/ens/expa/acc_regl.html). The protocol was validated by the Regional Ethics Committee on Animal Experimentation C2EA-02 and authorized by the French ministry with the reference number 14981-2018050417167566V3.The animal trial was conducted at the animal facilities of INRAE Herbipôle Experimental Unit UE1414 . Procedures on animals were carried out in accordance with the guidelines for animal research of the French Ministry of Agriculture and all other applicable national and European guidelines and regulations for experimentation with animals  were used for this study and were followed from birth to 64 days of age , one for protozoa enumeration, and the other was frozen at − 20 °C until microbiota analysis with qPCR and sequencing.Fecal contents were collected manually in the rectum of lambs and frozen at − 20 °C for further analysis.Blood sampling. Blood samples were collected from all lambs at day 2, 7, 28 and 55 of age through the jugular vein in dry collection tubes  by a qualified technician. Dry tubes were set for clotting for at least 1 h at room temperature, centrifuged at 4 500 rpm for 20 min at 4 °C and serum supernatants were stored at -80 °C until further analysis.Protozoa were enumerated in a Neubauer chamber under a microscope after staining with methyl green solution as previously described .DNA was extracted from at least 250 mg of rumen or fecal content using the Quick DNA Fecal/Soil Microbe kit . DNA yield and quality were determined after Nanodrop 1000 and Qubit spectrophotometric quantifications. DNA extracts were stored at -20 °C until analysis.E. coli virulence were also quantified in feces samples through the same qPCR method . PCR targets and primers are summarized in Supplementary Table i et al.  using thMicrobiota diversity and taxonomic composition were analysed by 16S/18S rRNA gene amplicon sequencing. DNA samples were quantified with a Qubit spectrophotometer to adjust concentrations to at least 20 ng/µL, and a volume of 30 µL per sample was sent to the Novogene sequencing platform . DNA sequencing was performed on a subset of samples from 8 lambs per group for each sampling time considered. The diversity and composition of rumen/fecal microbiota were studied using the high throughput sequencing Illumina MiSeq  method (2 x 250 nt paired ends). The primer sets used, and rDNA regions targeted are indicated in Supplementary Table https://www.R-project.org/) for the pipeline’s step of filtering, trimming, dereplication, to infer the sample composition and to remove chimera. The multiple-sequence alignment was performed using the DECIPHER R package [Bacterial taxonomic assignment was performed using the rANOMALY  pipeline package . The Sil package .All the diversity analyses were performed with the rANOMALY  pipelineThe serum Immunoglobulin G (IgG) concentration was analysed by radial immunodiffusion by CIAL Sud Ouest laboratory  from 1 ml lamb serum sample.SCFA concentrations were determined in rumen samples by gas chromatography, as previously described .At day 64, 5 lambs from each group were slaughtered in the experimental slaughterhouse of the INRAE Herbipôle facility .The different organs of the GIT tract were collected just after slaughter. The organ contents were withdrawn, and the organs were rinsed with tap water and weighted.Morphological measures were performed on the digestive organs, and rumen and intestinal biopsies were collected for transcriptomic study of targeted genes using RT-qPCR  followed by DNase digestion and Qiagen RNeasy column purification (Qiagen). The nucleic acid concentration and purity.were assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific). The RNA integrity was finally verified using an Agilent Bioanalyzer 2100 .C−ΔΔT method) [The gene expression analysis was performed by Helixio  as described below. Five hundred nanograms of total RNA were reverse transcribed with the “High-Capacity cDNA Reverse Transcription kit” (ThermoFisher Scientific) in a reaction volume of 80 µl, according to the manufacturer recommendations. The samples were incubated at 25 °C for 10 min, followed by 2 h at 37 °C, then the enzyme was inactivated at 85 °C for 5 min. A control was included (no reverse transcriptase) to verify no genomic DNA contamination. The SYBRgreen chemistry was used for qPCR quantification of the gene expression. The reaction mix contained 1X PowerUp™ SYBR™ Green Master Mix (2X) (ThermoFisher Scientific), 500 nM of forward and reverse primers Table , and 10  method)  and two  method) .Graphical representations and statistical analyses were performed using GraphPad Prism 9.0.2. The effect of rearing mode (ART or MOT) and Time factors and their interaction were evaluated using a linear mixed model with repetitions and multiple comparisons with Sidak’s adjustment.For some parameters, the data could not fit the chosen mixed model i.e., when the data were not normally distributed and the nonparametric Mann–Whitney (MW) test was thus applied to compare the ART and MOT groups at each time. Due to the sporadic detection of virulence genes, results were analysed as detection frequencies and statistical differences were tested via Fisher exact test. Statistical significance was determined at a p-value < 0.05 and trends were discussed when p < 0.10.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


On 12 November 2021, after a stagnation of 8.5 years, Japan decided to resume proactive recommendations for HPV vaccines. However, it is not known how the public reacted to the resumption of proactive recommendations for HPV vaccines, which are key elements in health communication that promote HPV vaccination. This study aimed to capture discussions on HPV vaccination on Twitter and to identify user characteristics, sentiments, discussed themes and their potential reach before and after 12 November 2021, when the Ministry of Health, Labour, and Welfare’s decision to resume proactive recommendation for HPV vaccines was first reported by the media in Japan. This study conducted a content analysis of tweets related to HPV vaccines posted on 11 November and 12 November 2021. Tweets were coded in terms of user characteristics, sentiments  and themes. Reach was measured by the number of retweets, likes and followers. A total of 3623 tweets were identified. The results showed that approximately 50% of health professionals and researchers tweeted neutral content about HPV vaccines. The most frequently discussed theme was the safety and side effects of HPV vaccines, which was accompanied by mainly negative sentiments. Although health professionals and researchers are influential on Twitter, half of them tweeted neutrally about HPV vaccines. Influential professionals, such as health professionals and researchers, are expected to disseminate accurate information to correct misinformation and recommend HPV vaccination on Twitter to overcome the HPV vaccination crisis, which is characterized by the low vaccination rates. Monitoring the spread of information about HPV vaccines, the users who disseminate, sentiments and themes discussed on Twitter, can be useful for understanding the public perception of HPV vaccines.Most Twitter users expressed neutral sentiments about HPV vaccines after news reports of Japan’s resumption of proactive recommendations. After the news reports, the most discussed theme was safety concerns regarding HPV vaccines, which were mainly tweeted negatively.Although health professionals and researchers gained more followers, approximately half of them tweeted neutrally about HPV vaccines. They are expected to disseminate accurate information and recommend HPV vaccination on Twitter.Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide . DespiteJapan reported one of the lowest HPV vaccine coverages among high-income countries . In 2013Twitter has been studied as a health research tool  who tweet about the HPV vaccine, and are there any differences in user characteristics before and after the news reports on the resumption of proactive recommendations for HPV vaccines?RQ2: Is there any difference in the sentiment of the tweets before and after the news reports, and any difference in reach (i.e. the sum of followers) by sentiment after the news reports?RQ3: Does the sentiment of tweets differ between users on Twitter? Is there any difference in reach (i.e. the sum of followers) by the type of users after the news reports?RQ4: What are the most frequently discussed themes on Twitter regarding the HPV vaccine before and after the news reports and is there any difference in sentiment?The first author (MT) applied and was approved for academic research on the Twitter application programming interface (API) through a developer account . Tweet dwakuchin’ and ‘yobouseshu’ in the search, which correspond to the following English terms: ‘vaccine’, ‘vaccination’ and ‘immunization’. The Japanese refer to cervical cancer and HPV vaccines as ‘shikyu keigan’ and ‘sikyu keigan wakuchin’. We applied a language filter to retain only Japanese tweets and used the keywords (((HPV OR ‘sikyu keigan’) AND (‘wakuchin’ OR ‘yobouseshu’ OR ‘yobou wakuchin’)) OR Gardasil OR Cervarix OR Silgard) on Twitter and ‘HPV wakuchin’ on Google Trends based on previous studies  of the total sample of tweets was randomly selected, and TN coded them to assess inter-rater reliability for all categories. All data coding was performed using Microsoft Excel .For RQ1, we manually classified users according to their usernames and self-descriptions, consistent with previous studies .A total of 3623 tweets were extracted, of which 349 were tweeted on 11 November 2021 and 3274 were tweeted on 12 November 2021. A total of 271 and 2657 unique users tweeted on 11 and 12 November, respectively. The number of tweets on 12 November was 9.38 times the number on 11 November.The total potential reach of these tweets (the sum of followers) was the highest at 36 169 806 users for neutral sentiment, followed by 9 978 113 users for positive sentiment and 1 478 480 users for negative sentiment.The total potential reach among users (the sum of followers) was the highest for reputable news sources at 25 481 438, followed by 14 644 538 for general public and 3 996 638 for health professionals.N = 3 430 984) of all health professionals’ followers, with 49.1% tweeting positively, 45.3% tweeting neutrally and 5.7% tweeting negatively regarding HPV vaccines. The top 12 researcher influencers had 87.6% (N = 715 001) of all researchers’ followers, with 50.0% tweeting positively, 33.3% tweeting neutrally and 16.7% tweeting negatively regarding HPV vaccines. The top 13 politician influencers had 94.8% (N = 1 035 150) of all politicians’ followers, with 38.5% tweeting positively, 61.5% tweeting neutrally and 0% tweeting negatively regarding HPV vaccines.This content analysis presented an overview of the user characteristics, sentiments and themes of public discussions about HPV vaccines, and their potential reach on Twitter before and after the news reports of the resumption of proactive recommendations for HPV vaccines. The tweet volume of the HPV vaccine increased 9.4-fold after the news reports, indicating the magnitude of public interest.Regarding user characteristics, general public who could not be classified into any specific category accounted for approximately 80% of users. Health professionals accounted for approximately 5% on both days which was lower than expected. Previous studies found that approximately 10% of English tweets related to HPV vaccines . This alThe sentiment analysis showed that negative tweets were 27.8% before the news of the resumption of recommendation, which was similar to previous studies (e.g. 25–43%)  among health professionals, researchers and politicians accounted for most of the followers. The median number of retweets and likes increased as the number of followers increased, suggesting that interaction and reach of Twitter discussions are strongly dependent on those influencers. Only half of the self-defined health professionals, researchers and politicians were positive towards HPV vaccines, whereas more than 40% (including influencers) were neutral. These results show a striking contrast with the results of a study reporting that 95.3% of advocacy users, including health professionals and research institutions, tweeted positively about HPV vaccines in English . Negative sentiment dominated 42.6% of the tweets with the theme of ‘safety and side effects’. Side effects were the most common theme in Japanese online information on HPV vaccines in 2016  tweeted neutrally about HPV vaccines. The most frequently discussed theme was concerned with the safety and side effects of HPV vaccines; it was mostly mentioned from a negative perspective. Influential professionals, including health professionals and researchers, are expected to provide peer-to-peer discussions and disseminate accurate information through tweets to correct misinformation and recommend HPV vaccination on Twitter and to contribute to overcome HPV vaccination crisis.daad153_suppl_Supplementary_Appendixs_1-2Click here for additional data file.


Considering the crucial role of soil diazotrophs and root arbuscular mycorrhizal fungi (AMF) in soil nutrient cycling during ecosystem restoration, diazotroph and AMF communities may be determined by slope position. However, the effect of slope position on diazotroph and AMF abundance, diversity, and community composition of karst ecosystems remains unknown. In this study, soil diazotrophs and root AMF characteristics on varying slope positions were assessed in a karst shrub ecosystem. The results displayed that the abundance of soil diazotrophs and root AMF diversity were significantly affected by slope position. Diazotroph abundance accompanied by soil nutrient and plant richness was higher on the lower slopes than the upper slopes, whereas root AMF diversity displayed the opposite trend. The soil diazotroph and root AMF community composition differed among the upper, middle, and lower slopes. The dominant taxa of soil diazotrophs and root AMF at the order level were Rhizobiales and Glomerales, respectively. Moreover, the diazotroph order of Nostocales and the AMF order of Paraglomerales were richer on the upper slopes than on the lower slopes. The slope position directly affected the plant diversity and soil nutrient distribution, indirectly affecting the diazotroph and AMF communities. Increased available nitrogen on the lower slope caused great diazotroph abundance by stimulating plant growth with sufficient carbohydrates. However, low soil nutrients and plant diversity but high plant root biomass induced more root AMF diversity on the upper slope than on the lower slope. Therefore, this study expands the knowledge of soil diazotroph and root AMF ecological functions along different slope positions during vegetation recovery for the successive stages of grass and shrub in the karst region. Diazotrophs and arbuscular mycorrhizal fungi (AMF) are important nutrient cycling regulators, particularly during nitrogen (N) progression ,2,3. FreSlope position governs the plant community, nutrient content, soil moisture, and light, which may be closely linked with the richness and composition of diazotrophs and AMF ,11,12. FBradyrhizobium and Glomus, respectively [Bradyrhizobium and Glomus) was stronger in karst forests than in non-karst forests [Karst ecosystems are widely distributed in southwest China . The soiectively ,24,25. T forests . HoweverThe aim of the study was to explore the distribution characteristics of soil diazotrophs and root AMF within the upper, middle, and lower slopes to assist in selecting the optimal methods for vegetation recovery. The soil diazotroph abundance, diversity, and community compositions were investigated. In addition, root AMF diversity and community composition were investigated under different slope positions.Pyracantha fortuneana, Vitex negundo, and Alchornea trewioides. Additionally, other shrubs, such as Tirpitzia ovoidea, Celtis biondii, and Mallotus barbatus were distributed in the study area. Grass, such as Pteridium aquilinum, Euonymus alatus, and Pogonatherum crinitum were also distributed along slope positions.Soil and root samples were collected from nine sites of shrub ecosystems from a typical karst catchment at the Chinese Academy of Sciences: Huanjiang Observation and Research Station for Karst Ecosystems  in the Guangxi Zhuang Autonomous Region, China. The study area for the karst peak-cluster depression is surrounded by mountains with a steep slope of approximately 30°. The calcareous soil (according to the Food and Agriculture Organization (FAO) soil classification system) in the study region was developed from a dolomite base. The site is characterized by a typical subtropical monsoon climate with an average annual temperature and precipitation of 13 °C and 800–1500 mm, respectively. The rainy season is mainly from May to August, while the dry season is mainly from November to April. This region was naturally restored from cultivated land to a shrub ecosystem over 25 years. The dominant shrubs were Three transects were selected at the experiment site, and each transect included three slope position with lower, middle, and upper slopes. Therefore, nine plots (10 m × 10 m) were included in this study (three slope positions × three transects). Plants grow well in the growing season; plant investigation and root samples were determined in July. To ensure comparability, soil samples were also collected in July. Each plot was divided into 25 grids of 2 m × 2 m, and a shrub diversity assessment was carried out in each plot. We also investigated grass community characteristics. Soil samples were collected using a soil drill at the vertex of each grid, and a total of 36 points at a depth of 0–20 cm were thoroughly mixed. Visible stones and residue were removed using a 2.0 mm mesh, and plant roots were collected. The fine roots (<2 mm) were kept at 4 °C within 24 h until rinsed two to three times with distilled water to remove the soil from the surface. Approximately 10 g of rinsed fine roots were placed in a 15 mL centrifuge tube and stored at −80 °C for the microbial index measurement. A portion of the soil sample was stored at 4 °C to analyze soil physicochemical properties, and the remaining soil was stored at −80 °C for DNA extraction. The samples were kept at 4 °C within 5 h prior to the −80 °C storage. AMF is crucial for the acquisition of nutrients, especially in the plant root due to the symbiotic relationship between the plant and AMF. The genetic diversity of coexisting rhizobia was low based on the plate culture and monoclones ; thus, wSoil pH was measured using a glass electrode with a soil-to-water ratio of 1:2.5. The soil organic matter (SOM) content was determined using the potassium dichromate oxidation method . Total n® SPIN Kit  and Plant DNA Extraction Mini Kit B , respectively. The nifH amplicon for diazotrophs was amplified using the specific primer pair nifH-F (AAAGGYGGWATCGGYAARTCCACCAC) and nifH-R (TTGTTSGCSGCRTACATSGCCATCAT). The reaction volume for 20 μL included 0.8 μL of each 10 μM forward and reverse primer, 10 ng of template DNA, 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.4 μL of FastPfu Polymerase, 0.2 μL of BSA, and added up to sterile water. The PCR condition was conducted at 95 °C for 3 min, then 40 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and final extension at 72 °C for 10 min.Root samples were ground in liquid nitrogen. The DNA was extracted from the soil and roots using a Fast DNAThe nested PCR was used to amplify the 18S rRNA gene fragments for root AMF community analysis. The primer set AML1 (ATCAACTTTCGATGGTAGGATAGA) and AML2 (GAACCCAAACACTTTGGTTTCC) were determined at the first PCR reaction and AMV4.5NF (AAGCTCGTAGTTGAATTTCG) and AMDGR (CCCAACTATCCCTATTAATCAT) was used to amplify at the second round PCR. The PCR reaction conditions and systems were described in our previous studies ,26.nifH and 18S rRNA genes were pooled and purified. After that, sequencing was performed using the Illumina MiSeq PE300 platform  at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. .PCR products for nifH gene sequences were processed in QIIME v2-2020.2, and the NifH Miseq Illumina Amplicon Analysis Pipeline (NifMAP) was used to remove sequences amplified from pseudogenes and homologs. The raw reads of both genes were imported into the QIIME environment. Amplicon sequence variants (ASVs) were denoised for raw sequences with the “q2-DADA2” plugin. The sequences of pseudogenes and homologs in nifH ASVs were then filtered against nucleotide-based HMM using the hmmsearch command in HMMER according to NifMAP. A naïve bayes classifier trained, with the PR2 and nifH reference database, was used to classify the ASVs of corresponding gene with the “q2-feature-classifier” plugin of QIIME2, respectively. All taxa were verified using the NCBI taxonomy database. Alpha diversity indices were calculated using normalized OTU tables in the R package “RAM” and further illustrated in the R package “ggplot2”. The ASV tables were further rarefied with the function “rarefy“ of the R package “vegan” and used to calculate alpha diversity indices with functions from the R package “RAM”.The 18S rRNA of AMF and The plant richness (R) and Shannon index (H′) were calculated as follows:S is the total number of species in each plot, and Pi is the proportional density of individuals of the ith species.R = the total number of plant species in each 10 m × 10 m plotsp < 0.05. Ternary plots were performed to identify the distribution of soil diazotroph and root AMF community composition across the upper, middle, and lower slopes. Random forest analysis was used to identify the main predictors of diazotroph abundance and root AMF diversity. The increase in mean square error (MSE) resulted in the need to quantify the relative importance of soil physiochemical property parameters and plant diversity using the ‘randomForest’ package [Significant differences in soil physiochemical properties, soil diazotrophs, root AMF, and plant diversity among slope positions were examined using one-way ANOVA and Duncan’s test at  package . StatistThe one-way ANOVAs of slope position showed that soil nutrients, soil diazotroph abundance, plant diversity, and root AMF diversity significantly differed among the lower, middle, and upper slopes. Specifically, TK, AN, AP, and AK were lower on the upper slope than on the lower slope . Soil pHAdditionally, soil diazotroph abundance was higher on the lower slope compared to the upper and middle slopes, while no difference in diazotroph richness and the Shannon index was observed between the lower, middle, and upper slopes. Moreover, both root AMF richness and the Shannon index were higher on the upper slope than on the middle and lowerslopes .Bradyrhizobium (74.4%) and Frankia (12.7%) were the most abundant taxa for soil diazotrophs, while Glomus (72.5%) and Paraglomus (10.7%) were the dominant genera for root AMF , Frankiales (12.7%), and Rhodospirillales (2.8%) were the most abundant orders. Three AMF orders were identified from plant roots. Root AMF taxa were dominated by Glomerales (73.7%) and Diversisporales (15%) a,b. At troot AMF c,d.Nostoc were higher on the upper slope compared to the lower slope , leading to variations in soil diazotrophs and root AMF communities ,32. ManyIn contrast, considering the close relationship between plant communities and AMF diversity in plant roots, the opposite results were obtained here. Although plant richness was enriched on the lower slope, lower root AMF richness and the Shannon index were observed on the middle and lower slopes compared with the upper slope . The sloUnderstanding the microbial compositions at different slope positions is essential to predict the adaptive strategies of soil diazotrophs and root AMF groups on the upper, middle, and lower slopes. Taxonomic profiling revealed that Rhizobiales and Glomerales were the most abundant orders, accounting for 77% and 74% of the total sequences for soil diazotroph and root AMF, respectively . This waNostoc were higher in the upper slopes than the lower slopes. This could be explained by the fact that Nostoc that belongs to Nostocales performs well in extreme situations [The slope position showed potential impacts on soil diazotroph and root AMF community compositions. Specifically, the observed species distribution was different within the upper, middle, and lower slopes. The relative abundances of the order Nostocales and genus tuations ,46, suchtuations . Generaltuations ,49,50. TA random forest model was constructed to better understand the main contribution of soil properties and nutrients. Many previous studies reported that soil environment conditions  and nutrients  were strongly related to diazotroph abundance and diversity ,25,46. IPlant richness, AP, and AN were the main contributors to root AMF richness and the Shannon index variations . The uppIn addition, as mentioned above, richer plant root biomass in the surface soil due to the shallow soil layer leads to poorer water-holding capacity on the upper slope than on the lower slope. Additional AMF species in the plant roots were selected by enhancing cooperative relationships among root AMF taxa when the plant root biomass was increased in the surface soil on the upper slopes; this emphasized the important effect of plant root biomass on root AMF diversity. Consequently, the deterministic assembly factors for the variations in soil diazotroph abundance and root AMF diversity can be partially explained by environmental heterogeneity along the slope gradients .This study expands our understanding of soil diazotrophs and root AMF communities in response to slope position during vegetation recovery. Soil nutrients and plant properties were the main factors driving the variation in community composition, and the diversity of diazotrophs and AMF. High N availability stimulates plant growth, thereby increasing diazotroph abundance, owing to sufficient litter and root exudate inputs. High light and low nutrient levels could promote AMF diversity in plant roots on the upper slope. Moreover, the slope position indirectly affected the diazotroph and AMF community composition. For example, the relative abundances of the order level for the diazotroph Nostocales and AMF Paraglomerales on the upper slope were richer than the lower slope. Overall, our results emphasize that slope position indirectly influences soil diazotroph abundance and root AMF diversity by regulating soil nutrients, microclimate conditions, and plant roots. However, future studies should evaluate their important roles in organic matter decomposition and N fixation along the slope gradients.


We sought to quantify the relationship between morning, afternoon or evening physical activity and consistency (e.g. routine) and risk of type 2 diabetes.A cohort of 93,095 UK Biobank participants (mean age 62 years) without a history of type 2 diabetes wore a wrist-worn accelerometer for 1 week. We converted accelerometer information to estimate metabolic equivalent of task (MET), summing MET h of total physical activity completed within three intra-day time segments . We quantified physical activity consistency as the SD of participants’ daily total physical activity. We ultimately associated each of the following with incident type 2 diabetes: (1) morning, afternoon or evening ‘time-segmented’ MET h per week; and (2) consistency. We also considered moderate-to-vigorous physical activity (MVPA) and vigorous physical activity (VPA) in association with type 2 diabetes incidence. p=7×10−8) and afternoon  but did not have evidence for evening physical activity  with type 2 diabetes. There was no difference between MET-measured morning and afternoon physical activity. Our substitution model highlighted the importance of adjusting for lifestyle factors (e.g. sleep time and diet); the effect of a substitution between afternoon and evening physical activity was attenuated after adjustment for lifestyle variables. Consistency of MET-measured physical activity was not associated with type 2 diabetes (p=0.07). MVPA and VPA were associated with decreased risk for type 2 diabetes at all times of the day.When considering MET as the physical activity measure, we observed protective associations of morning (HR 0.90 ,Total metabolic equivalents of physical activity in the morning and afternoon had a protective effect on diabetes risk and evening activity was not associated with diabetes. Consistency of physical activity did not play a role in decreasing risk for diabetes. Vigorous activity is associated with lower risk no matter the time of day of activity.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-023-06001-7. Physical activity is a preventive factor for type 2 diabetes but its timing and consistency  has been relatively unexplored. It has been shown that midday–afternoon but not evening physical activity is associated with lower risk of mortality compared with morning physical activity but the relationship with type 2 diabetes remains understudied . We soughttps://axivity.com/product/ax3]) were given randomly to 103,686 UK Biobank participants, who were asked to wear the device on their dominant wrist for a continuous period of 7 days. The raw accelerometer data were pre-processed by the Biobank Accelerometer Analysis software  followed by metabolic equivalent of task (MET) calculation as described by Doherty et al and Le Goallec et al ). To test for the associations of time-specific activity, we fitted a Cox model with separate terms for morning, afternoon and evening MET h, adjusting for age, sex, BMI, ethnicity, socioeconomic factors including education, household income before tax, and lifestyle factors, such as smoking status, alcohol intake, sleep duration, wake time and diet. In particular, the dietary information encompassed vegetable and fruit intake, meat and fish consumption, and bread type (grain or not) eaten by the study participants. Time and age were measured in years in the Cox model.We estimated the effect of physical activity timing substitution  using the effect estimates and covariances from the timing Cox model. We fitted three sets of models in the substitution analysis: (1) only adjusting for basic information ; (2) adding adjustment for socioeconomic factors (education and household income); and (3) adding adjustment for lifestyle factors . We also examined the associations stratified by sex and age .We also examined the association between MVPA and VPA and type 2 diabetes. We tested for improved model fit using time-specific rather than overall summed activity using an LRT. Finally, to test the association between total physical activity routine and type 2 diabetes, we fitted a separate model testing an individual-level SD of daily MET h over a week, adjusting for total weekly MET h and sex, age, ethnicity, BMI, education, household income, smoking status, alcohol intake, sleep duration and dietary information.1c compared with the entire UK Biobank cohort  Fig. . PopulatBoth morning and afternoon physical activity were associated with reduced risk of type 2 diabetes. Each additional MET h of physical activity in the morning was associated with 10% risk reduction of type 2 diabetes  had 24% and 34% reduction in risk of type 2 diabetes compared with participants who performed little to no morning physical activity (Q1) , but morning, afternoon and evening risk was dependent on sociodemographic and lifestyle adjustment factors (ESM Table p=0.0041) and 10%  reduction in type 2 diabetes risk, respectively, for model set 1 . For model set 2 (adjusting for variables in model set 1 plus education and household income), substituting one MET h of evening physical activity with an equivalent amount in the morning was associated with 9% reduction in risk ; an equivalent amount in the afternoon was associated with 13% reduction in type 2 diabetes risk . The morning–evening substitution for model set 3  was associated with 6% risk reduction  but afternoon–evening substitution was not associated with risk reduction (p=0.23) for model set 3.Furthermore, in substitution analyses, the substitution between morning and afternoon was not statistically significant for any of the models  and age . Women had a small relative benefit from afternoon physical activity when compared with men. For participants in all subgroups, morning and afternoon physical activity were associated with reduced type 2 diabetes risk. Evening physical activity, however, was not associated with risk reduction for female participants or participants aged over 65 years , demonstrating no association between the consistency of total physical activity and type 2 diabetes.Compared with participants who developed type 2 diabetes, participants who did not develop type 2 diabetes had higher total weekly MET h and similar daily MET h SD , associations for morning MET h became larger and more precise , and afternoon MET h become more precise. Second, we observed that the type of objective physical activity also matters in testing associations . Lifestyle has less of an impact on the association of VPA with diabetes. Adding lifestyle factors to the VPA models did not make a sizable difference in the substitution models for morning, afternoon and evening associations Below is the link to the electronic supplementary material.


After adjusting for the main risk factors , the risk of RD was found to be 3% lower than their counterparts . In conclusion, this study provides evidence for the association between maternal gestational Vitamin D3 supplementation and improved early respiratory outcomes in young children.This study aimed to evaluate the association between maternal gestational Vitamin D3 supplementation and early respiratory health in offspring. This was a population-based record-linkage study which used data from the French National Health Database System. Maternal Vitamin D3 supplementation consisted of a single high oral dose of cholecalciferol,  from the seventh month of pregnancy, according to national guidelines. In total, 125,756 term-born singleton children were included, of which 37% had respiratory illness defined as hospital admission due to respiratory causes or inhalation treatment up to 24 months of age. Infants prenatally exposed to maternal Vitamin D3 supplementation  were more likely to have a longer gestational age (GA) at birth (GA 36–38 weeks, 22% vs. 20%,  Pregnant women are a vulnerable population at high risk of vitamin D insufficiency or deficiency. Vitamin D deficiency (blood 25-hydroxyvitamin D [25(OH)D] levels below 50 nmol/L) is common during pregnancy worldwide; in France, the rate was observed to reach 68% ,2,3. ThiIn addition to its role in bone homeostasis and growth, there is evidence that vitamin D plays a biological role in immune function and fetal lung development ,9. AnimaAlthough their frequency has decreased worldwide, respiratory diseases remain a frequent cause of hospital admission and mortality in young children, particularly in those younger than 2 . MoreoveUsing the French National Health Database System, we aimed to evaluate the association between maternal Vitamin D3 supplementation during pregnancy and early respiratory outcome in a population of term-born infants living in the Provence–Alpes–Côte d’Azur (PACA) region of France.Programme de Médicalisation du Système d’Information, (PMSI)) and the National Health Care Insurance Database   of 0.64 [0.47–0.87]) ) ,34. Thesers (aOR 5% CI of 7–0.87]) . VDR ele7–0.87]) . Vitamin7–0.87]) .2D administration prevents lung injury associated with chorioamnionitis and restores lung structure in pups prenatally exposed to maternal gestational vitamin D depletion [Although there is a relationship between maternal or fetal vitamin D status and children’s respiratory outcomes, the preventive effects of maternal gestational Vitamin D3 supplementation is controversial. In rodents, perinatal 1,25(OH)epletion ,39. A meepletion . This fiOur findings provide additional evidence, although the dose of maternal Vitamin D3 supplementation was lower and the administration was later during pregnancy. A single maternal high dose of Vitamin D3  at the beginning of the third trimester (between GA 28 weeks and 32 weeks) is equivalent to approximately 1100 IU daily for 3 months. The pulmonary effects observed in our study may result from the pharmacokinetics profile and the timing of the supplementation protocol. After a single high dose of Vitamin D3, blood 25(OH)D levels significantly increased from day 2 and reached a steady state from days 15 to 21 during one month before declining . This raThe National Health Database System does not include data on relevant individual factors, including ethnicity, breastfeeding, home environment , and allergy antecedents, which could influence infant respiratory outcomes. However, some of these factors are linked to socioeconomic status, which was included in our analysis . MaternaThe main strength of this study is that it was based on the National Health Database System, which included data from all public and private hospitals, as well as on the consumption of medical drugs that are exclusively delivered by pharmacies in France. This database system provides robust and high-quality data, making it possible to link prenatal and postnatal pathologies with healthcare utilization in a large population. We used robust criteria to define respiratory disease and assess the use of an inhaled treatment that is considered to be a reliable marker of childhood wheezing pathology. Finally, the use of this system can be an alternative to observational clinical studies that are often difficult to conduct over a long term and to highlight associations in large populations.. Moreover, they should better define the optimal protocol, including the doses and the timing of supplementation.In this large French regional population-based record-linkage study, using the French National Health Database System, we found an association between high single-dose maternal vitamin D supplementation during the seventh month of pregnancy and lower odds of respiratory illness in children, as defined by hospital admission for respiratory reasons or the use of inhaled treatment in a population of term-born infants up to 2 years of age. Our study provides further insight into the biological role of vitamin D in lung and immune system development. Maternal vitamin D supplementation during pregnancy could be a strategy used to prevent early respiratory diseases in young children and reduce adverse pulmonary consequences in the long term. Given the study design, the study population, and limited data, particularly regarding maternal vitamin D status and dietary intake, the generalizability of our results may be questioned. Further studies should include maternal gestational vitamin D status, diet, and lifestyle investigations


The gut microbiota is closely related to parenteral noncommunicable diseases through intestinal immunity and plays an important role in the occurrence of diabetes and diabetic nephropathy. The aim of the study was to understand the gut–kidney axis by an analysis of gut microbiota composition among patients with biopsy-proven diabetic nephropathy (DN), patients with type 2 diabetes for more than 10 years without kidney damage (DM), and healthy controls (NC). Thirty-five DN patients, 40 DM patients and 40 healthy subjects matched by age and sex were enrolled between January 2022 and December 2022. Baseline information and clinical parameters were collected. 16S rDNA sequencing was performed to characterize the gut microbiome and identify gut microbes that were differentially abundant between patients and healthy controls. The relationship between the relative abundance of specific bacterial taxa in the gut and clinical phenotype and pathological indicators was evaluated. Substantial differences were found in the richness of the gut microbiota and the variation in the bacterial population among DN patients, DM patients and healthy controls. DM patients could be accurately distinguished from age- and sex-matched healthy controls by variations in g_Clostridium-XVIII (AUC = 0.929), and DN patients could be accurately distinguished from age- and sex-matched healthy controls by variations in g_Gemmiger (AUC = 0.842). DN patients could be accurately distinguished from age- and sex-matched DM patients by variations in g_Flavonifractor or g_Eisenbergiella . The gut microbiota was also closely related to clinical phenotypes and pathological indicators. The study of gut microbiota composition was explored to determine its relationship to the occurrence of DN and a long history of diabetes without kidney damage. The renal pathological progression of DN may be delayed by regulating changes in the gut microbiota. As a serious microvascular complication of diabetes, DN has become the main cause of end-stage renal disease (ESRD) worldwide4. There are about 500–1000 species of bacteria in the human gastrointestinal tract, with a number of 1014 CFU, which are vividly referred to as acquired "organs"5. They assist the host in maintaining normal physiological functions, including absorbing energy, producing vitamins and basic active molecules, biological antagonism, immune regulation. Its composition and number have maintained a dynamic balance with the host in evolution6. A large number of studies have shown that gut microbiota can participate in the occurrence and development of diseases by regulating host energy metabolism, systemic inflammatory response, and the secretion of intestinal hormones. Gut microbiota also play an important role in a variety of kidney diseases, and the endotoxins, proteins and some metabolites produced by them have certain effects on the kidney through the gut-kidney axis7. By observing the gut microbiota of patients with diabetic nephropathy confirmed by renal biopsy and patients with long-term diabetes without kidney damage, this study aimed to explore the imbalance pattern and functional changes in the two disease states and establish a classification algorithm model to distinguish the two by gut microbiota. We also explore the correlation between gut microbiota and clinical phenotypes and pathological indexes.With the increasing prevalence of diabetes, the number of patients with diabetic nephropathy (DN) has also increased significantly. The results of an epidemiological survey in China from 2015 to 2017 showed that the prevalence of diabetes in adults was 11.2%18–65 y of age;Diagnosed with type 2 diabetes;Diabetic nephropathy diagnosed based on renal puncture pathological examination;No evidence of primary renal disease;2;Estimated glomerular filtration rate(eGFR) ≥ 60 ml/min/1.73 mInformed consent can be signed.The retrospective study included 35 patients with diabetic nephropathy confirmed by renal biopsy, 40 patients with type 2 diabetes for more than 10 years without kidney damage, and 40 healthy subjects matched by age and sex. We enrolled patients and healthy controls in Shanxi Provincial People’s Hospital between January 2022 and December 2022. In the DN group, inclusion criteria were as follows:18–65 y of age;The duration of type 2 diabetes was more than 10 years;2 and urine albumin creatinine ratio (UACR) < 30 mg/g);No diabetic microvascular complications, including diabetic retinopathy and renal damage . The patients/participants provided their written informed consent to participate in this study.Fresh fecal samples were collected from the selected individuals. The fecal samples were discharged into a sterile stool box and transferred to a − 80 °C refrigerator for storage within 2 h after sampling. DNA was extracted from the samples using the QIAamp PowerFecal DNA Kit. Agarose gel electrophoresis was used to analyze DNA integrity, NanoDrop was used to measure the purity, and DNA concentration was accurately quantified with Qubit.High-fidelity DNA polymerase was used to amplify the V3-V4 variable region of DNA by two-step PCR amplification-specific amplification and the addition of tag and joint sequences. An FC Magnetic Beads Kit (Enlighten) was used to purify and recover the product. Qubit4.0 was used to quantify the purified library, Qsep100 was used to check whether the length of the library was as expected, and each sample was diluted to 4 nM. The hybrid library was prepared and denatured by DNA, and at least a 5% Phix library was added to balance the library polymorphism. Sequencing was carried out on an Illumina MiSeq sequencer using the PE300 strategy.The quality of the original data was controlled by QIIME2, low-quality sequences and joint sequences were filtered by Trimmomatic software, and chimeric sequences were removed by USEARCH software. OTU clustering was carried out by UPARSE software, and the bacterial 16S region was compared to the RDP database.Statistical analysis was performed using R software (version 4.0.2). Amplicon analysis included only microorganisms that existed in ≥ 12% of samples. Alpha diversity was used to measure the richness of microbial species in a single sample. Beta diversity was used to measure the similarity of microbiota composition among different samples. In this study, principal coordinate analysis (PCoA) was used to analyze the β diversity of the microbiota. Through PCoA, we observed whether there was an overall deviation of microbiota between the disease group and the healthy population. LEfSe, or linear discriminant analysis effect size analysis, can find biomarkers with statistically significant differences between groups. This method was used to compare the gut bacteria with statistically significant differences between each disease group and the normal control group. Random forest is an integrated machine learning method that can obtain the final result by establishing multiple decision trees and voting (average) on the prediction results of each decision tree, which has the same function as the decision tree to complete the classification and regression tasks. In this study, the random forest prediction model was established by omicStudio software. The classification efficiency of the model was evaluated by the area under the curve (AUC) of the working characteristic curve (ROC). The prediction performance of the model was analyzed. The discriminant value was low when AUC < 0.70, good when 0.7 ≤ AUC ≤ 0.9, and high when AUC > 0.9. All heatmaps were visualized by the package pheatmap (version 1.0.12). The clinical data of patients with DN and DM and healthy controls were expressed as the mean ± SD or median, and the numerical data were expressed as percentages. A t test or nonparametric test was used to compare continuous variables. Chi-square tests were used to compare categorical variables. The statistical analysis was performed using SPSS version 26.0.We included 35 patients with DN confirmed by renal biopsy with a median age of 54.09 years and 40 DM patients with a median age of 58.83 years. The baseline characteristics of the DN, DM and healthy control (NC) groups are summarized in Table According to the pathological grade of diabetic nephropathy, the severity of pathological changes was evaluated by glomerular volume (G), glomerular basement membrane (GBM), mesangial matrix (M), glomerulosclerosis (S) and interstitial lesions (I). G0 represents no significant increase in glomerular volume, and G1 represents an increase in glomerular volume. GBM1 represents mild thickening of the glomerular basement membrane, and GBM2 represents significant thickening of the glomerular basement membrane. M1 represents mild proliferation of the mesangial matrix, M2 represents significant widening of the mesangial matrix, and M3 represents the formation of one or more tuberous sclerosis (Kimmelstiel-Wilson nodules). S1 represents less than 50% glomerulosclerosis and S2 represents more than 50% glomerulosclerosis. I1 represents mild renal interstitial lesions, I2 represents moderate renal interstitial lesions, and I3 represents severe renal interstitial lesions. Among the 35 diabetic nephropathy patients, there were 5 cases of G0 and 30 cases of G1, 23 cases of GBM1 and 12 cases of GBM2, 4 cases of M1, 4 cases of M2 and 27 cases of M3, 30 cases of S1 and 5 cases of S2, 17 cases of I1, 16 cases of I2 and 2 cases of I3, as shown in Table A total of 844 OTUs were obtained at a 97% homology cut-off. The NC group showed the largest number of OTUs. As shown in the Venn diagram Fig. a, the nuP = 0.005, DM vs. NC Chao P < 0.001, DN vs. NC Chao P < 0.001), but no different diversity of OTUs was found in the three groups (Shannon P > 0.05) . The fecal microbiota of patients with DM and DN and the NC subjects was clearly separated along PCoA2 and PCoA1, which explained 11.58% and 12.93% of the total variation, respectively. Together, these data suggest that the fecal microbial structure was significantly altered in patients with DM and DN compared to that in NC subjects in the presence of OTUs.The Chao index of the OTU profile was used to evaluate gut microbiota richness, and the Shannon index was used to estimate community diversity. The differences in community richness were significant in the three groups (DM vs. DN Chao P < 0.05). The numbers of Christensenella, Clostridium-XIVa, Eisenbergiella, Flavonifractor and Clostridium-XVIII in the DN group were significantly increased , while the numbers of butyric-producing bacteria, Bacillus, Enterobacter, Trichospira and Rosacella were significantly decreased in the DM group and DN group (P < 0.05). At the genus level, Clostridium XVIII and Gemmiger were significantly decreased in the DM and DN groups  effect size (LEfSe) algorithm was used Fig. a. LEfSe ups Fig. b.Figure To illustrate the microbial signature of DM and DN and further explore the potential of the gut microbiome in DN identification, receiver operating characteristic (ROC) curves for classifying DM from NC, DN from NC and DN from DM were developed. We could detect DM individuals accurately based on only one genus (g_Clostridium-XVIII), as indicated by an area under the receiver operating curve (AUC) of up to 0.929 Fig. a. SimilaSpearman correlation analysis was used to further evaluate the correlation between gut microbiota differences and clinical characteristics. At the genus level, Gemmiger, Clostridium XlVb, Lachnospiracea incertae sedis and Vallitalea showed strongly negative correlations with glycosylated haemoglobin (HbA1c) and fasting blood glucose (FBG) levels, while Megasphaera had a positive correlation with HbA1c levels. Pseudomonas, Eisenbergiella and Flavonifractor showed markedly positive correlations with the urine albumin/creatinine ratio (UACR). Enterococcus, Clostridium XlVa and Eisenbergiella exhibited a negative correlation with the estimated glomerular filtration rate (eGFR) Fig. .Figure 4Correlations between differences in gut microbiota and pathological types were analyzed using spearman correlation analysis. At the genus level, Ruminococcus callidus and Clostridium viride showed strongly negative correlations with glomerular volume and positive correlations with glomerulosclerosis. Thickening of the glomerular basement membrane was positively correlated with Citrobacter werkmanii, Eisenbergiella tay, Klebsiella pneumoniae subsp. ozaenae, Ruminococcus callidus, Megamonas funiformis and Barnesiella intestinihominis. Flavonifractor plautii and Hungatella hatheway were positively correlated with mesangial matrix hyperplasia. Megasphaera micronuciformis showed a positive correlation with renal interstitial lesions Fig. . It is s8, diabetic nephropathy has become the main cause of chronic kidney disease9, which constitutes a major public health problem worldwide10. A large number of studies have shown that the gut microbiota is closely related to parenteral noncommunicable diseases through gut immunity13. Qin et al.14 and other studies15 found a significant decrease in butyrate production in diabetic patients. Wu et al.16 found that compared with healthy controls, the relative abundance of Streptococcus and Enterococcus in patients with IgAN was higher, while that of Bacteroides was lower. The changes in the gut microbiota of IgAN affected the metabolism and absorption of polyunsaturated fatty acids and activated the metabolic pathway of arachidonic acid, thus constructing the metabolic system of IgAN. Li et al.17 found that the genera Lactobacillus, Clostridium IV, Paraprevotella, Clostridium sensu stricto, Desulfovibrio, and Alloprevotella were enriched in the fecal samples of patients with CKD, while Akkermansia and Parasutterella were enriched in those of NC subjects.Diabetic nephropathy is a common microvascular complication in patients with diabetes. With the sharp increase in the number of diabetic patients18. As the imbalance pattern of gut microbiota in patients with metabolic diseases is greatly affected by regional factors, there are differences in the imbalance pattern in different regions19. Some studies have also found that the α diversity of the gut microbiota in patients with DM decreased20. The β diversity analysis showed that there were significant differences among the DM, DN and healthy control groups. Importantly, DM could be accurately distinguished from age- and sex-matched healthy controls by variations in g_Clostridium-XVIII (AUC = 0.929), and DN could be accurately distinguished from age- and sex-matched healthy controls by variations in g_Gemmiger (AUC = 84.2%). DN could be accurately distinguished from age- and sex-matched DM patients by variations in g_Flavonifractor or g_Eisenbergiella . These results show that the powerful function and practicability of data mining technology are reflected in the diagnosis and prediction of disease markers through a large number of multidimensional genome-related databases. Therefore, future research should continue to look for specific microbial markers for the diagnosis of DN.This study found that the α diversity index of patients in the DM and DN groups was higher than that in healthy people, which was consistent with the report of Larsen et al.21. Our findings demonstrated that healthy controls had more Gemmiger in the gut microbiota than DN patients. The correlation analysis between Gemmiger and biochemical indices also found that there was a negative correlation between Gemmiger and blood glucose level. Gemmiger are typically producing butyrate bacteria22, which are involved in the adjustment of the body’s reaction to inflammation. This study also found that Eisenbergiella was enriched in gut microorganisms of DN patients, and Eisenberg was positively correlated with urinary microalbumin. The correlation analysis between Eisenbergiella and renal pathological classification of DN showed that Eisenberg was positively correlated with glomerulosclerosis and glomerular basement membrane thickening. This bacterium was highly abundant in the intestinal tract of mice with colitis and might participate in intestinal inflammation23. Flavonifractor is a conditional pathogen that was more enriched in DN patients than in the NC and DM groups and synthesizes lipopolysaccharide (LPS). When LPS binds to the receptor complex in macrophages, it activates the signal cascade, resulting in the production of a large number of inflammatory cytokines, such as IL-1β, IL-6, TNF α, and IL-1224.Our study found that in healthy people, the content of Clostridium-XVIII was significantly higher than that in the diabetes group. Clostridium-XVIII has the ability to ferment and produce short-chain fatty acids (SCFAs) in the human intestinal tract, and SCFAs are generally considered to play a variety of important roles in maintaining human health. For example, they act as a special nutrition and energy component of the intestinal epithelium, protect the intestinal mucosal barrier, reduce the level of inflammation and enhance gastrointestinal motility25.Studies have shown that there is an interaction between intestinal microorganisms and the kidney, called the gut‐kidney axis, and the balance between intestinal symbiotic bacteria and pathogenic bacteria ensures the integrity of the intestinal barrier and normal renal function. Healthy kidneys communicate with the intestinal microbiota through cellular and molecular signals to ensure normal intestinal microbiota homeostasis. The imbalance of DN or gut microbiota will lead to the destruction of this balance, loss of the integrity of the intestinal barrier, activation of immune cells and secretion of cytokines, thus further worsening renal function and symbiotic relationships2 to rule out the effect of end-stage renal disease on gut microbiota26. In the DM group, we selected patients with diabetes course of more than 10 years without renal damage, and the difference between the two groups can better reflect the difference of gut microbiota in diabetes and diabetic nephropathy. A number of studies have found that pathological classification of diabetic nephropathy has an important guiding value for judging renal prognosis28. Therefore, we analyzed the correlation between gut microbiota and pathological indicators of diabetic nephropathy, and found that various gut microbiota play an important role in the pathological progression of diabetic nephropathy. It is suggested that regulating the changes of gut microbiota may delay the progression of renal pathology in diabetic nephropathy.Because diabetic nephropathy is mostly clinically diagnosed, many patients with diabetes complicated with chronic kidney disease are easily misdiagnosed as diabetic nephropathy, resulting in phenotypic heterogeneity. For the accuracy of phenotype, diabetic nephropathy patients confirmed by renal biopsy were selected for the DN group, and eGFR ≥ 60 ml/min/1.73 mHowever, this study is a cross-sectional study of a single-centre and small-scale population, and its results and conclusions have some limitations, so it is necessary to verify them in a follow-up multicentre population cohort. In addition, most of the patients in this study took different types of hypoglycaemic or antihypertensive drugs, so the effect of drugs on microbiota variation also needs to be considered.Our study explored the imbalance pattern and functional changes of gut microbiota in the two disease states, identified the dangerous strains that promote the progression of diabetic nephropathy in diabetic patients, and established a classification algorithm model to distinguish the two by gut microbiota, providing a certain basis for the diagnosis and prediction of diabetic nephropathy through gut microbiota. The follow-up research can try to restore microbial homeostasis by interfering with gut microbiota, and then protect the kidney by regulating the gut-kidney axis, bringing a new method for the treatment of diabetic nephropathy.


Atherosclerosis is a leading cause of mortality in the rapidly growing population with diabetes mellitus. Vascular interventions in patients with diabetes can lead to complications attributed to defective vascular remodeling and impaired healing response in the vessel wall. In this study, we aim to elucidate the molecular differences in the vascular healing response over time using a rat model of arterial injury applied to healthy and diabetic conditions.Wistar  and Goto-Kakizaki  rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 20 hours, 5 days, and 2, 4, and 6 weeks. Noninvasive morphological and physiological assessment of the CCA was performed with ultrasound biomicroscopy (Vevo 2100) and corroborated with histology. Total RNA was isolated from the injured CCA at each timepoint, and microarray profiling was performed . Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database, and Cytoscape software.P < .01; two-way analysis of variance) as well as exaggerated negative remodeling was observed after 2 weeks of injury in GK rats compared with heathy rats, which was confirmed by histological analyses. Bioinformatic analyses showed defective expression patterns for smooth muscle cells and immune cell markers, along with reduced expression of key extracellular matrix-related genes and increased expression of pro-thrombotic genes, indicating potential faults on cell regulation level. Transcription factor–protein-protein interaction analysis provided mechanistic evidence with an array of transcription factors dysregulated in diabetic rats.Significant increase in the neointimal thickness (In this study, we have demonstrated that diabetic rats exhibit impaired arterial remodeling characterized by a delayed healing response. We show that increased contractile smooth muscle cell marker expression coincided with decreased matrix metalloproteinase expression, indicating a potential mechanism for a lack of extracellular matrix reorganization in the impaired vascular healing in GK rats. These results further corroborate the higher prevalence of restenosis in patients with diabetes and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes. Moreover, the presented study provides the research community with the valuable longitudinal gene expression data bank for further exploration of diabetic vasculopathy. Vascular interventions causes injury to the arterial wall, which in turn induces a healing response to restore vessel wall homeostasis. However, in patients with diabetes, such interventions lead to exaggerated healing response and defective remodeling. There is a need to understand the molecular mechanisms underlying the defective healing response in diabetes. In this study, ultrasound biomicroscopy, histology, and microarray profiling were used to demonstrate the transcriptional and physiological changes at various timepoints following arterial injury in healthy Wistar and diabetic GK rats. This study also provides a database of longitudinal transcriptional changes for the research community to study vascular healing in diabetes. Microarray profiling of the injured left common carotid artery at each timepoint revealed defects in smooth muscle cell and immune cell signaling.•Take Home Message: Ultrasound biomicroscopy, histology, and microarray profiling provided physiological and molecular insights into the delayed vascular healing in Goto-Kakizaki rats.,The epidemic of diabetes mellitus (DM) is increasing worldwide, and DM is an independent risk factor for morbidity and mortality associated with cardiovascular disease (CVD).The major effects of uncontrolled hyperglycemia and insulin resistance in diabetes are manifested as microvascular and macrovascular complications. In large arteries, veins, and in the heart, increased release of free fatty acids from insulin resistant adipocytes results in increased reactive oxygen species (ROS) production and dysfunction of endothelial cells, leading to atherogenesis.The rat common carotid artery (CCA) balloon injury model is a commonly used experimental model to study intimal hyperplasia and restenosis. The model involves a mechanical stretch injury of the arterial wall and denudation of the endothelial lining of the CCA, which induces a healing response characterized by vascular SMC proliferation, extracellular matrix (ECM) accumulation, and rapid reendothelialization.Here, we aimed to combine US imaging, histology, and microarray analysis to systematically study the physiological and molecular changes throughout the healing of the vessel wall in the diabetic environment. We compared the transcriptomic profiles of injured arteries from healthy Wistar rats and diabetic GK rats to identify differentially expressed genes, altered pathways, and transcriptional changes along each timepoint throughout the healing process.A). The timepoints for euthanasia and analysis was selected based on the previous study performed in Sprague-Dawley rats,Male Wistar were purchased from Charles River (Scanbur Research A/S), and GK rats were bred in-house . Rats were housed in enriched cages at the animal facility and were constantly monitored by professional animal caretakers. The rats (n = 40 for Wistar and n = 40 for GK) were subjected to left common carotid artery balloon injury using 2F Fogarty balloons as previously describedNoninvasive morphological assessment was performed using US  at 2-, 4- and 6-weeks post-surgery. The US image analysis was performed using an offline imaging software (Vevolab 1.7). US images were used to measure lumen and total vessel diameter as described previously by Röhl et al.Upon euthanization, both the injured and uninjured  vessels were used for microarray and histological analyses. All animal experiments were approved by the Stockholm Ethical Board , and institutional guidelines for proper animal care were followed.RNA was extracted using Qiazol Lysis Reagent  and purified using the miRNeasy Mini kit . The RNA concentration was determined using the Nanodrop ND-1000 (Thermo Scientific), and the RNA quality was estimated using a Bioanalyzer capillary electrophoresis system (Agilent Technologies). Total RNA of appropriate quality and integrity  was used for microarray profiling with Affymetrix GeneTitan Rat Gene ST v1 arrays.Carotid arteries were fixed in 4% Zinc-formaldehyde for 24 hours and dehydrated in 70% ethanol. Then the tissues were embedded in paraffin blocks. Five-μm sections were obtained using a microtome. The sections were deparaffinized using Histolab Clear and rehydrated in gradually decreasing concentrations of ethanol. After washing, the sections were treated with Bouin's solution (Sigma-Aldrich). The slides were stained with Weigert's iron hemotoxylin (1:1) (Histolab), washed and stained with Biebrich's scarlet-fuschin solution (Sigma-Aldrich). After washing, the sections were stained with phosphomolybdic-phosphotungstic acid solution (1:1+2 volumes of distilled water) (Sigma-Aldrich) and then with Aniline Blue. The sections were washed with 1% acetic acid and dehydrated using increasing concentrations of ethanol. The sections were mounted using Pertex (Histolab) and scanned in an automated SlideScanner system (Scanscope CS).https://www.r-project.org/). The data was centered and scaled, and PCA was calculated. Differential gene expression analysis was performed using a two-sided multiple t tests , by comparing Wistar and GK rats for each timepoint. The statistically significant changes in gene expression at each timepoint were represented by a volcano plot. Genes important for vascular healing among the top 20 differentially up- and down-regulated genes were indicated on the volcano plot.Robust multi-array average normalization and batch effect correction of the microarray dataset was performed, and processed gene expression data was returned in log2-scale. Principal component analysis (PCA) was performed using the R software (https://david.ncifcrf.gov/). Briefly, a list of up-/down-regulated genes from each timepoint was used to generate gene ontology (GO)-biological processes with an EASE score of 0.0001 and gene count for each pathway of five. The resulting list of pathways were filtered with dispensability less than 0.3.Pathway analysis was performed using The Database for Annotation, Visualization, and Integrated Discovery (DAVID) online bioinformatic tool (https://maayanlab.cloud/Enrichr/) and STRING (https://string-db.org/) databases. Significantly up- and down-regulated genes from the pathway analysis were fed into Enrichr to obtain TF-PPIs for each timepoint. The top 10 significantly up- and down-regulated transcription factors from Enrichr were used to generate PPI network using the STRING database. These networks were imported to Cytoscape software (version 3.9.0), where the styles of the nodes were modified according to P-value and direction of fold change.Transcription factor protein-protein interaction (TF-PPI) analysis was performed using the web-based Enrichr (B). We observed that seven arteries from Wistar and 10 arteries from GK rats thrombosed in total at the end of the experiment. The vessel diameter of the intact arteries was significantly smaller in GK rats, as expected, because they correlate with the body weightA). However, the loss of the total vessel diameter was significantly more pronounced in diabetic animals compared with Wistar rats, indicating negative remodeling in these animals at 2, 4 and 6 weeks (A). The intima-media thickness was significantly increased in GK rats compared with Wistar rats at 4 weeks after injury (B). In parallel with stabilization of the vessel diameter, we could see continuous regrowth of the endothelial layer in Wistar rats.Morphological and physiological changes in response to carotid balloon injury in GK and Wistar rats were assessed by US , B. We o 6 weeks , A. The r injury , B. In pD and Histological analysis, using Masson-trichrome staining, confirmed that the neointimal thickness was greater in GK rats at 2, 4, and 6 weeks D and . Taken tA), indicating that the variation in the data is more complex. The first two principal components did not separate the expression profiles of uninjured and injured vessels at 0 hours in both Wistar and GK rats. However, the expression profiles could be grouped into early, intermediate, and late phases of healing by PCA analysis, as previously reported.The global gene expression profiles of Wistar and GK rats from each timepoint were compared individually in a principal component analysis (PCA). The PCA plot showed that only about 28% of the variations in gene expression between the groups were explained by the first two principal components , A, indiB, To further analyze the transcriptomic differences between the groups, differential gene expression comparing Wistar and GK rats was performed for each timepoint as visualized by volcano plots B, . GK ratsThe contribution of various major cell types in the vascular wall to vessel healing was assessed from microarray data. Previously recognized classical markers of SMCs, ECs, macrophages, lymphocytes, and platelets were queried for each timepoint in GK and Wistar rats and plotted as a heat map .A). However, in GK rats in later stages of the response, these markers increased significantly more than in Wistar rats (A and B). In addition, proliferation markers were also reduced during the remodeling phase in the GK rat vessels (E). Taken together, these observations establish that the increase in intimal hyperplasia in GK rats at 4 weeks . The endothelial progenitor cell marker Cd34 also decreased following injury and recovered at the end of the healing process in Wistar rats. In GK rats, the expression levels decreased at 6 weeks, indicating a defect in the production of endothelial progenitor cells (B).The endothelial markers Vwf and Podxl, decreased following vascular injury in both Wistar and GK rats, while they gradually increased after 2 weeks indicating a trend towards vascular homeostasis. However, Vwf expression was significantly increased in GK rats compared with that of Wistar rats , B. The or cells , B.C). Most lymphocyte genes increased at 20 hours after injury in both GK and Wistar rats (D). However, the costimulatory molecule Itgal markedly decreased during 2 weeks in GK rats (D).The macrophage marker Cd68, which peaked at 20 hours in Wistar rat vessels, peaked at 5 days in GK rats, with a significantly increased expression. Cd80, on the other hand, peaked at 5 days in both groups, but was also higher in GK rat vessels. Both these markers fell sharply in GK rats thereafter, decreasing more significantly than Wistar rats at 4 weeks , C. Mosttar rats , D. Howe GK rats , D.A). Interestingly, genes that promote coagulation, such as Serpine1 and Serpinb2, had distinctly higher expression in GK rats at various phases of healing (C). Although Serpine1 was increased in uninjured GK rats' vessels and 4- and 6-weeks injured vessels, Serpinb2 was abnormally increased at 20 hours post injury. The key adhesion molecule Itga3 was higher in uninjured GK rat vessels and was also consistently higher throughout the healing process (C).Increase in platelet expression was limited to 0 and 20 hours in GK and Wistar rats , A. Inte healing , C. Alth process , C.Taken together, these results show that SMC and macrophage gene expression are profoundly affected in GK rats compared with Wistar rats.B and B-C). In the intermediate phase, processes such as response to virus, integrin signaling, cell adhesion, and migration were enriched, indicating a delayed induction of inflammatory response. However, processes related to cell proliferation were repressed in this phase (C and D-E). The late phase of healing was characterized by SMC proliferation, cell survival, response to hormonal signaling, and response to mechanical stress. It is important to note that processes related to vessel remodeling, such as collagen fibril organization, ECM organization, and integrin signaling, were still repressed (D and F-G). Overall, the pathway analysis corroborated the gene expression changes and confirmed the aberrant induction of inflammatory response and SMC contraction.To get an insight into the consequences of the aberrant signaling in SMCs and immune cells towards overall vessel healing in GK rats, we used the significantly up- and down-regulated genes in GK rats to identify biologically enriched pathways by GO analysis. Certain pathways such as hypoxia signaling and angiogenesis were enriched immediately following injury and persisted until 4 weeks post injury . In the se C and , D-E. Thed D and , F-G. OvB). At 20 hours, TFs that induce proliferation and differentiation were upregulated . However, induction of Foxp3 and downregulation of Batf, Stat3, and Smad3 indicated a dampening of inflammatory response at this early stage of vessel wall healing (B). Increase in Gata1, Gata2, and Lmo2 expression signaled the beginning of the endothelium recovery, whereas inflammatory responses were still repressed  after 5 days of injury (D). The immune response to the injury was finally induced at 2 weeks with the increase of Sp1, Cebpb, Pax5, and Smad3. However, transcription factors that promote T-cell differentiation were suppressed through 20 hours  and 5 days  and activated only at 2 weeks  and continued through 4 weeks  and 6 weeks . It is important to note that, at this point, cell proliferation is strongly repressed by the downregulation of many cell cycle genes  and upregulation of transcriptional repressors  (C). At 4 weeks, these immune responses diminished as shown by the activation of Smad3, Jun, and Nr3c1 and repression of Stat3 and Stat5a. However, various other cell survival and proliferation markers were also activated  (F). This trend continued at 6 weeks as well, since proliferation markers were consistently upregulated  (D). In summary, the network of TF-PPIs at each timepoint confirmed that the typical healing responses were delayed in GK rats, and this dysregulation was controlled at the transcriptional level.To identify regulatory changes that potentially led to dysregulated vessel healing in GK rats, we performed analysis of TF-PPI interaction networks. The analysis showed that following injury, genes responsible for induction of proliferation were repressed at several levels, with upregulation of transcriptional repressors such as Zbtb7a, Ncor1, and Sox17 and with the repression of transcriptional activators such as Ncoa1, Fos and Neurod1 , B. At 2 healing , B. Incrf injury , D. The a, Atf2) , C. At 4, Ep300) , F. This Ctnnb1) , D. In sIn this study, we investigated temporal transcriptomic changes in the vessel wall that drive faulty arterial recovery in diabetic rats. Ultrasound analysis revealed that GK rats had an increased intimal hyperplasia in parallel with exaggerated negative remodeling, which was confirmed by histological analyses. Global transcriptomic analysis of the vessels in GK and Wistar rats revealed changes in expression patterns, which could explain the altered physiological and morphological phenotype.The PCA analysis showed that the biggest changes in the transcriptomic profiles of Wistar and GK rats were observed at 20 hours, 5 days, 2 weeks, and 4 weeks, with most profound differences at 4 weeks following injury, corresponding to the late stages during the remodeling phase. This was corroborated by the US and histological analysis, where lumen was significantly smaller in GK rats at 4 and 6 weeks after injury compared with Wistar rats.On the level of individual genes, classical contractile SMC markers decreased equally in both groups during the acute injury phase, but the increase in the remodeling phase in GK rats was much higher compared with Wistar rats. This observation is strengthened by the appearance of contractile markers and SMC-specific transcription factors as the top upregulated genes at 4 and 6 weeks. Indeed, it is known that luminal narrowing during early part of the healing is due to smooth muscle contraction.,Pathway analysis showed an upregulation of SMC proliferation at 4 weeks, further confirmed by the induction of transcription factors that promote SMC proliferationThe endothelial markers decreased, as expected, following injury to vessel wall, and gradually increased in expression after 2 weeks as the vessel wall approached homeostasis, in both GK and Wistar rats. However, von Willebrand factor (vWF), which is also a key molecule for platelet aggregation after tissue injury, was lower in intact vessels of GK rats, suggesting a possible endothelial dysfunction at baseline. As their expression levels increased towards the late phase of healing, there was a significant increase in vWF levels in GK rats at 4 weeks compared with Wistar rats. It is also noteworthy that the GK rats had an increased amount of thrombosis after surgery. On the contrary, the carotid diameter of the GK rats were relatively smaller compared with Wistar rats, which could also have contributed to increased thrombosis. The increase in vWF levels coincided with the specific increase of all platelet markers at 4 weeks in GK rats. Abnormal platelet activation is a feature of diabetes, and, in the context of restenosis, could lead to thrombosis in patients with diabetes.We observed an abnormal induction of innate and adaptive immune response during vascular healing in GK rats. Particularly, in the late phases of healing (at 4 weeks), macrophage markers reduced significantly in GK rats compared with Wistar rats. The lymphocyte marker expression did not change in GK rats. However, reduction in Itgal levels, but not Cd28 levels, shows that in GK rats, the adhesion of T cells to antigen presenting cells seem defective.Restenosis is characterized by a chronic inflammatory response and negative remodeling with SMC proliferation due to delayed re-endothelialization. Our observations in this study confirm these features are recapitulated in the balloon catheter injury of carotid arteries in diabetic rats. Future studies into the mechanisms of the dysfunctional build-up of SMCs during the remodeling phase and the aberrant regulation of macrophages throughout the injury could provide further insights into the pathogenesis of restenosis in patients with diabetes.It is important to note that, although GK rats generally have moderate increase in blood glucose levels, they also have relatively normal or moderately decreased insulin levels. This is because the changes in insulin levels are caused only by the decrease in β-cell mass and are not driven by obesity.It is also important to note that the study was conducted exclusively in male rats. Studies focused on investigating gender differences in restenosis have found that females have a significantly lower incidence of in-stent restenosis.When it comes to some advantages, this study can be seen as a global longitudinal encyclopedia of dysregulated arterial healing in diabetes, generating a powerful resource available for future explorations in this field. Moreover, the integration of morphological data from minimally invasive US with histological and transcriptomic data allows for direct correlation between the physiology of the vascular wall and molecular changes occurring in various cell types during the healing, thus improving the robustness of the observations.We systemically analyzed the morphological and physiological changes during vascular healing at various timepoints, together with longitudinal transcriptomic changes and their molecular drivers in diabetic GK rats. A striking difference was observed in the morphology and delayed healing in response to injury between GK and Wistar rats. Transcriptomic analysis revealed that, although Wistar rats had a healing response similar to that reported previously in healthy Sprague Dawley rats,Conception and design: SR, LM, ARAnalysis and interpretation: SN, ML, MKData collection: SRWriting the article: SNCritical revision of the article: SN, SR, ML, MK, LM, ARFinal approval of the article: LM, ARStatistical analysis: SNObtained funding: LMOverall responsibility: ARLM and AR contributed equally to this article and share co-senior authorship.