Verrucomicrobiota, Kiritimatiella, WCHB 41, and uncultured_rumen_bacterium were significantly enriched in the LZS group. KEGG pathway analysis found that LZS was significantly higher than the CK group in the Excretory system, Folding, sorting and degradation, and Immune system pathways (p < 0.05). The results of ELISA assay showed that the level of IL-10 was significantly higher in the LZS group than in the CK group (p < 0.05), and the levels of TNF-α and IL-1β were significantly higher in the CK group than in the LZS group (p < 0.05). LEfSe analysis revealed that the dominant flora in the large intestine segment changed from Bacteroidota and Gammaproteobacteria to Akkermansiaceae and Verrucomicrobiae after PAS addition to Hu sheep lambs; the dominant flora in the small intestine segment changed from Lactobacillales and Aeriscardovia to Kiritimatiellae and WCHB1 41. In conclusion, the addition of PAS to sheep diets can increase the number and types of beneficial bacteria in the intestinal tract, improve lamb immunity, and reduce intestinal inflammation. It provides new insights into healthy sheep production.Prickly Ash Seeds (PAS), as a traditional Chinese medicinal herb, have pharmacological effects such as anti-asthma, anti-thrombotic, and anti-bacterial, but their impact on gut microbiota is still unclear. This study used a full-length 16 s rRNA gene sequencing technique to determine the effect of adding PAS to the diet on the structure and distribution of gut microbiota in Hu sheep. All lambs were randomly divided into two groups, the CK group was fed with a basal ration, and the LZS group was given a basal diet with 3% of PAS added to the ration. The levels of inflammatory factors  in intestinal tissues were measured by enzyme-linked immunosorbent assay (ELISA) for Hu sheep in the CK and LZS group. The results indicate that PAS can increase the diversity and richness of gut microbiota, and can affect the community composition of gut microbiota. LEfSe analysis revealed that  Relevant inflammatory factors were then validated using ELISA kits. The aim was to complement the incomplete study of PAS on the intestinal flora of sheep and to provide a theoretical basis for the application of PAS to the regulation of intestinal health in ruminants.2.2.1.All experimental designs and procedures were approved by the Animal Care Committee of Gansu Agricultural University (GSAU-AEW-2020-0057). in accordance with the Guidelines for Animal Care and Experimental Procedures established by the Ministry of Science and Technology of the People’s Republic of China .2.2.During the study, 12 weaned male lambs  of Hu sheep were selected from Dongxiang County Dawan Breeding Farmers’ Specialized Co-operative Society . The nutrient composition of PAS is shown in 2.3.Genomic DNA was extracted from the contents of duodenum, jejunum, ileum, cecum and colon using the TGuide S96 Magnetic Bead Method Soil and Fecal Genomic DNA Extraction Kit  Co., Ltd.) according to the instruction manual, and the concentration of the DNA was determined by Nanodrop, and the purity of the DNA was determined by agarose gel electrophoresis, which was qualified and used for the amplification of full-length 16S rRNA gene amplification.After extracting the total DNA of the samples, specific primers with Barcode were synthesized according to the full-length primer sequences , and the products were purified, quantified, and normalized to form a sequencing library (SMRT Bell). The constructed libraries were first subjected to library quality control, and the qualified libraries were sequenced by PacBio Sequel II. Sequencing was performed by Biomarker Technologies .2.4.Corresponding intestinal tissues (18 samples) from the jejunum, ileum, and cecum were selected for enzyme-linked immunosorbent assay. Three inflammatory factors were measured by double-antibody sandwich ELISA provided by Jingmei Biotechnology Co., Ltd. : TNF-α (JM-07817S1), IL-1β (JM-00413S1), and IL-10 (JM-07814S1).2.5.After exporting the CCS (Circular Consensus Sequencing) file through smrtlink analysis software. Firstly, the Raw-CCS sequence data were obtained based on Barcode sequence identification, followed by CCS filtering using cutadapt (version 2.7) software , and finr < 0 means negative correlation, r > 0 means positive correlation, p > 0.05 means insignificant difference, *p < 0.05 means significant difference, **p < 0.01 means the difference is highly significant.Correlation analysis was performed using Spearman correlation coefficient (Spearman correlation); 3.3.1.We collected five intestinal segment sites  and obtained a total of 60 samples for sequencing. A total of 1,200,134 CCS sequences were obtained by Barcode identification, with at least 11,700 CCS sequences per sample and an average of 20,002 CCS sequences. First, we analyzed the number of features in the CK and LZS groups. The total number of intestinal features in the CK and LZS groups was 1,424, the number of shared OTUs was 1,296, and the number of observed features in the intestines of the CK and LZS groups was 1,361 and 1,359, respectively, with no significant difference . RarefacTo prove the accuracy of the analyzed results, we performed intestinal microbial diversity analysis. The results showed that the addition of PAS to the diet was able to influence gut microbial richness and diversity. The Shannon, Simpson, and Chao1 indices of the LZS group were higher than those of the CK group –F, indic3.2.PCoA and NMDS analyses revealed a significant separation of the microbial population structure in the gut of the LZS group from that of the CK group ,C. Next,uncultured_rumen_bacterium (11.95%) and Akkermansia (9.26%), and the dominant genera in the LZS group were uncultured_rumen_bacterium (25.67%) and Mycoplasma (10.18%). In addition to this, most of the annotated bacteria were more abundant in the LZS group compared to the CK group. For example, Mycoplasma , Christensenellaceae_R_7_group , Clostridium_sensu_stricto_1  and Romboutsia .In the analysis of microbial composition, we focused on exploring the top 10 bacteria at the phylum and genus level. The level of microorganisms at each taxonomic level was analyzed. At the phylum level , Firmicu3.3.Aeriscardovia, Streptococcus and Enterobacterales and were significantly higher than those in the LZS group . The LZS and CK groups contained taxa with significant differences in microbial communities for abundance, which may be related to the feeding of PAS. In order to investigate the effect of feeding PAS on intestinal metabolic pathways, we used a p-value of < 0.05 as a criterion for differential enrichment analysis. As shown in p < 0.05); whereas the LZS group was significantly higher than the CK group (p < 0.05) in Excretory system, Folding, sorting and degradation, and Immune system pathways were significantly higher in CK group (p < 0.05).Based on the results of cluster analysis, we performed LEfSe analysis on LZS and CK groups. It was found that the significantly enriched bacteria in the CK group were mainly 3.4.p < 0.05), and significantly higher in the small intestinal segment than in the large intestinal segment for the CK group and the LZS group for the ACE and Chao1 indices (p < 0.05). Additionally it was shown by the dilution curve that the sequencing data could be used; Finally, the PCoA analysis revealed that the differences explained by principal component 1 (PC1) and principal component 2 (PC2) were 65.91 and 6.83%, respectively ; The level of IL-10 in the intestinal tissues of sheep was significantly lower than that of the LZS group (p < 0.05). Inflammatory factors were correlated with the top 10 gut flora phylum and genus level abundance (p < 0.05) and negatively correlated (r < 0) with Verrucomicrobiota. IL-10 was significantly different (p < 0.05) and positively correlated (r > 0) with Verrucomicrobiota. TNF-α and IL-1β were significantly different (p < 0.05) and positively correlated (r > 0) with Actinobacteriota, Aeriscardovia, and Streptococcus. IL-10 was significantly different (p < 0.05) and negatively correlated (r < 0) with Actinobacteriota, Aeriscardovia, and Streptococcus.Changes of inflammatory factors  were measured by enzyme-linked immunosorbent assay ,B. The lbundance ,D. The r4.Pseudois nayaur) and other groups , and that the proportions of metabolic pathways of Digestive system and Infectious diseases:parasitic were significantly higher than those in the CK group (p < 0.05). The proportion of metabolic pathways in Digestive system and Infectious diseases:parasitic was significantly higher than that in LZS group (p < 0.05). It shows that PAS can indeed improve the immunity of Hu sheep lambs, reduce the Parasitic disease of Hu sheep lambs  and a significant decrease in IL-1β and TNF-α levels (p < 0.05) in the LZS group compared to the CK group. IL-10 prevents colitis by eliminating dysfunctional mitochondria and inhibiting mTOR signaling and inflammatory vesicle activation in macrophages  can be found below: NCBI - PRJNA1003863.The animal studies were approved by Animal Care Committee of Gansu Agricultural University (GSAU-AEW-2020-0057). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.DL: Methodology, Writing – original draft, Writing – review & editing. HY: Writing – review & editing. QL: Writing – review & editing. KM: Writing – review & editing. HW: Writing – review & editing. CW: Writing – review & editing. TL: Writing – original draft, Writing – review & editing. YM: Methodology, Writing – original draft, Writing – review & editing.


Trichoplusia ni (Lepidoptera: Noctuidae) that frequently feeds on glucosinolate-containing plants. Caterpillars feeding on diets with high concentrations of ITC experienced reduced survivorship and growth rates. High concentrations of ITC suppressed the appearance of several types of hemocytes and melanization activity, which are critical defenses against parasitic Hymenoptera and microbial pathogens. In terms of T. ni humoral immunity, only the antimicrobial peptide (AMP) genes lebocin and gallerimycin were significantly upregulated in caterpillars fed on diets containing high levels of ITC relative to caterpillars that were provided with ITC-free diet. Surprisingly, challenging caterpillars with a non-pathogenic strain of Escherichia coli resulted in the upregulation of the AMP gene cecropin. Feeding on high concentrations of plant toxins hindered caterpillar development, decreased cellular immunity, but conferred mixed effects on humoral immunity. Our findings provide novel insights into the effects of herbivore diet composition on insect performance demonstrating the role of specific plant defense toxins that shape herbivore immunity and trophic interactions.Insect herbivores frequently encounter plant defense molecules, but the physiological and ecological consequences for their immune systems are not fully understood. The majority of studies attempting to relate levels of plant defensive chemistry to herbivore immune responses have used natural population or species-level variation in plant defensive chemistry. Yet, this potentially confounds the effects of plant defense chemistry with other potential plant trait differences that may affect the expression of herbivore immunity. We used an artificial diet containing known quantities of a plant toxin  to explicitly explore the effects of a plant toxin on the cellular and humoral immune responses of the generalist herbivore  Plants have a diverse array of chemical defenses against insect herbivores. However, many herbivores have developed counter-defense mechanisms such as excretion, detoxification, and sequestration to overcome the negative effects of plant defense metabolites , 2. ThisJunonia coenia  and Ceratomia catalpa (Lepidoptera: Sphingidae) [Plutella xylostella (Lepidoptera: Plutellidae) and Pieris rapae (Lepidoptera: Pieridae) [Grammia incorrupta caterpillars against parasitoids [Plantago lanceolata, has been shown to enhance the cellular immunity of Anartia jatrophae against pathogens [Organisms that occupy a middle trophic level like caterpillars must balance the potentially conflicting bottom-up pressures of plant anti-herbivore defenses and top-down pressures from their own natural enemies . Hence, ingidae) , 11. On ieridae) , 12. In asitoids . Similarathogens . These cCaterpillars employ both cellular and humoral immune responses against invaders, such as parasitic wasps and microbial pathogens . PreviouBoth aspects of the innate immune system work to oppose foreign invaders. Hence, an understanding of caterpillar immune status requires the simultaneous study of both cellular and humoral immune functions. Furthermore, maintaining an elevated immune status is costly, thus caterpillars must devote nutrient resources towards immunity, which may compromise their ability to detoxify plant defensive chemistry . For thiTrichoplusia ni (Lepidoptera: Noctuidae), an herbivore with a broad host range, that commonly feeds on plants in the genus Brassica. Brassica produce glucosinolates as their anti-herbivore defense metabolites [T. ni [T. ni prefers to oviposit and feed on glucosinolate containing host plants, even when given a choice [T. ni.Here we studied abolites . Followiabolites . Isothios [T. ni , 27. Yeta choice  Hence, iT. ni larval performance and immune responses, we use artificial diets to which we added known quantities of 4-methylsulfinylbutyl isothiocyanate (4- MSOB -ITC or ITC). We examine how different concentrations of the isothiocyanate mixed in an artificial diet affects the weight, mortality, development, as well as cellular and humoral immune responses of T. ni caterpillars. Our study illustrates that certain plant toxins can strongly influence herbivore performance and modify innate immune capacity.As different plant species vary in more than just defensive chemistry, studies comparing plant species effects on caterpillar immunity can only make correlative associations between plant chemical defenses and herbivore immune responses. To determine whether isothiocyanates directly affect T. ni was maintained in an environmental chamber with conditions set at 25±5°C, a 16L:8D photoperiod, and 30% relative humidity for several generations on artificial diet before use in experiments following an established protocol [T. ni were provided with 10% honey water solution as a food source and allowed to mate inside a plastic container (3.78 L) that was covered with a sheet of paper towel on which T. ni adults laid eggs. The egg sheet was collected the next day, it was placed in petri dishes, and incubated at 25°C for three days until the T. ni eggs hatched. After hatching, first instar T. ni larvae were used in all experiments.A laboratory colony of protocol  (S1). BrTrichoplusia ni caterpillars primarily feed on cabbage that contains glucosinolates, the most abundant of which (approximately 95%) are aliphatic compounds like sinigrin and glucoraphanin. Hydrolysis of this compound produces allyl-isothiocyanate. The concentration of allyl-isothiocyanate  in Brassica plants can reach up to 3 μmol/g [® soufflé cups, Dart Container Corp.) on approximately 15 ml of artificial diet containing one of the four isothiocyanate concentrations (n = 30/treatment). After eleven days of feeding on their respective diet, caterpillars were weighed and returned to their cups. Caterpillars were maintained on the same diet until pupation when pupae were weighed (n = 22/treatment). Survival rates were measured by monitoring a separate group of caterpillars, which were reared under the same experimental conditions. The number of caterpillars that successfully pupated was recorded out of the initial number of first instars placed on each of the four treatment diets allowing us to calculate survival rates. (n = 210 caterpillars/treatment). The total development duration from egg to pupal stage was recorded (n = 21/treatment).3 μmol/g . Based o3 μmol/g  used in Cellular immunity To quantify the effect of 4- MSOB -ITC on the cellular immune status of T. ni, 11-day-old larvae (between first to late second instar) were collected from each of the isothiocyanate treatments (n = 10/treatment). Using a sterile needle, a lateral incision was made across the abdomen of each larva and the hemolymph was collected and added to a pre-chilled Eppendorf tube. To quantify the total number of hemocytes, the collected hemolymph was diluted with a fixed amount of phosphate buffer saline . Approximately 8 μL of diluted hemolymph was added onto a Neubauer hemocytometer and left to settle for 15 minutes, after which individual hemocytes were counted under the microscope  at 200x magnification. Individual hemocytes were identified morphologically by performing an FITC tagged phalloidin-DAPI staining using established protocols [n = 10/treatment).rotocols . Brieflyrotocols  (n = 10/n = 10/treatment).To measure the phenoloxidase activity, hemolymph from individual larvae was mixed with ice-cold PBS in a 1:2 ratio in an Eppendorf tube and centrifuged at 4000g for 15 min at 4°C. The supernatant was added to a micro plate well containing 20 μL of PBS and 50 μL of 2 mM L-Dopa as a substrate for phenoloxidase. The reaction was allowed to proceed for 30 minutes. The degree of melanization was recorded spectrophotometrically at 490 nm (Versamax multimode reader)  and then allowed to feed on their respective diet for 24 hours (n = 3/treatment). RNA was isolated by homogenization of samples in TRIzol reagent . RNA concentration and purity were measured using Nanodrop (A260/280 ~ 2). Reverse transcription was performed on 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR (qPCR) reactions were conducted and monitored with a CFX96 Real-Time System, C1000 Thermal Cycler (Bio-Rad) with the following cycling conditions: 95°C for 2 minutes, 40 repetitions of 95°C for 15 seconds followed by 61°C for 30 seconds, and then one round of 95°C for 15 seconds, 65°C for 5 seconds, and finally 95°C for 5 seconds. Each reaction well contained 10 μl of GreenLink No-ROX qPCR Mix (BioLink), 40 ng of cDNA template, forward and reverse primers (provided in S2) at a final concentration of 200 nM and ultrapure water to 20 μl total volume. A dissociation curve analysis was performed for all primer pairs, and all experimental samples yielded a single sharp peak at the amplicon’s melting temperature. All assays were run three times  and each experiments included technical replicates. Relative fold changes for each gene were set to 1 for the control treatment, which involved caterpillars kept on diet in the absence of ITC. For analysis, the comparative quantitation method (ΔΔCt) was used to contrast the different treatments, and transformed to absolute values with 2-ΔΔCt for obtaining relative fold changes.One-way analyses of variance (ANOVAs) were used to compare the different treatments, followed by Tukey’s post-hoc significance test at a level of 5%. Caterpillar survivorship was compared between the treatments with generalized linear model having binomial distribution with logit link function. All statistical analyses were performed using SPSS 28.0. Figures were made using Origin 2021b and BioRender software.3,116 = 251, P<0.001; P<0.001; P = 0.008; P = 0.002; F3,84 = 1.24, P = 0.299; Caterpillar performance was highest on an ITC-free diet (control), with minimal mortality and greater larval weight compared to caterpillars that fed on diets containing higher levels of ITC . Over anF3,36 = 6.62, P = 0.001; F3,36 = 5.22, P = 0.004; F3,36 = 5.56, P = 0.003; F3,36 = 9.2, P = 0.0001; F3,36 = 6.45, P = 0.001; F3,36 = 3.2, P = 0.01; The total hemocyte count in circulation decreased by 28% when caterpillars consumed diets containing 3 μmol/g of ITC compared to the control diet  and gallerimycin , was found to be significantly upregulated in caterpillars which had been fed on diet containing 2 μmol/g of ITC in comparison to control diet without ITC  was compared and the expression two AMP genes, hout ITC . When caT. ni. The detoxification of ITC requires conjugation to glutathione, which is a high energy consumption process and may participate in the trade-offs that we observed . Furthermore, if the caterpillars feed on a diet that contains higher concentrations of ITC, a higher level of free ITC will not be conjugated, which may lead to the reduced larval performance [T. ni feeds on glucosinolate containing plants, where the caterpillars grow slowly, which may render them more vulnerable to their natural enemies [T. ni. It is conceivable that exposure to higher levels of ITC triggered a compensatory response in the caterpillars, leading to physiological adjustments that allowed them to achieve comparable pupal weights despite the toxin’s presence. Whether this adjustment comes with a trade-off associated with adult stage will be our next endeavor.Several studies show the correlation between total or individual glucosinolate levels with herbivore physiology and performance by using plant natural populations. These findings do not exclude the impact of other unquantified factors such as physical defenses, morphology, leaf toughness, and plant nutritional quality like protein, free amino acids, carbohydrates, antioxidants and various other macro- or micro-nutrients contributing to the observed effect –34. Our formance , 27. Des enemies , 36. IntT. ni is negatively affected by the consumption of isothiocyanate (ITC), supporting the ‘immunocompromised host hypothesis’ [Plutella xylostella and Pieris rapae have shown that higher glucosinolate concentrations are correlated with enhanced cellular immunity against parasitic wasps at the cost of reduced larval weight and development success [T. ni caterpillars showed similar trends in weight and development as specialist herbivores like P. xylostella and P. rapae, their cellular immunity was negatively affected. This apparent difference could also be due to the presence of other nutrients like carbohydrates, proteins and free amino acids. In Spodoptera littoralis, higher protein-to-carbohydrate ratios and the availability of free amino acids were linked to improved antibacterial responses and increased phenoloxidase activity [T. ni immunity and whether a higher protein content and free amino acid diet can rescue the downregulated immune status are topics that require further investigation.Our data also show that the cellular immunity of othesis’ , 14, 37.othesis’ . Hence, othesis’ . These o success , 30. Altactivity –42. WhetT. ni cellular immune response was negatively correlated with glucosinolate content, we examined if this was also the case with the humoral immune response. Humoral immunity acts together with cellular immunity to eliminate foreign invaders including parasitoids and pathogenic microbes. Our data indicate that the consumption of a diet containing 2 μmol/g of ITC does elevate the expression level of the AMP-encoding genes lebocin and gallerimycin, which are produced upon activation of IMD signaling  [E. coli downregulated the expression level of both AMP genes. Surprisingly, this result is contrary to the known function of lebocin, which has been shown to possess antibacterial properties through the agglutination of E. coli cells in Manduca sexta (Lepidoptera: Sphingidae) [T. ni by increasing the transcriptional levels of certain AMP genes such as lebocin, but the combined presence of plant and E. coli toxins may perturbs the activation of the Toll and Immune Deficiency (IMD) pathways that regulate AMP production and activity. Other reasons for this apparent difference could be due to the mode of E. coli introduction. Previous studies have conducted feeding experiments where the caterpillars were exposed to artificial diet supplemented with bacteria [cecropin in T. ni was downregulated by ITC, infection with E. coli elevates its expression level in comparison to the respective control. This indicates that the detoxification of ITC does suppress cecropin expression but fails to perturb the activation pathway in response to infection with non-pathogenic bacteria.Because the esponse) . Howeveringidae) . Maybe tbacteria , 46, whicecropin and gallerimycin constitute an efficient T. ni host immune response against parasitoids or microbial pathogens deserves further attention. Nonetheless, the current study is original in terms of demonstrating the impact of glucosinolates on caterpillar immunity. To our knowledge, this is the first report to reveal how the glucosinolate metabolites negatively affect the development as well as modulate cellular and humoral immunity of a generalist caterpillar. This opens a new avenue of investigation to further explore similar effects conferred by toxic plants on the efficiency of various physiological processes of major agricultural insect pests.Overall, our data suggest that the presence of plant toxin negatively affects two important life-history traits: caterpillar development and immunity. Whether the observed elevated numbers of plasmatocytes and expression levels of


Nagaya, a collective housing program, was established in Soma City, Fukushima, Japan to address social isolation among older adults and support their long-term health. This study aimed to identify characteristics of individuals in Nagaya and examine the sustainability of this initiative.Following the Great East Japan Earthquake, the living environment of socially isolated older adults has become a pressing concern. In response, Nagaya, emphasizing their characteristics, the continuity of their stay in Nagaya, and their care certification levels, using data up to December 31, 2022. We employed Kaplan–Meier curves to analyze the duration for which residents continued to reside in Nagaya and the time leading up to the requiring care-level certification.We conducted a retrospective analysis of residents who were relocated to Nagaya after the disaster, 30 people (46.2%) continued to live there, 21 (32.3%) died during their stay, and 14 (21.5%) moved out. The overall duration of occupancy averaged 6.39 years (SD 3.83 years). The proportion of requiring care-level certification occurrences per person-year was 0.0577 for those without care certification and 0.3358 for those with requiring support level at the time of moving in.Of 65 people who moved to Nagaya-style communal housing may offer suitable living environments for older adults with diverse needs during disasters and serve as a valuable tool for developing public policies in aging societies.In summary,  In addiIn developed countries with rapidly aging populations, addressing the health, housing, and caregiving concerns of socially isolated older adults is becoming a critical public health issue. In 2020, Japan had an aging rate of 28.6%, with 15.0% of older men and 22.1% of older women living alone . China iIn this context, learning from approaches employed to support isolated older people after the Great East Japan Earthquake provides insights for addressing challenges in rapidly aging regions. Prolonged evacuations following the 2011 Great East Japan Earthquake, tsunami, and nuclear accident exacerbated the isolation among older people, adversely affecting their health. There was a subsequent rise in the occurrence of chronic diseases and an elevated risk of receiving care certification . As the Idobata-Nagaya, a shared living housing model in Soma City. Soma City, which is located 40–50 kilometers from the Fukushima Daiichi Nuclear Power Plant, had a notably high aging rate in 2010, with 25.3% of its population aged over 65 years. The earthquake and subsequent tsunami resulted in 486 deaths in the area, leading more than 2000 people to evacuate to temporary housing , which was designed as a hub for daily social interaction  and requiring long-term care levels 1 and 2 (moderate disability) and goes up to requiring long-term care levels 3–5 (severe disability), with a total of 7 stages  changes in requiring support/long-term care levels over time. To assess retention, the number of years from moving in to a departure event was calculated by Kaplan–Meier curves. Regarding care level changes, residents without care level certification and those with certified requiring support levels at the time of moving in were targeted. Kaplan–Meier curves were calculated using years until a requiring long-term care level of 1 or higher certification event. Censorship occurred when residents died or left Nagaya. Only those who turned 65 years of age during the study period were included in the care-level change analysis since those under 65 years of age have different care certification requirements. This study was approved by the Ethics Review Committee of the Fukushima Medical University . As this was an anonymized, information-based study, research consent was obtained using an opt-out approach.We investigated (1) residents’ characteristics; (2) retention status in 33.1Nagaya began operations in April 2012, 65 people moved into Nagaya  died during their stay; and the remaining 14 (21.5%) left Nagaya. The main reason for leaving was moving to live with relatives (eight people), followed by health-related hospitalization or relocation to a facility (five people). The average overall duration of stay was 6.39 years (SD: 3.83 years), with a median of 7.04 years (interquartile range [IQR]: 3.82–9.08 years), ranging from 21 days to 10.7 years. Regarding the duration of stay, five people left within one year; four people died during the period, and one person left for admission to another facility. Six people left within three years, four people died during this period, and two people moved to live with relatives. Eleven people left within 5 years, with four dying during the study period, four moving to live with relatives, and three leaving for facility admission. The remaining 39 individuals continued to reside for over 5 years. Of the 65 people, 30 (46.2%) continued to reside in 3.3At admission, 40 of the 62 people who were 65 years and older during the observation period had no requiring support/care certification, 10 had a requiring support level, and 12 had a requiring long-term care level  . The annNagaya; one resident started at requiring long-term care level 3 but improved to requiring long-term care level 2 after 2 years. One individual was certified as requiring long-term care level 2 at move-in, requiring long-term care level 1 a year later, and requiring support level 2 after 2 years. At move-in, the last person had requiring long-term care level 2 but was certified as requiring long-term care level 1 a year later.4Nagaya, disaster collective housing, 11 years after the community was established. The community welcomed not only independent older people but also those who needed care or were very old and struggling to live alone. Many residents continued living in the community for a long period, and the duration from moving to requiring care was maintained for an extended period. The study suggests that the Nagaya environment encourages residents to interact with the community, enabling many residents to maintain their health and continue their independent daily lives. The insights gained in this study can be applied to housing issues for isolated older individuals after other disasters and may also be relevant to housing issues for older isolated individuals in aging societies.We observed a cohort of residents in Nagaya fostered an environment for residents to interact and support each other  can be found in the article/The studies involving humans were approved by Fukushima Medical University Ethics Review Committee Chair, Clinical Research Review Committee for Guidelines Chair, General Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.TA: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. HS: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. NM: Writing – review & editing. NI: Writing – review & editing. MoT: Writing – review & editing. YK: Writing – review & editing. AO: Writing – review & editing. YN: Writing – review & editing. CY: Writing – review & editing. TZ: Writing – review & editing. MS: Writing – review & editing. MaT: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.


Renal allograft fibrosis is one of characteristic causes of long-term renal function loss. The purpose of our study is to investigate the association between fibrosis-related genes single nucleotide polymorphism (SNPs) and kidney function in 5 years after kidney transplantation.A total of 143 recipients were eligible for screening with 5-year follow-up information and SNP sequencing information from blood samples were included in this study. Minor Allele Frequency (MAF) and Hardy–Weinberg Equilibrium (HWE) analysis was conducted to identify tagger single-nucleotide polymorphisms (SNPs) and haplotypes. SNPs associated with the fifth year chronic kidney disease (CKD) staging were screened by SPSS and the “SNPassoc” package in RStudio and used for subsequent prediction model construction.A total of 275 renal transplant-related SNPs identified after target sequencing analysis. 64 Tagger SNPs were selected, and two SNPs (rs13969 and rs243849) were statistically significant for stage of CKD in 5 years. Finally, a model based on Gender, Age, rs1396, and rs243849 was constructed by multivariate linear regression analysis. Additionally, this model has a good performance in predicting uremia five years after kidney transplantation.Two SNPs (rs13969 and rs243849) were identified to be significantly associated with long-term renal allograft function. Based on this, a prediction model for long-term allograft function was established containing Gender, Age, rs1396, and rs243849. However, an independent cohort should be enrolled to validate the predicting performance.The online version contains supplementary material available at 10.1186/s12920-023-01686-6. End-stage renal disease happens as the final and most severe stage of chronic kidney disease (CKD), with poor prognosis and high medical cost. Although transplantation of kidneys is the preferred treatment option for most end-stage renal diseases , the faiFibrosis of the transplanted kidney clearly requires the involvement and interaction of various types of cells, including the activation of mesangial and fibroblast cells, the epithelial-mesenchymal transition (EMT) in tubular epithelium, the infiltration of monocyte/macrophage and T-cell as well as cell apoptosis . In receVarious physiological and pathophysiological processes are regulated by genetic factors, delivering an orientation for Intervention of renal allograft fibrosis at early stage. The genetic variation known as single nucleotide polymorphism (SNP) have an impact on gene functions of progression of disease. Some common single-nucleotide polymorphisms  in the human decrease the dopamine D2 receptor gene which have a correlation with the inflammation and fibrosis in human renal [In the process of fibrosis, diverse cytokines are secreted, for instance, TGF-β and its ligand TGFBR bind to activate transcription factors of the SMAD family, which can regulate the expression of proteins in cells and affect a series of kidney fibrosis processes, including Tubular Atrophy, EMT . Based oThe research we conducted is divided into two parts. Firstly, we follow up the patients in the cohort over time to obtain clinical information and determine the long-term trend in renal function. Then, comparative research of 274 SNPs of 7 fibrosis-related gene was performed in kidney transplant recipients with next-generation sequencing (NGS) application. Lastly, we formulate a signature of SNPs to predict prognosis of kidney allografts.This was a retrospective case–control study which contains 143 participants with available follow-up information during 5-years-term periods. The participants underwent kidney transplantation surgery between 1st February 2010 and 1st December 2015, at the First Affiliated Hospital with Nanjing Medical University. The included recipients met the following inclusion criteria: (1) recipients who were more than 18 years or less than 60 years, (2) recipients who either experienced stable serum creatinine levels  for at least 3 months or were diagnosed with chronic allograft dysfunction (CAD) by laboratory and pathological examinations, and (3) recipients with follow-up for more than 5 years after kidney transplantation. The exclusion criteria included are as follows: (1) recipients who did not meet the inclusion criteria; (2) recipients with severe heart, liver, or lung disease or chronic viral infections; and (3) recipients diagnosed with pregnancy and lactation.We followed up the clinical manifestation such as eGFR, urea nitrogen, and urine protein levels of patients as endpoint of allograft function at 1, 3, and 5 years. The eGFR calculated by modified glomerular filtration rate estimating equation for Chinese patients , is usedBasiliximab or antihuman thymocyte immunoglobulin was used as induction therapy at kidney transplantation. In all participants, calcineurin inhibitors such as tacrolimus or cyclosporin A were administered as immunosuppressive regimens during the maintenance period. Detail immunosuppression protocols were elaborated in this previous paper . The dosOur previous study described the detailed procedures of sample collection and the TS steps . PeripheP value will be excluded for further analysis. We used one-way analysis of variance (ANOVA) and chi-square test to compare the clinical variables in comparison with 3 groups sectionalized by CKD stages. Ordinal logistic regression analysis was used to select candidate clinical variables, which was considered as confounders in the following covariance analysis to select Tagger SNP related with long-term renal function, by using SPSS 26.0 software . A genotype association analysis of tagger SNPs was performed using the dominant model , recessive model , additive model (major homozygotes vs. heterozygotes vs. minor homozygotes), HET model (major homozygotes vs. heterozygotes) and HOM model (major homozygotes vs. minor homozygotes) by the RStudio version 4.0.5 software  implemented by the “SNPassoc” package version 2.0–2. Significant SNPs with long-term renal function were selected by taking the intersection of the results calculated by SPSS and “SNPassoc” package. Furthermore, general linear models (GLMs) were used to test associations between eGFR in the fifth years after the transplant and genotypes, by using SPSS 26.0 software . P < 0.05 was considered statistically significant and the adjustment of P-value by the number of genetic variants was performed in LD and variants association analysis.Unless otherwise specified, data are presented as mean + Standard Deviation (SD). The Hardy–Weinberg equilibrium (HWE), Minor Allele Frequency (MAF) and the linkage disequilibrium (LD) blocks were calculated using the Haploview version 4.2 software . Genetic variants with MAF < 0.05 and/or HWE less than Adjusted-2 in year 1 to 86.89 ± 41.13 ml/min/1.73m2 in year 5. By year 5, the proportion of patients with stage CKD1 had decreased from 55.2% to 43.4%. As a result, kidney function declines over time. A Kaplan–Meier (K-M) survival curve was used to demonstrate the overall renal function changes in the cohort over a 5-year period in Fig. P > 0.05) in Table P = 0.01, Gender: P = 0.009).In total, 143 recipients (including 38 women) were enrolled in this retrospective cohort study. In this study, the mean age of the patients was 36.87 ± 9.71 years, with a range of 10 to 59 years. Before kidney transplantation, none of the patients was detected with PRA. Primary kidney transplantation was performed on all recipients. Other baseline characteristics of recipients, including recipient weight, the presence of DGF, and incidence of AR before kidney transplant, and immunosuppressive protocols are presented in Table Further analysis of SNPs was conducted for 7 genes related to fibrosis Table S. In TablP = 0.001; gender: OR = 2.365, P = 0.033; incidence of AR episode: OR = 0.437, P = 0.034; Table P value = 0.05). It was concluded that rs13969 and rs243849 were statistically significant for stage of CKD. Finally, we get rs13969 and rs243849 by taking the intersection between the ANCOVA result of SPSS and the multiple inheritance models result of “SNPassoc” package in RStudio , glomerulosclerosis/renal tubulointerstitial fibrosis, diabetic nephropathy, polycystic nephropathy, and renal carcinoma . The serFor our study, we included 143 recipients who met our exclusion criteria and had full 5-year follow-up information. The Modified glomerular filtration rate estimating equation, which is more suitable for Chinese people, was used to estimate the eGFR value of patients through the clinical information of patients, for the subsequent CKD staging of kidney transplantation patients [Fibrosis of the transplant kidney is characterized by pathological manifestations such as glomerular sclerosis, renal tubule atrophy, interstitial fibrosis of the renal tubule, and inflammatory cell infiltration, capillary remodeling and other pathophysiological features. It is now accepted that kidney fibrosis is formed as a result of the body’s healing response . After iMMPs belong to a 23-member family of endopeptidase, which contain zinc, are dependent on calcium, and can degrade and remodel the proteins that form the extracellular matrix(ECM) , whose eHowever, as the common final outcome of CKD, fibrosis in transplanted kidneys is not impacted only by accumulation degradation of ECM, but also by inflammation and vascular remodeling. Inflammatory cells are recruited by MMPs in the early stages of inflammation. Collagen fragments created by MMP-9, could recruit neutrophils and stimulate them to release more MMP-9 . MeanwhiEven so, there are still limitations in our entire research process. First of all, the description of the mechanism of action described in this article is not specific enough. Secondly, even though our predictive model is equipped with satisfactory performance in our dataset, the sample size in our dataset is relatively small. However, the SNP data in our study are from peripheral blood, thus the predictive model can be applied to determine the likelihood of eGFR through timely blood tests, which suggests our model has an outstanding classification performance. Our predictive model needs to be investigated further in clinical work.In our study, MMP9 rs13969 and MMP2 rs243849 was found to be statistically associated with the eGFR in the 5 years after renal transplantation. Moreover, a prediction model for long-term allograft function containing gender, age, rs1396, and rs243849 was established. However, an independent cohort should be enrolled to further validate the prediction efficacy of this model in the prognosis of long-term allograft function.Additional file 1: Supplementary Figure 1. Linkage disequilibrium results of detecting tagger SNPs in the fibrosis-related genes.Additional file 2: Table S1. The genes related to fibrosis.Additional file 3: Table S2. Genetic information on SNPs of fibrosis-related genes.Additional file 4: Table S3. Results of HWE and MAF analysis.Additional file 5: Table S4. Results of LD analysis.Additional file 6: Table S5. The risk score of high-risk group and low-risk group.


Thymic hyperplasia patients were predominantly female (p = 0.049) and young (p < 0.001). The most common concomitant AD in this study was a thyroid-associated AD, which was associated with thymic hyperplasia (p < 0.001), Osserman type I MG (p < 0.001), and young age (p < 0.001). Thymectomy had a good therapeutic effect on MG complicated by AD, and there was a close correlation between surgery, thymus, MG, and ADs.Myasthenia gravis (MG) is an autoimmune disease (AD), and patients with MG often have other types of ADs. We analyzed the prognosis of patients with MG complicated by AD after thymectomy. A retrospective analysis was performed for patients with MG complicated by ADs treated surgically in our center over the past 22 years, and their general condition and follow-up data were collected and analyzed. 33 patients were included totally. 28 patients displayed improvement or even complete recovery of MG, and 23 of 36 ADs revealed improvement or even complete recovery. The prognosis of MG is significantly correlated with the duration of postoperative follow-up time ( Myasthenia gravis (MG) is an autoimmune disease (AD) caused by specific autoantibodies in the postsynaptic membrane of neuromuscular junctions. MG patients have a 22% risk of other ADs during their lifetime compared to 9% in the MG-free general population . PreviouThymectomy or thymomectomy is one of the most effective ways to treat MG , 4. The Our team's previous studies have found that surgical treatment of patients with thymoma accompanied by non-MG AD effectively relieves their condition . TherefoWe reviewed 460 patients who received MG treatment at Peking Union Medical College Hospital from 2000 to 2022. The inclusion criteria were MG with at least one AD and surgical treatment (thymectomy/thymomectomy). Patients were divided into thymoma and non-thymoma groups according to the presence or absence of thymoma. Preoperative MG was classified according to the Osserman classification . The patWe categorized the MG postoperative status into the following classifications: complete stable remission (remission without Prednisone), pharmacological remission (remission with medium/low dose Prednisone), stable, and depraved. In this study, we defined complete stable remission (remission without Prednisone) and pharmacological remission (remission with medium/low dose Prednisone) as improved.U tests were used to analyzing the correlation between groups. Clinical significance was set at p < 0.05.SPSS software (version 26.0) was used to perform rank correlation analysis. Pearson's correlation and Mann–Whitney In total, 33 patients were included in this study, including 11 males and 22 females, with a median age of 38 (15–76) years. Regarding the pathological diagnosis of the enrolled patients, 16 were diagnosed with simple thymic hyperplasia and 17 with thymoma. Two surgical approaches were used: median thoracotomy and video-assisted thoracoscopic surgery (VATS). There were no major perioperative complications or deaths in this group of patients. The median follow-up period in this review was 95 (5–235) months. At the last follow-up, only one patient died of septic shock nearly ten years postoperatively, and no recurrences or metastases existed in patients with a pathological diagnosis of thymoma.Among the 33 patients included, MG's Osserman classification was distributed from I to IIB, and different doses and frequencies of pyridostigmine bromide were used preoperatively to control symptoms. Some patients were also treated with a combination of hormones (prednisone/methylprednisolone/prednisone) before surgery. There were 30 patients with one AD and 3 with two ADs. Among the comorbid ADs, thyroid-related AD (TRAD) was the most common, with 17 patients having predominantly concomitant AD as TRAD, including Graves' disease, hyperthyroidism, hypothyroidism, and Hashimoto's thyroiditis. Besides TRAD, the other types of immune diseases included irritant dermatitis, pure red cell aplasia, allergic rhinitis, Henoch–Schönlein purpura, glomerulonephritis, connective tissue disease, asthma, primary biliary cirrhosis, and urticaria . However, this feature was not observed in the prognosis of autoimmune diseases . In addition, we found that patients with thymoma had significantly greater postoperative muscle weakness relief . In addition, we found that patients with larger thymoma had significantly greater postoperative MG relief . Using the Mann–Whitney U test, we found that among the enrolled patients, thymic hyperplasia was correlated with female gender (p = 0.049), younger age (p < 0.001), and TRAD (p < 0.001)  and associated with Osserman's type (p = 0.049) . We found that the longer the time after the operation, the better the relief of MG. In patients with thymoma, the larger the diameter of the tumor, the better is the postoperative MG improvement. Among the enrolled patients with thymus, those with hyperplasia were predominantly female and young. The most common comorbid autoimmune disorder was TRAD, which was associated with thymic hyperplasia, Osserman type I MG, and young age.


Before the Coronavirus Disease 2019 (COVID-19) pandemic in France, undocumented migrants had a higher risk than general population for being admitted to the intensive care unit (ICU) because of acute respiratory failure or severe infection. Specific data concerning the impact of COVID-19 on undocumented migrants in France are lacking. We aimed to analyze the mortality and respiratory severity of COVID-19 in this specific population.We retrospectively included all undocumented adult migrants admitted in French ICUs from March 2020 through April 2021 using the French nationwide hospital information system . We focused on admissions related to COVID-19. Undocumented migrants were compared to the general population, first in crude analysis, then after matching on age, severity and main comorbidities. The primary outcome was the ICU mortality from COVID-19. Secondary objectives were the incidence of acute respiratory distress syndrome (ARDS), the need for mechanical ventilation (MV), the duration of MV, ICU and hospital stay.p < 0.001). Although ICU mortality was comparable after matching , the incidence of ARDS was higher among undocumented migrants ) 1.25 (1.06–1.48); p = 0.01). Undocumented migrants needed more frequently invasive MV (OR (CI) 1.2 (1.01–1.42); p = 0.04 than general population. There were no differences between groups concerning duration of MV, ICU and hospital length of stay.During the study period, the rate of ICU admission among patients hospitalized for COVID-19 was higher for undocumented migrants than for general population (463/1627 (28.5%) vs. 81 813/344 001 (23.8%); During the first waves of COVID-19 in France, undocumented migrants had a mortality similar to the general population but a higher risk for ICU admission and for developing an ARDS. These results highlight the need for reinforcing prevention and improving primary healthcare access for people in irregular situation.The online version contains supplementary material available at 10.1186/s13613-023-01197-8. The part of undocumented migrants (UM) admitted to French intensive care units (ICUs) has been constantly increasing during the last decade  despite Our primary objective was to compare the ICU mortality from COVID-19 of undocumented migrants and general population. Secondary objectives were to compare the severity of the respiratory disease, as reflected by the rate of acute respiratory distress syndrome (ARDS), the need for invasive mechanical ventilation and the duration of MV, ICU and hospital stay.1st wave: from March 1st to May 31st 20202nd wave: from September 1st to December 31st 20203rd wave: from January 1st to April 30th 2021.All the adult patients admitted to French hospitals during the study period were identified using the French nationwide hospital information system , which collects administrative and medical information for all hospital stays regardless of hospital funding. Diagnosis-related groups are recorded according to ICD-10 criteria and life-supporting treatments according to the French classification of medical interventions (Classification Commune des Actes Médicaux). The study periods corresponded to the first months of COVID-19 pandemic in France, usually called “waves”:Only hospital stays in conventional ward  or intensive care unit were considered. Patients admitted for ambulatory care were excluded. In case of inter-hospital transfers , the 2 stays were considered as a single one with a duration equal to the sum of the hospitalization durations in the different hospitals. Consequently, a patient transferred from one ICU to another in a different hospital was considered as a single hospitalization resuming the information gathered during the two stays.Undocumented migrants and general population were identified into the study population using the French health insurance system. The agency within the statutory health insurance system that funds inpatient care is recorded as usual health insurance . For undocumented residents who entered France less than or at least 3 months earlier, the device is called, respectively, “urgent care” or “state medical aid”. State medical aid ensures a free takeover of all type of healthcare. Urgent care ensures a free takeover for acute healthcare only. Patients benefiting from both device can have free COVID-19 testing and treatments.All data are anonymized and are accessible to accredited researchers committed to following database-use guidelines .All patients admitted to the hospital during the study period were identified, and their health insurance status was recorded. Patients covered by the usual health insurance system formed the general population. Patients covered by either of the systems for undocumented residents  formed the undocumented migrants group. Patients not covered by any of the French statutory health insurance systems were excluded.Inside this study population, we focused on COVID-19 related hospitalizations. Patients hospitalized for COVID-19 were identified using the following ICD-10 codes: U07.10; U07.11; U07.14; U07.15. We then compared the characteristics and outcomes of patients admitted to the ICU for COVID-19 according to the undocumented migrants status .For each patient admitted to the ICU for COVID-19, we used standardized forms with extraction from nationwide hospital information system to collect the following data; admissions within the past year, admission from home or another hospital department, geographical location of the hospital, type of hospital , and some specific comorbidities from the modified Charlson Comorbidity Index  and ElixFor patients in the ICU, the evolution towards ARDS was noted. Life-supporting interventions such as high-flow nasal oxygen (HFNO), non-invasive ventilation (NIV), invasive mechanical ventilation (MV), septic shock, renal replacement therapy (RRT), extracorporeal membrane oxygenation, extracorporeal CO2 removal were derived from coding acts  and the need for invasive mechanical ventilation in ICU, duration of MV, ICU and hospital lengths of stay.t test with Welch’s correction if applicable for quantitative ones. We also computed standardized differences (SDs) which is an effect-size method to identify between-group differences that, unlike P values, are not influenced by sample size. Values greater than 0.10 are considered as clinically significant [We first performed a crude analysis on the whole population admitted in ICU comparing undocumented migrants to the general population. The data were described as mean ± SD or median [interquartile range] after assessment of distribution normality for quantitative variables and frequencies and percentages for qualitative ones. We used Chi-square test or Fisher exact test to compare both populations among qualitative variables and Student’s nificant .To study ICU admission rates, we performed a matched-control analysis among patients admitted in ICU. Migrants were matched to general population patients (exact matching of 1 migrant for up to 5 non migrant) on the following criteria: age (± 5 years), sex, Simplified Acute Physiology Score version II (SAPSII) without the age points (± 10), type of hospital, geographical area on the French territory (department), COVID-19 “wave”, COPD, comorbidities .Weights were applied to normalize the distribution of patients due to unequal matching numbers across matched migrants. Migrant people with no matched-control eligible were removed from the analysis. The remaining data formed the matched cohort, within which we compared the distribution of covariates between migrants and general population, by computing the SDs and taking into account weights.To assess associations between migrant status and outcomes, we fit Generalized Estimating Equation (GEE) models for repeated measures in the matched cohort, using logistic models for binary outcomes and linear log-normal-distributed models for quantitative outcomes. Associations were evaluated by computing odds ratios (ORs) for binary outcomes and back-transformed beta coefficients for quantitative outcomes, both with their 95% confidence intervals (95% CIs).® . All tests were two-sided, and P values smaller than 0.05 were considered to indicate statistically significant differences.The statistical analysis was performed using PROC GLIMMIX in SASFigure p < 0.001). The rate of ICU admission among COVID-19 patients was higher for undocumented migrants than for general population (463/1627 (28.5%) vs. 81.813/344.001 (23.8%); p < 0.001). Conversely, for patients not hospitalized for COVID-19, the rate of ICU admission was lower for undocumented migrants than for general population (1.303/38.099 (3.4%) vs. 416.449/5.774.239 (7.2%); p < 0.001).During the study period, 4.1% (1.627/39.726) of the hospitalizations among undocumented migrants were due to COVID-19 as compared to 5.6% (344.001/6.118.240) in the general population . The incidence of ARDS was higher among undocumented migrants ) 1.25 (1.06–1.48); p = 0.01). Undocumented migrants needed more frequently invasive MV (OR (CI) 1.2 (1.01–1.42); p = 0.04 than general population whereas general population had a more frequent recourse to NIV . There were no differences between groups concerning duration of MV, ICU and hospital length of stay.Patient’s outcomes are detailed in Table The incidence of septic shock and acute kidney failure with the need for renal replacement therapy were comparable in the general population and in migrants (458 (27.2%) vs. 121 (28.7%) patients; SMD = 0.0354 and 128 (7.9%) vs.33 (7.8%) patients; SMD = 0.0003).We describe here the first series specifically focusing on undocumented migrants’ critical illness during the 1st waves of COVID-19 in France. Our results show that undocumented migrants had a mortality similar to the general population but were more frequently admitted to the ICU and developed more ARDS.Our primary objective was to determine if undocumented migrants exhibited an excess of mortality from COVID-19 as compared to general population. This had been suggested by a previous French report  with a sWe also aimed to compare the severity of the respiratory disease, as reflected by the rate of ARDS and the need for invasive MV. We found that, even after adjustment, undocumented migrants had a higher incidence of ARDS, which was described in only one Koweitian series before ours . A possiIn our cohort, undocumented migrants had higher rates of ICU admission. This was described in other European Union countries , 6 with The main limitation of our study is its retrospective design. Furthermore, differences in region of birth, educational level, economic status, reasons for leaving the home country make the undocumented migrants population a very heterogeneous one, calling for caution when extrapolating results. Due to the study design, we could not collect some data of importance such as ICU non-admission decisions, especially at the beginning of pandemic when the strain on the healthcare system was particularly high, or specific SARS-CoV-2 treatments. Lastly, we did not report vaccination status. A reduced access to COVID-19 vaccines has been shown among migrants . HoweverDuring the first waves of COVID-19 in France, undocumented migrants had a mortality similar to the general population but a higher risk for ICU admission and for developing an ARDS. These results highlight the need for reinforcing prevention and improving primary healthcare access for people in irregular situation.Additional file 1. French Classification of Medical Acts (CCAM: Classification Commune des Actes Médicaux) for ICU supportive therapies.


Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin  were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available.To assist in the advancement of the large-scale production of safe IMPORTANCEMycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies. Mycoplasma pneumoniae, have also become important cellular models for systems biology , beef heart infusion and peptones (PPLO), pancreatic digest of casein, beef extract broth, or animal serum. The use of animal serum in culture medium entails several disadvantages, including low batch-to-batch reproducibility, undefined composition, possible interference with downstream processing, and a considerably increased cost of the medium. In addition, the use of both animal serum and raw materials derived from animals has important safety concerns, since these may contain viruses, antibiotics, endotoxins, and other bioactive molecules. Therefore, the availability of a culture medium that (i) is safe and free of animal components, (ii) supports robust growth, and (iii) allows serial passaging is fundamental in Mycoplasma vaccine development or other Mycoplasma-based therapies  or totally free of animal-derived components (vB13), capable of supporting growth of up to 60 to 70% of the biomass obtained with rich medium. The new formulations allowed serial passaging and maintained a cell physiology state without major proteomic or transcriptional changes. We also performed single-omission experiments to define strictly essential components required for growth, suggesting a minimal medium formulation that could also assist certain metabolic studies. Finally, we evaluated the performance of the developed medium in Mycoplasma hyopneumoniae, which is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs for which inactivated whole-cell vaccines are commercially available  a serum-free medium (vB10) containing bovine serum albumin (BSA) as a lipid carrier and PPLO and (ii) an animal component-free medium (vB13) in which BSA and complex animal extracts were completely removed . DependiM. pneumoniae possesses a low number of amino acid permeases and transporters, but possesses a peptide importer operon (oppBCDF) as well as aminopeptidases, suggesting that peptides may be an important source of amino acids , perhaps due to its role in oxidative defense . Howeverecursors . While Recursors . These rM. pneumoniaein vivo , which results in the formation of the glycolytic intermediate dihydroxyacetone phosphate and hydrogen peroxide  and the FA kinase MPN547 (FakA)  . We reas7 (FakA) . Support lipases  and a su lipases . In facte medium , 44–46. d medium , suggestd medium . Strikind medium . AlthougMycoplasma capricolum culture  is essential in M. pneumoniae, suggesting that this bacterium is limited on amino acid transporters and depends on the peptide import system  medium by adding each component individually and a set of CMRL variants lacking a specific group of compounds, namely, amino acids, vitamins, inorganic salts, nucleotide precursors, and other components (listed in Table S2). As expected, substitution of the commercial CMRL present in vB13 with the hm-CMRL resulted in a similar growth performance . Except t system . Neverth2 tissue culture flasks. Cultures were started with a 1:200 dilution of inoculum, and growth was monitored over time by measuring both the protein and DNA biomass during 96 h of culture. The two quantification methods showed that growth performance was very similar in the vB10  and protein (r = 0.89) levels . Differences in gene expression were larger at the proteomic levels, but not statistically significant . Although quality assessment of the final bacterial product is required, these results show a minor impact in gene expression, supporting vB13 medium as a suitable and potential formulation for the production of M. pneumoniae-based therapies.To examine the physiological effect of growing ) levels . At the Mycoplasma growth or of nutritional requirements might differ among different species. To test this, we assessed the performance of vB13 to grow M. hyopneumoniae, for which inactivated whole-cell vaccines are available . Although M. hyopneumoniae can ferment glucose, its metabolism produces less lactic acid than M. pneumoniae. Consequently, we removed HEPES, allowing us to assess qualitative growth by monitoring color change.We wondered whether this medium formulation could be universal for vailable –30. We failable –. TherefoM. hyopneumoniae growth performance (M. hyopneumoniae lacks the thymidylate synthetase gene (thyA), suggesting that synthesis of dTMP may depend on the activity of the thymidine kinase (tdk)  and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), which improved culture consistency. Finally, we found that the addition of extra vitamins impaired growth  . In factactivity . Since Re of BSA . Similard growth . OverallM. hyopneumoniae vaccine production upon the process of industrial optimization.To further assess growth performance, we scaled up the growth conditions to 30 mL and performed growth curve analyses by measuring DNA biomass over time. As shown in M. pneumoniae. We provide several medium formulations that can be chosen depending on the application, including a medium composition free of animal components. We showed that depending on the Mycoplasma species, further optimization might be required, as illustrated by the cultivation of M. hyopneumoniae, an important swine pathogen with available vaccines. The results presented in this study could assist in the large-scale production of Mycoplasma-based therapies in the absence of animal components upon industrial optimization, contributing to the development of safer and cheaper products.In this study, we developed a serum-free medium capable of supporting robust growth of M. pneumoniae strain M129 and the nonpathogenic strain J of M. hyopneumoniae were cultivated in different serum-free medium formulations and compared to modified Hayflick , and the pellet was resuspended in 30 mL of fresh medium. Finally, the cell suspension was stored at −80°C in aliquots of 400 μL until needed.For For each experiment, an aliquot was thawed to avoid repeating freezing and thawing cycles. To measure the inoculum concentration, one aliquot was used to quantify the protein biomass using the Pierce bicinchoninic acid (BCA) protein assay kit.M. pneumoniae, we established a workflow method to monitor the growth of multiple medium formulations in a 96-well-plate format incubated in a Tecan Spark plate reader at 37°C and atmospheric CO2 conditions. A duplicate plate was cultured in parallel under standard growth conditions using a cell culture incubator set at 37°C and 5% CO2. Each medium was tested at least in duplicate wells containing 200 μL of medium inoculated with 1.5 μg of a frozen stock as starting inocula (approximately 1:100 dilution). Medium performance was monitored by growth curve analyses based on the growth index method previously described . Then, the protein biomass for each well lysate was measured in duplicate using the Pierce BCA protein assay kit.To optimize the formulation of the serum-free medium in M. hyopneumoniae, even using shaking conditions. Determination of cell biomass gain by protein quantification also resulted in high background signals, likely due the nonadherent nature of this bacterium. Consequently, we set up the following culture conditions to estimate growth and aid the medium optimization process. For each experiment, 3 culture replicates of 4 mL each containing the tested medium or Friis medium were inoculated with frozen stocks (1:200 dilutions). Cultures were incubated at 37°C in an orbital shaker (180 rpm) in 15-mL conical tubes, and the total DNA biomass was estimated after 96 h of culture as follows. The 4-mL bacterial culture was harvested by centrifugation , and the pellet was directly lysed with 300 μL of tissue and cell lysis solution plus proteinase K from the MasterPure DNA purification kit (Lucigen). DNA was extracted following the recommendations of the kit manufacturer, and DNA was measured using a fluorometric method using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Invitrogen).Culture conditions in the 96-well-plate format were not optimal for growing M. pneumoniae and M. hyopneumoniae is detailed in Table S1 and Table S5, respectively.The formulation of different medium versions for M. pneumoniae and compared to Hayflick rich medium as follows. Several tissue culture flasks of 25 cm2 containing 5 mL of medium were inoculated with frozen stocks (1:200 dilution) and incubated at 37°C and 5% CO2. Protein and DNA biomass were then measured at different time points  as follows. For each time point, cells were scraped from the flasks in the culture medium, and 1 mL of cell suspension was harvested by centrifugation . This was performed in duplicate to obtain samples for both protein and DNA measurements. For protein biomass quantification, the cell pellet was washed twice with PBS and lysed with 100 μL of lysis buffer  prior to duplicate protein measurements using the Pierce BCA protein assay kit. For DNA biomass quantification, the cell pellet was directly lysed, and the DNA was extracted using the MasterPure DNA purification kit (Lucigen) following the recommendations of the kit manufacturer. Finally, extracted DNA for each time point was measured using a fluorometric method using the Qubit dsDNA HS assay kit (Invitrogen).Growth curve analysis of vB10 and vB13 medium formulations was performed in larger volume cultures for M. hyopneumoniae, 125-mL Erlenmeyer flasks containing 30 mL of medium were inoculated in triplicate with frozen stocks (1:200 dilutions) and incubated at 37°C in an orbital shaker set at 180 rpm. Then, 1 mL of culture sample was collected at different time points , and cells were harvested by centrifugation  prior to DNA quantification following the methods described above.To evaluate in more detail the performance of certain medium formulations for 2 containing 5 mL of medium were grown for 3 to 5 days. Then, cells were scraped from the flasks into the same culture medium and 200 μL of cell suspension diluted in 5 mL of fresh medium to start a new culture. This process was repeated for up to 10 consecutive passages.To test whether the vB10 and vB13 medium formulations could support growth after consecutive passages, we performed passaging experiments as follows. Cultures in tissue culture flasks of 25-cmM. pneumoniae was grown in duplicate in tissue culture flasks containing 5 mL of Hayflick medium or animal component-free medium vB13. After 72 h of culture at 37°C and 5% CO2, the culture medium was changed with fresh medium, and the cells were further incubated for 6 h prior to RNA extraction. RNA was isolated using the miRNeasy kit (Qiagen) following the manufacturer’s instructions, including the in-column DNase I treatment. The quality of RNA (amount and integrity) was assessed using a BioAnalyzer device (Agilent), and the transcriptome sequencing (RNA-seq) libraries were prepared at the CRG Genomics Unit using the TruSeq stranded mRNA sample prep kit v2 (p kit v2 . SequencM. pneumoniae M129 (NCBI accession no. NC_000912.1). Differential expression analysis was performed using edgeR v3.12.1 .Processing of sequencing reads was performed as described  using th v3.12.1 –59 with v3.12.1 –, and folM. pneumoniae was grown in duplicate in tissue culture flasks of 75 cm2 containing 20 mL of Hayflick medium or animal component-free medium vB13. After 72 h of culture at 37°C and 5% CO2, the culture medium was changed with fresh medium, and the cells were further incubated for 6 h. At this point, cells were washed twice with PBS, scraped from the flasks, and centrifuged at 13,100 × g for 10 min. The pellet was resuspended in lysis buffer  and disrupted using a Bioruptor sonication system (Diagenode) with an on/off interval time of 30/30 s at high frequency for 5 min. Cell lysates were spun, and the extracted protein was quantified using the Pierce BCA protein assay kit. Finally, samples were digested with trypsin using the filter-aided protocol (FASP), and they were analyzed using an LTQ-Orbitrap Velos Pro mass spectrometer  coupled to an EASY-nLC 1000 . Peptides (1 μg) were loaded onto the 2-cm NanoTrap column with an inner diameter of 100 μm packed with C18 particles of 5-μm particle size (Thermo Fisher Scientific) and were separated by reversed-phase chromatography using a 25-cm column with an inner diameter of 75 μm, packed with 1.9-μm C18 particles . Chromatographic gradients started at 93% buffer A (0.1% formic acid in water) and 7% buffer (0.1% formic acid in acetonitrile) B with a flow rate of 250 nL/min for 5 min and gradually increased to 65% buffer A and 35% buffer B in 120 min. After each analysis, the column was washed for 15 min with 10% buffer A and 90% buffer B.m/z 445.1200. The acquisition was performed in data-dependent acquisition (DDA) mode, and full mass spectrometry (MS) scans with 1 microscan at a resolution of 60,000 were used over a mass range of m/z 350 to 2,000 with detection in the Orbitrap. Auto gain control (AGC) was set to 1E6, and dynamic exclusion (60 s) and charge state filtering disqualifying singly charged peptides were activated. In each cycle of DDA analysis, following each survey scan, the top 20 most intense ions with multiple-charge ions above a threshold ion count of 5,000 were selected for fragmentation. Fragment ion spectra were produced via collision-induced dissociation (CID) at a normalized collision energy of 35%, and they were acquired in the ion trap mass analyzer. AGC was set to 1E4, and an isolation window of 2.0 m/z, an activation time of 10 ms, and a maximum injection time of 100 ms were used. All data were acquired with Xcalibur software v2.2. Digested bovine serum albumin (New England Biolabs) was analyzed between each sample to avoid sample carryover and to ensure stability of the instrument. QCloud  mass analyzer prior to the analyses, and an internal calibration was performed using the background polysiloxane ion signal at . QCloud  was usedM. pneumoniae open reading frames (ORFs) of >19 amino acids . For peptide identification, a precursor ion mass tolerance of 7 ppm was used for the MS1 level, trypsin was chosen as the enzyme, and up to three missed cleavages were allowed. The fragment ion mass tolerance was set to 0.5 Da for MS2 spectra. Oxidation of methionine and N-terminal protein acetylation were used as variable modifications, whereas carbamidomethylation on cysteines was set as a fixed modification. The false-discovery rate (FDR) in peptide identification was set to a maximum of 5%. Peptide quantification data were retrieved from the “precursor ion area detector” node of Proteome Discoverer (v2.0) using 2 ppm mass tolerance for the peptide extracted ion current (XIC). The obtained values were used to estimate the protein abundances with the average of the three most intense unique peptides per protein. Fold changes (log2) were calculated for those proteins detected under both growth conditions, and a two-tailed t test assuming unequal variances was performed when signal was detected in all replicates. Multiple-test correction was applied using the Benjamini-Hochberg method .Acquired spectra were analyzed using the Proteome Discoverer software suite  and the Mascot search engine  . The datg method , and folE-MTAB-10111 and E-MTAB-12308 for samples grown in Hayflick and vB13 animal component-free medium, respectively. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (PXD037431.The RNA sequencing data have been deposited in the ArrayExpress repository with the data set identifiers he PRIDE  partner