Background: Adipose tissue engineering may provide 3D models for the understanding of diseases such as obesity and type II diabetes. Recently, distinct adipose stem/stromal cell (ASC) subpopulations were identified from subcutaneous adipose tissue (SAT): superficial (sSAT), deep (dSAT), and the superficial retinacula cutis (sRC). This study aimed to test these subpopulations ASCs in 3D spheroid culture induced for adipogenesis under a pro-inflammatory stimulus with lipopolysaccharide (LPS).Methods: The samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery (Protocol 145/09).Results: ASC spheroids showed high response to adipogenic induction in sSAT. All ASC spheroids increased their capacity to lipolysis under LPS. However, spheroids from dSAT were higher than from sSAT (p = 0.0045) and sRC (p = 0.0005). Newly formed spheroids and spheroids under LPS stimulus from sSAT showed the highest levels of fatty acid-binding protein 4 (FABP4) and CCAAT/enhancer-binding protein-α (C/EBPα) mRNA expression compared with dSAT and sRC (p < 0.0001). ASC spheroids from sRC showed the highest synthesis of angiogenic cytokines such as vascular endothelial growth factor (VEGF) compared with dSAT (p < 0.0228). Under LPS stimulus, ASC spheroids from sRC showed the highest synthesis of pro-inflammatory cytokines such as IL-6 compared with dSAT (p < 0.0092).Conclusion: Distinct physiological properties of SAT can be recapitulated in ASC spheroids. In summary, the ASC spheroid from dSAT showed the greatest lipolytic capacity, from sSAT the greatest adipogenic induction, and sRC showed greater secretory capacity when compared to the dSAT. Together, all these capacities form a true mimicry of SAT and hold the potential to contribute for a deeper understanding of cellular and molecular mechanisms in healthy and unhealthy adipose tissue scenarios or in response to pharmacological interventions. Adiposeironment .In a 3D cell culture, the spatial arrangement of cell-to-cell and cell-to-extracellular matrix has a positive impact on crucial cell processes such as proliferation, differentiation, and various other cellular signaling events . ASCs hain vitro fat-on-a-chip 3D systems may be produced with critical features for functional assays of adipokine secretion, glucose uptake, and lipolysis  resident immune cell populations have contributed to immune–metabolic research  and to tipolysis .Recently, our research group described distinct ASC subpopulations dwelling in distinct subcutaneous adipose tissue (SAT) microenvironments: superficial (sSAT), deep (dSAT), and the superficial retinacula cutis (sRC) . These Ain vitro   supplemented with 2% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin (Sigma). The ASC monolayer was maintained in culture until reaching confluence, with medium changes made every 3 days. Subsequently, the monolayer was released from the culture plastic with 0.125% trypsin (Gibco) and 0.78 mM ethylenediamine tetraacetic acid (Gibco) to prepare ASC spheroids for adipogenic induction and further analysis. All ASC spheroids were formed from the ASC monolayer at the second passage. One representative donor of the adipose tissue samples was included in all analyses.Abdominal SAT was obtained during aesthetic plastic surgeries in Brazil , as described previously (n = 6) . The exc6 cells were seeded into the chamber of the non-adhesive hydrogel resuspended in a medium composed of DMEM supplemented with 50 ug/mL ascorbic acid , 1.25 ug/mL human albumin , 100 ug/ml penicillin and 100 ug/ml streptomycin, and ITS 1X (Lonza). For adipogenic induction, ASC spheroids were maintained in DMEM supplemented with 50 ug/mL ascorbic acid , 1.25 ug/mL human albumin (Farma Biagini SPA), 100 U/ml penicillin and 100 ug-ml streptomycin, and ITS 1X , and the components of adipogenic induction were 10 μM insulin  0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, and 200 μM indomethacin . At week 1 of adipogenic induction, the supplementation of the medium was changed, and only 10 μM insulin  was maintained as a component of adipogenic induction for up to 5 weeks. A part of ASC spheroids induced for adipogenesis was maintained under 0.5 ug/mL lipopolysaccharide (LPS) .ASC spheroids were produced using micro-molded non-adhesive hydrogel (2% agarose in NaCl 0.9%) with 800 μm diameter in each of the 81 circular recesses  following the manufacturer’s protocol. Approximately 2 × 10The diameter of the spheroids was measured from the newly formed spheroids at weeks 1 and 2 of adipogenic induction and under LPS stimulus using an optical microscope (Leica DMI 6000 B) equipped with a Leica DF 500 digital camera as described . We randLDH release was determined using the commercial non-radioactive colorimetric CytoTox 96 Cytotoxicity Kit . The assay was performed from the spheroids’ culture supernatant. Briefly, 30 μL of the supernatant was transferred to a 96-well plate, followed by the addition of 30 μL of the substrate. After 20 min of incubation at room temperature and in the absence of light, 30 μL of the stop solution was added to each well. The intensity of staining was proportional to the number of cells with plasma membrane disruption. The optical density (OD) was measured using a Biotek Synergy H4 microplate reader  at 490 nm. The assay was performed using eight replicates from the supernatant of 162 spheroids obtained from two independent experiments.At week 5 of adipogenic induction, ASC spheroids from sSAT, sRC, and dSAT were washed in PBS and were transferred to 4% paraformaldehyde in PBS at room temperature for 1 h. Subsequently, induced ASC spheroids were washed in PBS and placed in successive baths of 15% and 30% sucrose solution at room temperature for 24 h each. The ASC spheroids were embedded in optimal cutting temperature  and maintained in a −80°C freezer until sectioning. Sections of 10 μm were obtained using a cryostat (Leica DMI 6000 B) and subsequently collected onto 0.01% poly-L-lysine-coated slides (Sigma). The slides with the sections were stored in a freezer at −20°C until staining. Cryosections were left at room temperature for 15 min and stained with 1 mg/mL Nile red (Sigma) diluted (1:50) in PBS. The cell nucleus was stained with 0.5 μg/mL Hoechst. The images were obtained with the aid of a fluorescence microscope (Leica DMI 6000B)  with LAS AF software . The laser microscope was programmed to stimulate at a range of 640–720 nm. Two independent analyses were evaluated, with a total of 324 spheroids of each sample obtained from four independent experiments.The lipolysis assay on ASC spheroids from sSAT, sRC, and dSAT was performed using the Lipolysis Colorimetric Assay Kit (Sigma), according to the manufacturer’s instructions. Briefly, the first step consisted of preparing the standard curve. The samples (ASC spheroids) were washed with PBS and subsequently washed twice with 200 𝜇L of Lipolysis Wash Buffer. After washing, 150 𝜇L of Lipolysis Assay Buffer was added. The reaction mix was prepared and added to each well and mixed using a horizontal plate shaker and incubated for 30 min at room temperature in dark. The absorbance was measured at A570 excitation through the espectrofotômetro Synergy H4 hybrid reader. Three independent analyses in triplicate were evaluated with a total of 81 spheroids of each condition of each sample obtained from three independent experiments.vs. newly formed), ASC spheroids at week 1 of adipogenic induction were relativized with ASC spheroids at week 5 of adipogenic induction (fold change week 1 of adipogenic induction vs. week 5), and ASC spheroids of adipogenic induction were relativized with ASC spheroids of adipogenic induction under LPS at week 5 (fold change of adipogenic induction vs. LPS) to compare the difference between each pair of groups. Two independent analyses were evaluated in triplicate for each gene. RNA samples were isolated from 162 spheroids of each sample obtained from four independent experiments.The expression levels of fatty acid-binding protein 4 (FABP4) and CCAAT/enhancer-binding protein-α (C/EBPα) genes of ASC spheroids from sSAT, sRC, and dSAT were measured on the newly formed spheroids, at weeks 1 and 2 of adipogenic induction and under LPS stimulus, by quantitative polymerase chain reaction (qPCR). The RNeasy Mini Kit  was used for RNA extraction according to the manufacturer’s instructions. The qPCR was performed using the AgPath-ID ™ one-step RT-PCR kit . Briefly, 1.5 μl total RNA (15 ng/μl) and amplified Master Mix composed of 0.4 μL of 25x RT-PCR enzyme mix, 0.67 μl of detection enhancer, and 5 μL of 2x RT-PCR buffer and completed with RNase-free water to a final volume of 10 μL of the Master Mix solution. Subsequently, we performed the analysis of genes using specific primers and specific TaqMan probes . The analyzed genes were FABP4 (HS01086177_m1) and CEBPα (HS00269972_m1). Ribosomal protein L (RBL) (Hs99999902_m1) was set as the reference gene. For data analysis, the quantification cycle (Cq) value was determined, and specific gene expression was normalized to the reference gene using the ΔΔCq method. ASC spheroids at week 1 of adipogenic induction were relativized with the newly formed ASC spheroids . After 24 h of culture medium change to remove adipogenic inducers and LPS, the supernatant of all spheroid samples was harvested and frozen at −80°C. The determination of proteins was carried out using the Luminex xMAP technology based on a magnetic beads panel for recognition of human MIP-1β, IFN-y, interleukin-1ra (IL-1ra), IL-5, GM-CSF, TNFα, RANTES, IL-2, IL-1β, Eotaxin, bFGF, VEGF, PDGF-BB, IP-10, IL-13, IL-4, MCP-1, IL-8, MIP-1a, IL-10, G-CSF, IL-15, IL-7, IL-12p70, IL-17ra, and IL-9  using the instrument Bio-Plex MAGPIX (Bio-Rad Laboratories Inc.). The concentration of each analyte was quantified using the software xPONENT v3.1 . The results were expressed in picograms per milliliter (ρg/ml). One independent analysis was evaluated in quadruplicate from 162 spheroids of each sample obtained from four independent experiments.p < 0.05. GraphPad Prism 6.0 software was used .To compare the data between sSAT, sRC, and dSAT in the analysis of diameter, the one-way ANOVA analysis test  was used followed by multiple comparisons post-test (Dunn’s test). The Omnibus normality test by D'Agostino and Pearson revealed that the lipolysis assay data are normally distributed, so the one-way ANOVA analysis test was performed followed by Sidak’s multiple comparisons test. To compare the data between sSAT, sRC, and dSAT in the PCR analysis, the two-way ANOVA analysis test was performed followed by the Tukey’s multiple comparisons test and in the multiplex analysis, the one-way ANOVA analysis test was performed followed by the Dunn’s multiple comparisons test. To compare the control group and the induced group or the group stimulated with LPS with the group not stimulated with LPS of each tissue—sSAT, sRC, and dSAT— Student’s t-test was used. The results in the graphs were realized with 95% confidence interval  and expressed as mean ± standard deviation. Differences were considered statistically significant when p = 0.0001) or presence of LPS stimulus (p < 0.0001) (p = 0.0348) and dSAT (p = 0.0464) showed a decrease in their sphericity at week 5 of adipogenic induction exclusively under LPS stimulus compared with the newly formed spheroids  . Phase c 0.0001) . The ASC 0.0001) . The ASCpheroids .The induced ASC spheroids from sSAT, sRC, and dSAT showed similar levels of LDH in the absence or presence of LPS , revealiThe induced ASC spheroids from sSAT showed a majority of unilocular cells with a more rounded morphology (arrow), with a preferential location in their center as well as cells located in the periphery more fibroblastic (arrowhead) . Inducedp = 0.0268), sRC (p = 0.0157), and dSAT (p = 0.0116) showed an increase in their lipolytic capacity compared with spheroids maintained in the absence of LPS. Interestingly, the induced ASC spheroids from dSAT under LPS stimulus showed the highest lipolytic capacity compared with sSAT (p = 0.0045) and sRC (p = 0.0005)  .FABP4 and CEBPα gene expression in ASC spheroids from sRC and dSAT compared with newly formed spheroids from the respective samples (p < 0.0001) (p < 0.0001)  . Curious 0.0001) .p < 0.0001). Interestingly, only ASC spheroids from sSAT showed an increase up to 5 million-fold in their gene expression . At week 5, the highest secretion was detected for CCL10 compared with spheroids from dSAT (p < 0.0257). At weeks 1 and 5 of adipogenic induction, VEGF secretion in ASC spheroids from sRC was higher than that from dSAT . No difference was found in the secretion of IL-2, IL-5, and IL-7 among the three types of ASC spheroids, as well as no difference in secretion at weeks 1 and 5 of induction (data not shown).The induced ASC spheroids from sSAT, sRC, and dSAT significantly decreased the secretion at week 5 of adipogenic induction of the following cytokines: IL-1Rα , IL-6 F, IL-8 F, IL-12p7p < 0.001, IL-8 .Induced ASC spheroids from sRC showed higher secretion compared with sRC in the absence of LPS stimulus of IL-6  p < 0.0001, IL-8  p < 0.001, IL-10  p < 0.04 IL-2P70  p < 0.016, MCP-1  p < 0.0158, VEGF  p < 0.0001, CCL5  p < 0.055, CCL10  p < 0.00nd CCL11  p < 0.05the IL-6  p < 0.00IL-12p70  p < 0.002, MCP-1  p < 0.0172, VEGF  p < 0.0228, CCL5  p < 0.00nd CCL10  p < 0.00Recently, we showed, for the first time in scientific literature, the presence of ASCs in a poorly explored human SAT Q11 microenvironment, the superficial retinacula cutis (sRC) . In thisin vivo (Under stimulus, the ASC spheroids undergo proliferation and differentiation processes, triggering migration and selection events that can grow in a different structure, which leads to a symmetry break. In this way, these spheroids can acquire a certain degree of architectural complexity to mimic the organization of organs in vivo . Inducedin vivo , minibrain vivo , or chonin vivo . On the in vivo . SimilarThe scientific literature describes visceral adipose tissue as being more lipolytic than SAT . The SATDuring adipogenic differentiation, the master regulator genes PPARγ and CEPBα are expressed in the stage in which cells accumulate lipids in their cytoplasm as unilocular or multilocular droplets. Both the genes induce transcription of many adipocyte genes and play a role in maintaining the adipocyte phenotype . SurprisIn this study, the ASC spheroids were stimulated with LPS to mimic an inflammatory condition observed in some diseases such as obesity. Interestingly, we observed that all ASC spheroids showed higher expression of FABP4 and CEPBα when exposed to LPS stimulus. Furthermore, induced ASC spheroids from sSAT maintained their higher expression of CEPBα and FABP4 compared with sRC and dSAT. The systemic inflammatory scenario inhibits adipogenic differentiation in obese patients , mainly The cytokine IL-6 is a pleiotropic inflammatory cytokine , for whiIn addition, the inflammatory stimulus provided by LPS triggered an increase in VEGF, CCL10, and CCL11 secretion of ASC spheroids from sRC. TNF-α increases the expression of the chemokine CCL5 and its receptor CCR5 in adipose tissue  and in tin vivo studies are needed to attest this hypothesis.Interestingly, besides MCP-1, the induced ASC spheroids from sRC showed higher secretion of IL-6 and IL-12p70 under LPS stimulus compared with dSAT. Stromal cells, including ASCs, when exposed to an inflammatory scenario, have the ability to polarize, moving from an anti-inflammatory to a pro-inflammatory phenotype (Silva et al.; The main limitation of the study was that all experiments were carried out with one representative donor adipose tissue sample. The pilot experiments were performed with samples from more than one donor; however, it was observed that the samples presented the same profile of biological responses. This homogeneity of samples is probably due to the exclusion criteria used in this study.ASCs from sSAT, sRC, and dSAT were able to self-assemble to generate spheroids in a scalable model of the 3D cell culture. Compared to the dSAT, ASC spheroids from sSAT and sRC are in favor of stimulating adipogenesis and angiogenesis, respectively. While the inflammatory stimulus triggers lipolysis in all ASC spheroids, dSAT is the most lipolytic subpopulation. Together, all these capacities result in a true mimicry of SAT and hold the potential to contribute for a deeper understanding of cellular and molecular mechanisms in healthy and unhealthy scenarios in response to drug stimulus. This 3D spheroid model also opens opportunities for evaluating individual responses in personalized medicine. Last, but not the least, the present study contributes and emphasizes the importance of considering the superficial and deep subcutaneous adipose tissue as two distinct adipose tissue depots and also revealed the potential relevance of the retinacula cutis, a cell niche of SAT not yet well-studied in the scientific literature.


For patients with T2aN0 stage IB lung adenocarcinoma, benefits of adjuvant chemotherapy remain controversial. Here, we aimed to evaluate such benefits.This retrospective cohort study was conducted on the database of the National Taiwan Cancer Registry. We analyzed patients with T2aN0 stage IB lung adenocarcinoma (re-classified by AJCC 8th edition) diagnosed during the period from January 2011 to December 2017. They were divided into two groups: (1) group 1: tumor <=3 cm with visceral pleural invasion (VPI); (2) group 2: tumor >3 cm, but <=4 cm. Overall survival (OS) and cancer specific survival (CSS) were evaluated. Risk factors for survival were determined.A total of 2,100 patients with T2aN0 stage IB lung adenocarcinoma  were enrolled for study. The proportions of patients receiving adjuvant chemotherapy in group 1 and 2 were 39.1% and 68.6%, respectively. Amongst group 1 patients, adjuvant chemotherapy was not an independent risk factor for OS and CSS. Amongst group 2 patients, high-grade histologic findings and receiving sublobar resection were two risk factors for poorer survival. Adjuvant chemotherapy was also associated with an OS (adjusted hazard ratio (aHR), 0.52; 95% confidence interval (CI), 0.38-0.72; P<0.001) and CSS  benefit regardless of the presence or absence of risk factors.For patients with T2aN0 stage IB lung adenocarcinoma, adjuvant chemotherapy improved OS and CSS in those with tumors >3 cm, but <=4 cm.For patients with tumors <=3 cm with VPI, adjuvant chemotherapy had no survival benefit. Lung cancer is by far the leading cause of cancer-related death . CompletSeveral randomized clinical trials reported the efficacy of adjuvant chemotherapy following surgery in patients with resectable lung cancer –8. The pth edition  performance status, histologic types, tumor size, tumor stage, smoking status, histologic grade, visceral pleural invasion (VPI), extent of resection, adjuvant treatment, status of N2 stations dissection, and types of health care institution. Sublobar resection refers to wedge resection and segmentectomy. Histologic grade was grouped into low grade (well or moderately differentiated) and high grade (undifferentiated or poorly differentiated). The staging system of lung cancer before 2018 was conducted according to the AJCC staging system 7 edition .th edition  is the length of time from the date of cancer diagnosis to the date of death due to any cause, or to the date of last follow-up. Cancer-specific survival (CSS) is the length of time from the date of cancer diagnosis to the date of death from the disease. Disease-free survival (DFS) is the length of time from primary treatment for the cancer to the date of disease recurrence or death. Survival status was determined based on the national death certificate database from the Department of Statistics, Ministry of Health and Welfare, Taiwan, and the status was updated until December 31, 2020. OS and CSS of patients were estimated using the Kaplan–Meier method, whereas the inter-group differences were assessed using the stratified log-rank test. Associations between clinicopathologic variables and outcomes were assessed using Cox proportional hazards regression model. The strength of association was presented as the Hazard ratio (HR) and 95% confidence interval (CI). In this study, we used the two-tailed test, and the significant level was set at P <0.05. All analyses were performed using SAS, version 9.4 statistical software (SAS Institute Inc).We analyzed 2,100 patients with T2aN0 stage IB lung adenocarcinoma. There were 1,265 (60.2%) patients having tumors 3 cm or less, and 835 (39.8%) patients having tumors larger than 3 cm, but 4 cm or less in size. Amongst patients with tumors 3 cm or less, 495 (39.1%) patients received adjuvant chemotherapy, whereas patients with tumors larger than 3 cm, but 4 cm or less in size, 573 (68.6%) received adjuvant chemotherapy. The details of the patient characteristics were shown in Group 1: tumor <=3 cm with VPIThe characteristics of patients with tumors 3 cm or less is shown in Group 2: tumor >3 cm, but <=4 cmThe characteristics of patients with tumors larger than 3, but 4 cm or less in size are shown in Group 1: tumor <=3 cm with VPIIn the multivariable Cox proportional hazard model, age >65 to 74 years old, tumor size larger than 2 cm, but <=3 cm, and being treated in regional hospitals were identified as independent prognostic factors for OS Table 2AGroup 2: tumor >3 cm, but <=4 cmIn the multivariable Cox proportional hazard model, age >65 to 74 years old, high grade histologic findings, smoking habit, and received adjuvant chemotherapy were identified as independent prognostic factors for OS. Receiving sublobar resection was identified as a prognostic factor in the univariate analysis Table 2BGroup 1: tumor <=3 cm with VPIAs shown in In group 1, OS and CSS between patients with and without adjuvant chemotherapy were similar . Amongst patients with tumors 2 cm or less, adjuvant chemotherapy was not associated with improved DFS . Amongst patients with tumors larger than 2 cm, but <=3 cm, we again found no benefit of adjuvant chemotherapy on DFS .With regard to group 2, adjuvant chemotherapy was not associated with improved DFS . Amongst patients with risk factor, adjuvant chemotherapy was not associated with improved DFS . Amongst patients without risk factor, we found no benefit of adjuvant chemotherapy on DFS .In this study, we found that the benefit of adjuvant chemotherapy was associated with tumor size amongst patients with T2aN0 stage IB lung adenocarcinoma. Adjuvant chemotherapy improved survival for those with tumors larger than 3 cm, but 4 cm or less in size. For patients with tumors 3 cm or less with VPI, adjuvant chemotherapy had no survival benefit.Tumor size is a topic of research in predicting the benefit for resectable lung cancer patients treated with adjuvant chemotherapy. For tumors larger than 4 cm, a survival advantage has been reported in association with adjuvant chemotherapy , 19, 25.For tumors 3 cm or less with VPI, we found no survival benefit with adjuvant chemotherapy. In a previous study using the Surveillance, Epidemiology and End Results (SEER) database, adjuvant chemotherapy does not improve survival in patients with tumors 4 cm or less with VPI. However, that study did not perform exploratory analysis focusing on tumors 3 cm or less . Pathak For patients with tumors larger than 3 cm, but 4 cm or less in size, we found that adjuvant chemotherapy had improved their OS and CSS even in the absence of risk factors. In a cohort study based on NCDB, patients with tumors larger than 3 to 7 cm were analyzed to evaluate the role of adjuvant chemotherapy. In subgroup analysis, there were 10,587 patients with tumors >3 cm, but <=4 cm and 1,608 (15.2%) of whom had received adjuvant chemotherapy. Adjuvant chemotherapy is associated with improved OS in the population with a hazard ratio of 0.75  . In aforEGFR mutation in lung cancer patients in Taiwan is higher than that in the western population . On the other hand, in medical centers, patients with tumors >3 cm, but <=4 cm more likely to have received adjuvant chemotherapy as compared with those having tumors 3 cm or less (74.1% vs 36.7%). The inconsistency of treatment strategies across health care institutions may have introduced selection bias for adjuvant chemotherapy.In conclusion, for patients with T2aN0 stage IB lung adenocarcinoma, the benefit of adjuvant chemotherapy depended on tumor size. Adjuvant chemotherapy within 8 weeks following surgery improved survival in those with tumors larger than 3 cm, but 4 cm or less in size. For patients with tumors 3 cm or less with VPI, adjuvant chemotherapy had no survival benefit.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Research Ethics Committee of the National Taiwan University (NTU-REC No.202101HM030), with waiver of informed consent owing to the lack of personal information and use of secondary data in the study. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for observational studies was used in the revision of this article. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Study concepts: P-HL, G-CC; Study design: C-JC, G-CC, Y-HH, J-ST; Data acquisition: C-JC, Y-HH, K-CC, K-HH; Quality control of data and algorithms: P-HL; Data analysis and interpretation: C-JC, P-HL, Z-RZ, C-HC; Statistical analysis: C-JC, W-CL; Manuscript preparation: P-HL; Manuscript editing: J-ST, T-WL; Manuscript review: G-CC, T-YY, J-YH. All authors contributed to the article and approved the submitted version.


This bioactive glass ceramic  was incorporated by weight  into commercially available GICs (Maxxion R and Fuji IX GP). Surface characterization was made by SEM (n = 3), EDS (n = 3), and FTIR (n = 1). The setting and working (S/W time) times (n = 3) and compressive strength (CS) were analyzed (n = 10) according to ISO 9917-1:2007. The ion release (n = 6) was determined and quantified by ICP OES and by UV-Vis for Ca, Na, Al, Si, P, and F. To verify cell cytotoxicity, stem cells from the apical papilla (SCAP) were exposed to eluates  and analyzed 24 h post-exposure. Antimicrobial activity against Streptococcus mutans  was analyzed by direct contact for 2 h (n = 5). The data were submitted for normality and lognormality testing. One-way ANOVA and Tukey’s test were applied for the working and setting time, compressive strength, and ion release data. Data from cytotoxicity and antimicrobial activity were submitted for Kruskal–Wallis’ testing and Dunn’s post hoc test (α = 0.05). Among all experimental groups, only those with 5% (wt) of Biosilicate® showed better surface quality. Only M5% showed a comparable W/S time to the original material (p = 0.7254 and p = 0.5912). CS was maintained for all Maxxion R groups (p > 0.0001) and declined for Fuji IX experimental groups (p < 0.0001). The Na, Si, P, and F ions released were significantly increased for all Maxxion R and Fuji IX groups (p < 0.0001). Cytotoxicity was increased only for Maxxion R with 5% and 10% of Biosilicate®. A higher inhibition of S. mutans growth was observed for Maxxion R with 5% of Biosilicate® (less than 100 CFU/mL), followed by Maxxion R with 10% of Biosilicate® (p = 0.0053) and Maxxion R without the glass ceramic (p = 0.0093). Maxxion R and Fuji IX presented different behaviors regarding Biosilicate® incorporation. The impacts on physico-mechanical and biological properties were different depending on the GIC, but therapeutic ion release was increased for both materials.This study investigated the influence of incorporating Biosilicate Glass ionomer cement (GIC) is commonly employed in minimally invasive dental procedures, such as atraumatic restorative treatment (ART) ,2, due tThe fluoride release of these materials is inadequate to promote remineralization of caries-affected dentin after an ART procedure , despite4 and condenses into a silica-rich layer. Ca2+ and PO43− migrate back to the surface of that silica-rich layer, forming an amorphous calcium phosphate layer that crystallizes to form hydroxyapatite.The incorporation of bioactive glass (BAG) into glass ionomer cements has been shown to induce the remineralization of dentin ,19,20,21® is a bioactive glass ceramic that achieves high crystallinity and mechanical properties through controlled crystallization while maintaining its bioactivity [® is a bioactive glass ceramic composed of 23.75% Na2O, 23.75% CaO, 48.5% SiO2, and 4% P2O5. It is available in two forms: BS-1P, which has one crystalline phase of sodium calcium silicate (Na2CaSi2O6), and BS-2P, which has two crystalline phases, Na2CaSi2O6 and sodium calcium phosphate (NaCaPO4) [Bioactive glasses exhibit notable bioactivity, but their mechanical properties are low . Thus, bactivity . BiosiliNaCaPO4) .® has been effectively utilized in the medical and dental fields for over 20 years. Its applications include treating dentine hypersensitivity [This material is highly bioactive, osteoconductive, osteoinductive, non-cytotoxic, and non-genotoxic, and it shows antibacterial properties . Biosilisitivity , servingsitivity , and funsitivity . It may ® on the setting behavior, mechanical properties, ion release, cytotoxicity, and antimicrobial activity against S. mutans of commercially available conventional and high-viscosity GICs. Thus, the research hypotheses tested are: (I) Biosilicate® incorporation into GIC will affect their setting behavior and mechanical properties; (II) Biosilicate® incorporation into GICs increases their ion release, (III) biocompatibility, (IV) and antimicrobial activity.This study aims to evaluate the effects of Biosilicate® microparticles (D50 = 5 μm) were synthesized by melting followed by heat treatment [® and the powder portions of each GIC were weighed using a 0.1 mg analytical balance . Then, Biosilicate® was manually incorporated into Maxxion R and Fuji IX powders in concentrations of 5, 10, or 15% wt. ® were used as control groups. M0% and F0% were manipulated following each GIC manufacturer’s instructions. For experimental groups F5%, F10%, and F15%, the P/L ratio was 1.8/1 (wt/wt) to improve handling conditions . The P/L ratio for Maxxion R groups was kept at 1.5/1 (wt/wt).Biosilicatereatment . Two gla®) were individually applied onto a carbon tape mounted on plastic stubs using a plastic rod and sputter-coated with gold  [n = 3), and stored for 24 h in relative humidity (100%). Following this, the cylindrical specimens were split off using a chisel and a hammer, finished and polished using #320 and #400 sandpaper with 99° ethanol, and stored for 24 h in a desiccator  at 37 °C. The specimens were mounted on the stubs, as previously described. A scanning electron microscope was used  and images were taken at magnifications of 1000×, 5000×, operating at 15 kV with a ray width of 25–30 nm and working distance of 10–15 mm. Energy-dispersive X-ray spectroscopy (EDS) was performed to qualitatively estimate the number of ions  on the cross-section (as previously described) of the cylindrical specimens (n = 3). The specimens were fixed on carbon tape over a plastic stub, coated with carbon , and analyzed using 15 kV with a working distance of 10 mm, spot size of 25 mm, and ×500 magnification. The images obtained in the SEM were used for qualitative analysis of the GIC surfaces, as were the spectra obtained by the EDS evaluation.The powders of each material   to evalun = 1). The cements were mixed, immediately placed and kept in the spectrometer, and curves were obtained after 1, 5, and 60 min [−1 in the region of 4000 to 400 cm−1 , though only the spectral range of interest (1800 to 900 cm−1) was represented. FTIR spectra were baseline-normalized using a specific software  and plotted in statistical software . The peaks of interest were searched for in the range of 1700 to 1715 cm−1 for poly-carboxylic acid and 1400 to 1640 cm−1 for poly-carboxylate salts [To investigate GIC setting dynamics, FTIR spectra were collected over time  were prepared according to ISO 9917-1:2007 using custom silicone molds (4.00 mm in diameter and 6.00 mm in height). Cements were mixed and placed into molds between two glass slides protected by a transparent polyester film (Mylar®). Specimens were removed from the molds after 30 min and stored at 100% relative humidity in an incubator at 37 °C for 24 h. The experiments were conducted using a universal testing machine  with a load of 50 N/min and a speed of 0.75 mm/min. The compressive strength (CS) was calculated (MPa) according to the formula:p represents the load (Newton) exerted by the load cell until the sample failure, and D is the diameter of each specimen (mm), measured using a digital caliper .Eighty (80) cylindrical specimens (n = 6) were made as previously described for CS and stored in an incubator at 37  °C for 1 h. The specimens were removed from the molds and placed in 20 mL of deionized water (DI) in plastic tubes  [Forty-eight (48) cylindrical specimens (n = 3) of 5.00 mm in diameter and 1.00 mm in thickness were made using polyvinylsiloxane (PVS) molds and allowed to set for 1 h at 37 °C. Subsequently, specimens were added to 300 μL of cell culture media for 24 h at 37 °C (surface area/mL was 1.8 cm2/mL) [TM Green assay was performed to assess the cytotoxicity of GICs on hSCAPs (RP-89 cell line). Cells were seeded at 10,000 cells/well in a black opaque-walled 96-well plate and maintained in α-MEM  supplemented with 10% fetal bovine serum , 1% L-glutamine, and 1% penicillin/streptomycin  at 37 °C and 5% CO2. After 24 h, the cell culture media were replaced by GIC-conditioned media. Cytotoxicity was evaluated using the Promega CellTox Green® assay at 24 h post-exposure, as per the manufacturer protocol. Fluorescence was read on a Spark® multimode microplate reader  with excitation/emission wavelengths of 485 nm/520 nm. Cells exposed to the provided lysis buffer were considered positive controls, while cells incubated in cell culture media alone were negative controls. Data were reported as percentage of dead cells in a population, relative to the positive control set arbitrarily to 100%.The cytotoxicity was measured as part of the biocompatibility assessment. Twenty-four specimens ( cm2/mL) . The stu cm2/mL)  and exhi cm2/mL) ,41. Thusn = 5) of 15.00 mm in diameter and 1.00 mm in thickness were made using custom PVS molds. After 1 h setting at 37 °C, each specimen was placed into a well of a 6-well plate and sterilized under UV light for 20 min per side. Streptococcus mutans Clarke  was inoculated in BHI broth  and incubated at 35 ± 2 °C supplemented with 5% CO2 for 24 h. Then, the overnight inoculum was centrifuged and resuspended to an optical density of 600 nm (OD600) = 1.0, approximately 1 × 109 colony-forming units/mL (CFU/mL) in dilute BHI (1:500 BHI in sterile DI water). Bacterial suspension was diluted 100-fold, and 50 μL of the diluted bacterial suspension was added to each specimen surface (approximately 5.0 × 105 CFU/specimen). Then, a UV-sterilized plastic coverslip (15.00 mm in diameter) was aseptically placed onto each disk and incubated at 100% relative humidity at 25 ± 2 °C. After 2 h of direct contact, 950 μL of sterile phosphate-buffered saline (PBS) was added to each specimen/well, and the plate was placed on an orbital shaker  at 150 rpm for 2 min to remove bacteria from the surface of the specimen. Then, liquid from each well (1 mL) was collected and serially diluted for plating onto BHI agar. Plates were incubated at 35 ± 2 °C + 5% CO2 for 48 h. The colonies formed were counted using a colony counter  and CFU remaining after surface exposure was calculated.Forty specimens (p < 0.05), Kruskal–Wallis’ test was recommended to detect differences in group medians (p < 0.05). The multiple comparison post hoc test developed by Dunn was used to identify differences between experimental groups.Statistical analysis was performed using GraphPad Prism 9 version 9.5.0 (November 2022). The Shapiro–Wilk test was utilized to examine the normality and lognormality of cell viability data with a significance level of 0.05. The compressive strength and ion release presented normal distribution, and the data were submitted to one-way ANOVA followed by a Tukey’s multiple comparisons test. Due to the non-normal distribution of cell viability and antimicrobial data (® (® particles have a sphere-like form and are clustered on large particles (hexahedron shape), indicating a greater particle size distribution. The particles were categorized based on their shapes [® C. All mar shapes .® concentrations (white arrows). −1. As the materials set, the intensity of the –COOH groups of polyacrylic acid (1715 cm−1) decreases and reorganizes as COO-  groups (1400–1640 cm−1), as indicated by the dashed lines. The loading graph reveals that these two bands vary significantly at 1, 5, and 60 min of setting. The neutralization reaction appeared at 1170 (Si-O), 940 (O–H) cm−1, and with the silica gel (Si-OH) at 1060 cm−1.p = 0.7254 and p = 0.5912). There was no significant difference in working time between M5% and M10% (p = 0.1314). M15% working and setting times were significantly shorter than M0% (p = 0.0072 and p = 0.0071). All experimental groups for Fuji IX (p = 0.1150) and F10% (p = 0.9017) regarding working time. However, F15% had the shortest setting time, followed by F5% and F10%, which were similar (p = 0.8043). Fuji IX C,D had s® to Maxxion R (p < 0.0001). The F10% group did not differ from the F5% and F15% groups. However, F5% was significantly higher than F15% (p = 0.0126). Fuji IX groups exhibited a higher compressive strength than Maxxion R, with a maximum value of 149 MPa compared to 75 MPa for Maxxion R.axxion R A did notaxxion R B, the co® added, the greater the amount of the studied ions released, except for the Al ion released by Maxxion (3.1.3).Compared to Fuji IX groups, Maxxion R A releasep = 0.2086). F0% released the lowest amount (3 ppm).All Maxxion R groups B differeM15% released an average of 60 ppm of Al ions, whereas M5%, M0%, and M10% did not differ statistically, releasing approximately 100 ppm C. The hip = 0.9981). M0% released the lowest amount (19 ppm). For Fuji IX (p < 0.0001): F15% (25 ppm) > F10% (7 ppm) > F5% (4 ppm) > F0% (1 ppm), with a decreasing amount of Si ions released as the concentration decreased.M15% released significantly more Si ions than the other Maxxion R groups  did not differ statistically from F0% (p = 0.4751). F10% (1 ppm) was statistically higher than F0% and F5% (p < 0.0001). F15% released the highest amount of P ions among Fuji IX groups (9 ppm) (M15% released the most significant amount of P ions (40 ppm), whereas M0% released the lowest amount (11 ppm). M5% and M10% were not statistically different (approximately 30 ppm) E. No P i (9 ppm) K.® into Maxxion R increased F ion release by a factor of 4 and by a factor of 2.5 for Fuji IX. M15% released the highest amount of F ion (40 ppm), followed by M10% and M5% (35 ppm); the latter two did not differ statistically. M0% presented a significantly lower (5 ppm) F ion release (p < 0.0001). The highest amount was observed for F15% (7 ppm), which did not differ statistically (p = 0.8187) from F10% (6.3 ppm), as shown in The incorporation of Biosilicate release F. Fuji Ip < 0.0001) and M15% (p < 0.0001). M5% appears to be the most cytotoxic since it was the only treatment significantly different from the negative control (p = 0.0009). The next most cytotoxic group was M10%, which did not differ from M5% (p > 0.9999). Among those Maxxion R groups with Biosilicate®, only M15% did not differ from M0% (p = 0.0541). A tendency toward decreased cytotoxicity when concentrations above 5% of Biosilicate® have been incorporated was observed. In the Fuji IX groups .The percentage of dead cells was calculated relative to the lysis buffer (positive control) set to 100%. M5% presented a significantly higher percentage of dead cells compared to M0% . M5% consistently reduced S. mutans by 4- to 5-logs compared to all other groups. No colonies (zero) were observed on the plates, meaning that less than 100 CFU/mL remained after direct contact with M5% (indicated by the black diamond in p = 0.0053) and M10% (p = 0.0093) showed statistically reduced CFUs after exposure compared to the N group, although there was no significant difference among M0%, M10%, and M15%. No significant reduction in CFUs was found for all Fuji IX groups when compared to each other or when compared to the N group . Regarding Fuji IX groups (p > 0.0001). Fuji 5% trended towards being the most effective in antimicrobial effects among Fuji IX groups, presenting a 29.4% (±10.7) survival compared to the no specimen group. F0% and F10% presented an intermediate percentage of survival, respectively, 29.4 (±14.9) and 60.9 (±5.3). F15% increased S. mutans survival by 8.1% (±5.9), but none of these differences reached statistical significance.The percentage of survival of X groups B, no sig®  to Maxxion R  and Fuji IX (high viscosity) glass ionomer cements. The first research hypothesis was accepted, as the incorporation of Biosilicate® in GICs impacted their mechanical properties and setting times. The setting and working times of M5% were found to be comparable to those of M0% in conventional GIC (Maxxion R). Maxxion with 5% Biosilicate® was well tolerated. However, the addition of 10% Biosilicate® resulted in a significant increase in setting time, while the addition of 15% Biosilicate® resulted in shorter setting times. The setting time of GIC with bioglass increased with the increase in the amount of bioglass added [This study aimed to evaluate the effects of adding Biosilicatess added ,19, wher® and GIC. The polycarboxylate salts in the range of 1400 to 1640 cm−1 [−1 range corresponds to calcium carboxylate (-COOCa2+), the 1459 cm−1 range corresponds to aluminum carboxylate (-COOAl3+), and the 1552 cm−1 range corresponds to sodium carboxylate (-COONa2+) [The FTIR spectrum indicated slight structural modifications when comparing the peaks obtained to the conventional glass ionomer cement setting mechanism, as reported by other researchers ,43,44. W640 cm−1  were alsCOONa2+) ,32,34.® in Fuji experimental groups may have impeded the hydration of polyacrylic acid (PAA), leading to a decrease in PAA ionization and a subsequent reduction in the leaching of cationic ions due to an acid attack on the glass. This, in turn, may have resulted in a reduction in the formation of crosslinks within the matrix.The use of GICs with distinct powder and liquid phase compositions may result in diverse effects on the analyzed properties and should therefore be taken into consideration. Fuji IX powder contains fluoroaluminosilicate glass particles and lyophilized PAA. The acid–base reaction is initiated by ionizing PAA in the powder with water. Maxxion powder contains fluoroaluminosilicate and calcium fluoride, while the liquid component consists of polycarboxylic acid, tartaric acid, and water. The inclusion of BiosilicateIn addition, it has been reported that mixing PAA and glass can facilitate the hydration process, particularly in the presence of unstable glass phases. The dental GIC typically employs the G338 glass system, which consists of three amorphous glass phases: an alumino-silicate (phase 1), a Ca-F-rich (phase 2), and a Ca-F-P-rich (phase 3). The alumino-silicate phase is the most resistant to acid attack by PAA among these phases . When co® had no impact on the compressive strength of the Maxxion groups. However, the P/L ratio appears to be crucial for the CS of the Fuji IX experimental groups . A decline in CS was anticipated for these groups.For Fuji IX, the P/L ratio was modified from 3.6/1 to 1.8/1 (wt/wt) for enhanced handling conditions. The P/L ratio, polyacid concentration, and powder particle size affect the working and setting times and mechanical properties ,46. SincIn addition to differences in the powder compositions of Maxxion R and Fuji IX, it is important to mention that the brand-specific polyacid type can influence the physico-mechanical properties of the GICs . Convent3+ in certain bioglass formulations has resulted in an increase in the compressive strength (CS) of glass ionomer cements (GICs) when compared to formulations lacking Al3+ [® contains sodium oxide (23.75% by wt) and no aluminum. The partial replacement of GIC powder with bioactive glass ceramic resulted in a reduction in the amount of Al3+. Aluminum ions (Al3+) enhance the strength of the GIC matrix by forming stronger three-dimensional bonds compared to calcium ions (Ca2+) [Studies have shown that the inclusion of Aling Al3+ . Sodium-ing Al3+ ,20,50. Bs (Ca2+) . Any imbs (Ca2+) .+ during acid–base reactions, which can negatively affect cement setting reactions, prolong setting time, and compromise mechanical properties [® by %wt; thus, an imbalance in the Al:Si ratio should not be discarded in this case.Bioactive glasses with a high sodium content may release Naoperties ,49. In aoperties ,53,54. TAccording to ISO 9917-1:2007, this material must have a minimum working time of 1.5 min and a maximum setting time of 6 min. F0% complied with ISO 9917 in the setting time, while M0% did not meet the requirements. F0% and F5% were the only groups that met the ISO 9917-1:2007 requirement of having CS values greater than 100 MPa. Maxxion groups did not meet this requirement, indicating a need for adjustments to be made to the experimental cements. Testing the reaction between different polyacids and bioglass or glass ceramic-containing GIC could enhance setting behaviors and mechanical properties. The initial reaction in conventional glass ionomer cement involves the neutralization of the aqueous polyacid and glass powder [® may cause charge repulsion, leading to a decrease in the binding of aluminum ions to the polymer chains [3+) and the polymeric acid. This results in improved hydrolytic stability and mechanical properties of cement [The presence of calcium and sodium ions in Biosilicater chains ,50. Thisr chains ,56, whicr chains . This alf cement .® were also observed in the experimental cements. These concerns are frequently noted in GICs that contain bioglass [All experimental groups exhibited crack lines, unreacted particles, and rough surfaces. Small particle clusters of Biosilicatebioglass . Fuji IXbioglass ,58. Lowe® concentrations resulted in greater ion release from GICs, thus supporting the acceptance of the second research hypothesis. Overall, the release of Na, Si, P, and F increased with increasing Biosilicate® concentrations. The Ca ion release was significantly higher in Fuji IX, at F15%, and in Maxxion R, at M10% and M15%, respectively. Aluminum release was significantly lower in the M15% group, suggesting that Biosilicate® at this concentration may have reduced the release of aluminum. The descending order of ion release profiles for Maxxion R groups was Na > Al > Si = P = F > Ca. For Fuji IX, the order was Na > Si > Al = P = F > Ca. Maxxion R groups released more ions than Fuji IX groups, as shown in Higher Biosilicate2F and NaF, while Fuji IX does not. Larger cations  have been found to disrupt the glass network more efficiently, leading to a reduction in oxygen density and an expansion of the glass network. This expansion facilitates the diffusion of smaller cations  through the glass [®-derived Ca.The Al release range of Maxxion R groups (60 to 105 ppm) was significantly higher than that of Fuji IX groups (<1 to 8 ppm), likely due to the distinct compositions of the two cements. Specifically, Maxxion R contains Cahe glass . Thus, i® resulted in a 4-fold increase in fluoride release for Maxxion R and a 2.5-fold increase for Fuji IX, as shown in ® to both GICs resulted in a proportional increase in therapeutic ion release. This indicates that these materials have the potential to remineralize both enamel and dentin.The liberation of fluoride ions is a key advantage of GICs, as previously stated ,9. Fluor® was rejected. No significant differences in cytotoxicity were found among the Fuji IX groups. However, the rate of cell death was significantly higher in M5% and M10% compared to M0%. The high alkalinity of Biosilicate® in Fuji IX may account for this, as the glass ceramic appears to neutralize the acidity in these groups with a 1.8/1 ratio (g/g), resulting in a similarity to F0% (ratio 3.6/1 (wt/wt)). The minimal release of Al ions from Fuji IX groups may contribute to their non-cytotoxic nature.Incorporating a biological aspect into exploratory research is worthwhile. Multiple assays, including cytotoxicity, cell viability, attachment, morphology, migration, and differentiation, can facilitate the attainment of this objective. This study evaluated biocompatibility via an in vitro cytotoxicity assessment. The third hypothesis regarding the enhancement of biocompatibility by BiosilicateThe Maxxion R groups exhibited a significant release of ions, specifically Al and Na, in contrast to the other groups. M5% demonstrated the highest cytotoxicity, possibly due to its release of 105 ppm of Al. The increase in Al concentration  is associated with a decrease in the percentage of live cells and an increase in the percentage of early apoptotic cells, late apoptotic cells, and dead cells . Aluminu® would lead to greater inhibition of S. mutans, was also rejected. S. mutans to levels below our limit of detection (less than 100 CFU/mL). M0% and M10% also significantly killed S. mutans compared to the no-disk control (N), but this killing was not significantly different from that of M15%. M5% released more Al (105 ppm) and may have contributed to the antimicrobial killing observed in the direct contact assay due to a higher amount of free Al on its surfaces. Aluminum has been observed to exhibit synergistic properties with fluoride, resulting in an increased bactericidal effect on S. mutans by inhibiting ATPase [3 complex in the enzyme’s catalytic site [S. mutans [The fourth research hypothesis, that a higher amount of Biosilicateg ATPase . The postic site ,67. ATPa. mutans .S. mutans (NCTC 10449) growth after exposure to F−, Al3+, and SiO32−, at respective concentrations of 8.6 ppm, 6.8 ppm, and 9.7 ppm has been reported previously [−, 105 ppm of Al3+, and 19 ppm of SiO32−. The synergistic effect of these ions may have enhanced the bactericidal activity against S. mutans. Al is the primary contributor to the antimicrobial effect observed on the surface of M5%, with F and Si playing a minor role.A significant reduction in eviously . In the ® and two distinct GICs. Insufficient information was provided by the manufacturer regarding the composition and quantity of elements in the matrix, making it difficult to understand the interaction between the elements studied. The absence of compositional data required a surface analysis using energy-dispersive X-ray spectroscopy (EDX).Lastly, this was a prospection and comprehension study designed to better comprehend the potential benefits of the interaction between Biosilicate®, and employing alternative methods for incorporating glass ceramic into GIC, such as using a molar ratio instead of %wt, automated methods for better homogenization of powders, and different formulations of polyacid with co-polymers instead of PAA.Further research is necessary to thoroughly explore the topic of improving the ion-releasing and antimicrobial properties of GICs, despite the promising advancements discussed in this article. This will require the use of different glass and ceramic compositions. The study proposes various methods for enhancing the properties of GIC, including altering their composition by increasing aluminum and decreasing calcium and sodium, testing different particle sizes of Biosilicate® at a weight percentage of 5% (wt) did not significantly alter the setting and mechanical properties of Maxxion R in this study. However, it did enhance ion release and antimicrobial activity. The addition of Biosilicate® did not alter the cytotoxicity of Fuji IX, but it led to an increase in the release of therapeutic ions such as Si, P, Na, and Ca. Fuji IX’s mechanical properties and setting behavior were affected by incorporating Biosilicate®.Incorporating Biosilicate


Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake. M. pneumoniae, a model organism for a minimal cell, has a dedicated protein, namely P116, to specifically extract essential lipids. The structure of P116 has a previously unseen fold, resembling a left-handed baseball glove forming a huge hydrophobic cavity. Mycoplasma pneumoniae is a facultative intracellular human pathogen that causes community-acquired pneumonia that can result in severe systemic effects1. Unlike other respiratory pathogens, there is no approved vaccine against M. pneumoniae2. Mycoplasma species lack a cell wall and have the smallest known genomes3. M. pneumoniae, with a 816-kb genome, is a model organism for a minimal cell4. Many of the metabolic pathways that are required to synthesize essential products are absent, which makes uptake by specialized mechanisms necessary. In fact, M. pneumoniae cannot synthesize several of the lipids that are important components of the cell membrane, such as sphingomyelin, phosphatidylcholine and cholesterol5. Instead, it must take up lipids from the host environment, and it adapts its membrane composition depending on the medium in vitro8. Cholesterol in particular, which is present in only a few prokaryotes, is essential for M. pneumoniae cells and several other Mycoplasma species6. It is the most abundant lipid in the membranes, accounting for 35–50% of the total lipid fraction6. Comprehensive studies on other cholesterol-utilizing bacteria are largely lacking; the best characterized organism in this group is Mycobacterium tuberculosis, for which it has been proposed that an ABC transporter homolog and other genes from the mce4 operon are involved in cholesterol uptake9. M. tuberculosis uses cholesterol as a carbon source, enabling long-term infections with the bacteria10. It has been shown that M. pneumoniae survive long-term in cholesterol-rich atherosclerotic plaques11. For other clinically relevant bacteria that use cholesterol, like Borrelia burgdorferi or Helicobacter pylori, the uptake mechanism remains elusive12. To date, it is unclear how Mycoplasma spp. uptake lipids from the environment.M. pneumoniae cells and is strongly immunogenic, thus making it a promising target for therapeutics13. Despite the essential role of P116, the M. pneumoniae genome contains only a single copy of mpn_213 (gene encoding P116), and, on average, only 34 copies of the protein are present in M. pneumoniae14. By contrast, the most immunogenic protein, P1, is not essential, has multiple gene copies present in the genome15, and has a 20-fold-higher copy number14. To elucidate the role of P116, we first determined the structure of the ectodomain by single-particle cryo-electron microscopy (cryoEM). To the best of our knowledge, this structure represents a previously uncharacterized fold (with no matches in the Protein Data Bank) featuring a uniquely large hydrophobic cavity that is fully accessible to solvent. Using mass spectrometry, we identified several different lipids (including cholesterol) bound to P116, some of which are essential, matching observed densities in the hydrophobic cavity. On the basis of these findings, we describe the mechanism by which Mycoplasma spp. extract lipids from the environment and possibly also deposit them in their own membrane, thus explaining the essential role of P116 in the survival of M. pneumoniae cells.In this work, we report the structural and functional characterization of P116. This protein was originally reported to contribute to host-cell adhesion. Furthermore, P116 is an essential protein for the viability of M. pneumoniae (residues 30–957) was overexpressed in Escherichia coli and purified by His-tag affinity and gel filtration chromatography  was determined by single-particle cryoEM at 3.3-Å resolution (according to the gold-standard criterion of Fourier shell correlation (FSC) = 0.143; Table 7 was det3. The cavity is completely hydrophobic but is fully accessible to the solvent . The N-terminal and core domains of P116 superimpose for 126 equivalent residues , suggesting that P116 might have been generated by duplication of an ancestor domain. The common secondary structural elements in the N-terminal and core domains consist of a β-sheet and the two helices preceding the β-sheet Fig. . The corFor the inner part of the P116 core domain, the cryoEM maps show prominent elongated densities (with a length of 10–19 Å and a width of 4 Å) that fill most of the hydrophobic areas Fig.  and 8. TMycoplasma species, including M. genitalium and M. gallisepticum. The amino acids lining the hydrophobic cavity are largely conserved   or with high-density lipoprotein (HDL) and then re-purified them by affinity chromatography. Medium containing FBS is a common growing broth for ids Fig. .The structures of the original P116, empty P116 and refilled P116 samples appear predominantly as homodimers. In all cases, the homodimer exhibits substantial flexibility. Most prominently, the arc diameter of the empty structure is approximately 10 nm smaller than that of the original and refilled structures. In addition, a wringing motion is visible in the refilled structure: each monomer is twisted in the opposite direction along the axis perpendicular to the dimer axis by ~80°, and bends up to 20°, depending on its cargo Fig. . In all 3H]cholesterol) or tritium-labeled cholesteryl oleate as a representative of cholesterol esters  and sphingomyelin (SM), as well as sterols and cholesterol  and liquid chromatography electrospray ionization coupled with tandem mass spectrometry (LC-ESI–MS/MS) analysis. We identified more than 500 lipid species in the samples and found striking differences between the original, empty and refilled P116 samples Fig. . In the rol Fig. .To analyze lipoprotein carryover in the FBS-refilled P116, we conducted an additional proteomics LC–MS/MS experiment , and further experiments are required to better characterize the attachment of P116 with the M. pneumoniae membrane  and HDL. Further, the ligands have been identified as essential lipids for the survival of the cells. In fact, we found a high specificity towards cholesterol, PC and SM, which are the most abundant membrane lipids in M. pneumoniae8. Crosslinking mass spectrometry studies indicate one weak amino acid-pair interaction between P116 and MPN161 (a protein of unknown function)22. Thus, although the involvement of other proteins in incorporating the extracted lipids into the Mycoplasma membrane cannot be excluded, it appears likely, given the observed conformational states upon lipid extraction, that P116 is also responsible for incorporation, thus P116 is responsible for the complete uptake  and the other monomer faces the environment . This wringing motion can be repeated in a continuous manner. In this way, P116 could undergo a rolling movement on the Mycoplasma membrane, thus facilitating the transport of cholesterol and other essential lipids in an apparently simple way for lipid transporters  while excluding others (TGs). This understanding of bacterial lipid uptake presents potential opportunities for treatment of mycoplasma infections and may for the first time2 enable the development of a vaccine against M. pneumoniae.The research complies with all relevant ethical regulations. The experimental procedures to immunize mice and obtain monoclonal antibodies were approved by the Ethics Committee on Animal and Human Experimentation from the Universitat Autonòma de Barcelona under the document CEEAH 1002R3R2R.M. pneumoniae M129 strain was grown in cell culture flasks containing SP4 medium and incubated at 37 °C and 5% CO2. Surface-attached mycoplasmas were collected using a cell scraper and resuspended in SP4 medium. To grow mycoplasma cells on IBIDI eight-well chamber slides, each well was seeded with about 1 × 105 colony-forming units and incubated for 12–24 hours in 200 μL SP4 supplemented with 3% gelatin.The 25 were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 μg mL−1 gentamicin (complete RPMI). Hybridomas were selected in complete RPMI supplemented with HAT medium and BM-Condimed (Sigma Aldrich).NSI myeloma cellsmpn_213 gene from M. pneumoniae were amplified from synthetic clones using different primers for each construct: P116F30 and P116R957 for P116(30–957); P116F13 and P116R957 for P116(13–957); P116F212 and P116R862 for P116(212–862); and P116W681 to generate variant P116 W681A. PCR fragments were cloned into the expression vector pOPINE  to generate constructs, with a carboxy-terminal His-tag. Recombinant proteins were obtained after expression at 22 °C in B834 (DE3) cells (Merck), upon induction with 0.6 mM IPTG at an optical density at 600 nm (OD600) of 0.8. Cells were collected and lysed by French press in binding buffer  and centrifuged at 49,000g at 4 °C. Supernatant was loaded onto a HisTrap 5 ml column  that was pre-equilibrated in binding buffer and elution buffer . Soluble aliquots were pooled and loaded onto a Superdex 200 GL 10/300 column  in a protein buffer (20 mM TRIS-HCl pH 7.4 and 150 mM NaCl).Regions corresponding to the To obtain empty P116, 2.6% Triton X-100 was added to the protein sample and incubated for 1.5 hours at room temperature. Subsequent purification followed the same methodology described above, but also included a wash step with the binding buffer plus 1.3% Triton X-100, followed by extensive washing with at least 20 column volumes of wash buffer  before eluting the samples from the column. P116 was concentrated with Vivaspin 500 centrifugal concentrators  to a final concentration of >0.5 mg/mL.To refill P116 with lipids, the empty protein was incubated with approximately 1 mL FBS per mg P116 for 2 hours at 30 °C while still bound on the column. After extensive washing with at least 40 column volumes of wash buffer, elution and concentration were performed as described above.g for 24 hours with an analytical fixed-angle rotor . The amount of cholesterol and apolipoprotein A1 were determined enzymatically and by an immunoturbidimetric assay, respectively, using commercial kits adapted for a COBAS 6000 autoanalyzer . Radiolabeled HDLs were prepared in the following way: 10 μCi of either  free cholesterol or cholesteryl oleate (Perkin Elmer) were mixed with absolute ethanol, and the solvent was dried under a stream of N2. HDL  was added to the tubes containing the radiotracers, as appropriate, and then incubated for 16 hours in a 37 °C bath26. The labeled HDLs (both 3H-cholesterol-containing and 3H-cholesteryl oleate-containing HDLs) were re-isolated by gradient density ultracentrifugation at 1.063–1.210 g/mL and dialyzed against PBS through gel filtration chromatography. Specific activities of 3H-cholesterol-containing and 3H-cholesteryl oleate-containing HDLs were 1,221 and 185 counts per minute (cpm)/nmol, respectively. The cholesterol transfer to P116 (1 g/L) was measured after adding either [3H] free cholesterol-containing or [3H]cholesteryl oleate-containing HDL (0.5 g/L of APOA1) and incubation for 2 hours at 37 °C. HDL and P116 were separated by a HisTrap HP affinity and size-exclusion columns . Human APOA1 levels were determined in both the HDL and purified P116 fractions by an assay  that used anti-APOA1 antibodies that react with the antigen in the sample to form antigen–antibody complexes, which, after agglutination, were measured turbidimetrically in the COBAS 6000 autoanalyzer (Supplementary Table Human HDL (density 1.063–1.210 g/mL) was isolated from plasma of healthy donors through sequential gradient density ultracentrifugation, using potassium bromide for density adjustment, at 100,000Molecular weights were measured from P116 samples using a Superose 6 10/300 GL  column in a Prominence liquid chromatography system (Shimadzu) connected to a DAWN HELEOS II multi-angle light scattering (MALS) detector and an Optilab T-REX refractive index (dRI) detector (Wyatt Technology). ASTRA 7 software (Wyatt Technology) was used for data processing and analysis. An increment of the specific refractive index in relation to concentration changes (dn/dc) of 0.185 mL/g  was assumed for calculations.All samples were mixed in a 1:1 ratio with sDHB  matrix solution (50 mg mL in 50% acetonitrile (ACN), 50% water, and 0.1% trifluoroacetic acid). Subsequently, 1-μL aliquots of the mixture were deposited on a BigAnchor MALDI target (Bruker) and allowed to dry and crystallize at ambient conditions.m/z in linear positive mode for intact protein measurements and in the mass range of 100–1,600 m/z in reflector positive mode for lipid measurements. The Compass 2.0 (Bruker) software suite was used for spectra acquisition and processing.MS spectra were acquired on a rapifleX MALDI-TOF/TOF  in the mass range of 20,000–120,000 27 and submitted to LC–MS/MS analysis using a nanoElute (Brukrt) system, equipped with C18 analytical column ), coupled to a timsTOF Pro 2 mass spectrometer (Bruker).Lipid samples, with an equivalent of 10 µg of protein, were extracted using a modified MTBE/Methanol extraction protocolSamples were loaded directly onto the analytical column with twice the sample pick-up volume with buffer A. Lipids were separated on the analytical column at 60 °C with a flow rate of 400 nL/minute, with the following gradient: 1% B for 1 minute, 1 to 30% B in 2 minute, 30 to 51% B in 4 minutes, 51 to 61% B in 5 minutes, 61 to 70% B in 5 minutes, 70 to 99% B in 5 minutes and constant 99% B for 13 minutes. This was followed by column re-equilibration with buffer A  with 10 mM ammonium formate and 0.1% FA) and buffer B  with 10 mM ammonium formate and 0.1% FA).m/z and a mobility window from 0.55–1.95 Vs/cm2.Lipids eluting from the column were ionized online using a captive spray ion source and were analyzed in two replicates for positive and negative mode using DDA-PASEF with a ramp time of 100 ms and 3 PASEF-MS/MS events. Spectra were acquired over the mass range from 50–1,550 28)) with default processing parameters for timsTOF data. Identified lipids were aligned to a pooled control sample and filtered by blank abundances (sample intensity/blank intensity >5). Intensities were normalized by mTIC, exported and further analyzed in R using the lipidr package29.Raw data were converted into ibf files and analyzed using the MS-DIAL lipidomics pipeline . Subsequent proteolytic digests were performed using S-TRAPs (Protifi), according to the manufacturer’s instructions. Peptides were further desalted and purified on Isolute C18 SPE cartridges  and dried in an Eppendorf concentrator (Eppendorf).After solubilization in 0.1% formic acid (FA) in ACN/water , samples were subjected to LC–MS/MS analysis on a nanoElute (Bruker) system, equipped with C18 analytical column ) coupled to a timsTOF Pro 2 mass spectrometer (Bruker).Samples were loaded directly onto the analytical column with twice the sample pick-up volume with buffer A. Peptides were separated on the analytical column at 60 °C with a flow rate of 500 nL/minute, with the following gradient: 2 to 35% B in 17.8 minutes, 35 to 95% B in 0.5 minutes and constant 90% B for 2.4 minutes with buffer A (0.1% FA in water) and buffer B (0.1% FA in acetonitrile).m/z and a mobility window of 0.85–1.3 Vs/cm2Peptides eluting from the column were ionized online using a captive spray ion-source and analyzed in DDA-PASEF mode with a cycle time of 100 ms and 4 PASEF-MS/MS events. Spectra were acquired over the mass range of 100–1,700 30. Raw files were recalibrated, search parameters automatically optimized and searched against the combined Uniprot reference proteomes for M. pneumoniae, E. coli and Bos taurus .Data analysis was performed in FragPipe 18 using MSFragger 3.5 for database searchesThe database search space was restricted to tryptic peptides with a length of 7–50 amino acids, allowing for up to two missed cleavages and with a minimum of one unique peptide per protein group. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine, as well as N-terminal acetylation, were set as variable modifications. Percolator was used to estimate the number of false positive identifications, and the results were filtered for a strict target false discovery rate (FDR) < 0.01.For single-particle cryoEM, a 3.5-µL drop of purified P116  or P116 mixed with HDL  was applied to a 45 s glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science), and plunge-frozen in liquid ethane  at 100% relative humidity, 4 °C, a nominal blot force of –3, and a wait time of 0.5 seconds, with a blotting time of 12 s. Before freezing, Whatman 595 filter papers were incubated for 1 hour in the Vitrobot chamber at 100% relative humidity and 4 °C.31) at a nominal magnification of ×130,000 (1.05 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan). For P116, P116 refilled and P116 with HDL, a total of 4,376, 4,019 and 3,114 micrographs with 34, 29 and 30 frames per micrograph and a frame time of 0.2 seconds were collected. The camera was operated in dose-fractionation counting mode with a dose rate of ~8 electrons per Å2 s−1, resulting in a total dose of 50 electrons per Å2 s−1. Defocus values ranged from –1 to –3.5 µm.Dose-fractionated movies of P116, P116 refilled and P116 mixed with HDL were collected with SerialEM v3.8  at a nominal magnification of ×105,000 (0.831 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios G2 electron microscope (Thermo Scientific), equipped with a BioQuantum-K3 imaging filter (Gatan), operated in zero loss peak mode with 20 eV energy slit width. In total,15,299 micrographs with 50 frames per micrograph and frame time of 0.052 seconds were collected. The K3 camera was operated in counting mode with a dose rate of ~16 electrons per A32) was used to process the cryoEM data, unless stated otherwise. Beam-induced motion correction and CTF estimation were performed using CryoSPARC’s own implementation. Particles were initially clicked with the Blob picker using a particle diameter of 200–300 Å. Particles were then subjected to unsupervised 2D classification. For the final processing, the generated 2D averages were taken as templates for the automated particle picking; for the processing of P116 with HDL, no template picking was performed. In total, 3,463,490, 4,532,601 particles, 2,930,863 particles and 262,981 particles were picked and extracted with a binned box size of 256 pixels for P116, P116 empty, P116 refilled and P116 with HDL, respectively. False-positive picks were removed by two rounds of unsupervised two-dimensional classification. The remaining 1,324,330 particles (P116), 1,140,275 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used to generate an ab initio reconstruction with three classes followed by a subsequent heterogeneous refinement with three classes. For the final processing, 1,315,362 particles (P116), 633,332 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used. For the remaining particles, the beam-induced specimen movement was corrected locally.CryoSPARC v3.2 , 4 Å (P116 empty), and 3.5 Å (P116 refilled) . From this stock, a 3.5-µL drop was applied to a (45 s) glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science), and plunge-frozen in liquid ethane  at 100% relative humidity, 4 °C, and a nominal blot force of –1, with a wait and blotting time of 10 seconds.31) at a nominal magnification of ×105,000 (1.3 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan). The total dose per tomogram was 120 e−/Å2, and the tilt series covered an angular range from −60° to 60° with an angular increment of 3° and a defocus set at −3 µm. Tomograms were reconstructed by super-sampling SART33 with a 3D CTF correction34.Tilt-series were recorded using SerialEM v3.8  structurally equivalent residues. Some residues at the N end of the N-terminal domain were difficult to identify and were represented as alanines in the final model. The whole P116 model was then refined using Phenix, and the final refined structure was deposited in the EMDB under code EMD-15274 . Only those clones with supernatants revealing a single 116-kDa band in protein profiles and also exhibiting a consistent fluorescent staining of M. pneumoniae cells were selected and used in this work. Polyclonal sera were obtained by cardiac puncture of properly euthanized mice just before splenectomy and titred using serial dilutions of the antigen. The titer of each polyclonal serum was determined as the half-maximal inhibitory concentration (IC50) value from four parameter logistic plots and was found to be approximately 1/4,000 for both sera. Polyclonal anti-P1 antibodies were obtained by immunizing two BALB/C mice with recombinant P1 proteins39, respectively, as described above. The titers obtained for polyclonal anti-P1 antibodies were approximately 1/2,500 and 1/3,000, respectively.Two BALB/C mice were serially immunized with four intraperitoneal injections, each containing 150 μg of recombinant P116 ectodomain (residues 30–957) in 200 μL of PBS with no adjuvants. The last injection was delivered 4 days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells40, with several modifications. Cells were washed with PBS containing 0.02% Tween 20 (PBS-T) prewarmed at 37 °C, and each well was fixed with 200 μL of 3% paraformaldehyde (wt/vol) and 0.1% glutaraldehyde. Cells were washed three times with PBS-T, and slides were immediately treated with 3% BSA in PBS-T (blocking solution) for 30 minutes. The blocking solution was removed, and each well was incubated for 1 hour with 100 μL of the primary antibodies diluted in blocking solution. For P116 and P1 polyclonal sera, we used a 1/2,000 dilution; a 1/10 dilution was used for monoclonal antibodies from hybridoma supernatants. Wells were washed three times with PBS-T and incubated for 1 hour with a 1/2,000 dilution of a goat anti-mouse Alexa 555 secondary antibody (Invitrogen) in blocking solution. Wells were then washed three times with PBS-T and incubated for 20 minutes with 100 μL of a solution of Hoechst 33342 10 μg/μL in PBS-T. Wells were finally washed once with PBS-T and replenished with 100 μL of PBS before microscopic examination. Cells were observed by phase contrast and epifluorescence in an Eclipse TE 2000-E inverted microscope (Nikon). Phase contrast images, 4′,6-diamidino-2-phenylindole  and Texas Red  epifluorescence images were captured with an Orca Fusion camera (Hamamatsu) controlled by NIS-Elements BR software (Nikon).The immunofluorescence staining of mycoplasma cells on chamber slides was similar to previously describedM. pneumoniae cells growing on IBIDI eight-well chamber slides. Before observation, medium was replaced with PBS containing 10% FBS and 3% gelatin prewarmed at 37 °C. A similar medium has been used to test the effect of P1 antibodies on mycoplasma adhesion and gliding motility41. After incubation for 10 minutes at 37 °C and 5% CO2, the slide was placed in a Nikon Eclipse TE 2000-E inverted microscope equipped with a Microscope Cage Incubation System (Okolab) at 37 °C. Images were captured at 0.5-second intervals, for a total observation time of 10 minutes. After the first 60 seconds of observation, the different antibodies were dispensed directly into the wells. The frequencies of motile cells and detached cells before the addition of antibodies were calculated from the images collected between 0 and 60 seconds of observation. The frequencies of motile cells and detached cells after the addition of antibodies were calculated from the images collected in the last minute of observation.The effect of anti-P116 antibodies and anti-P1 polyclonal serum on mycoplasma cell adhesion was investigated by time-lapse cinematography of Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-023-00922-y.Supplementary InformationSupplementary Tables 1 and 2 and the captions and legends of the Supplementary Movies.Reporting SummaryPeer Review FileSupplementary TablesSupplementary Tables 3 and 4Supplementary Video 1Microcinematography adding P1 polyclonal antibodies.Supplementary Video 2Microcinematography adding P1116 polyclonal antibodies.Supplementary Video 3Control microcinematography.Supplementary Video 4Density map of the P116 homodimer.Supplementary Video 5Ribbon model of P116.Supplementary Video 6Hydrophobicity map of the P116 homodimer.Supplementary Video 7Cross-section of one P116 monomer.Supplementary Video 8Ribbon model of a P116 monomer  with the unaccounted elongated densities.Supplementary Video 9Conformational change of P116 top view.Supplementary Video 10Conformational change of P116 distal view.Supplementary Video 11Conformational change of P116 distal view with ligands.Supplementary Video 12Flexibility of P116.


The crystal structure, morphology and photoluminescence properties have been investigated in detail. BLMT:Mn4+ phosphor has two strong and wide excitation bands in the range of 250–600 nm, which matches well with a near-UV or blue chip. Under 365 nm or 460 nm excitation, BLMT:Mn4+ emits an intense FR light ranging from 650 to 780 nm with maximum emission at 704 nm due to 2Eg → 4A2g forbidden transition of Mn4+ ion. The critical quenching concentration of Mn4+ in BLMT is 0.6 mol%, and its corresponding internal quantum efficiency is as high as 61%. Moreover, BLMT:Mn4+ phosphor has good thermal stability, with emission intensity at 423 K keeping 40% of the room temperature value. The LED devices fabricated with BLMT:Mn4+ sample exhibit bright FR emission, which greatly overlaps with the absorption curve of FR absorbing phytochrome, indicating that BLMT:Mn4+ is a promising FR emitting phosphor for plant growth LEDs.Far-red (FR) emitting LEDs are known as a promising supplement light source for photo-morphogenesis of plants, in which FR emitting phosphors are indispensable components. However, mostly reported FR emitting phosphors are suffering from problems of wavelength mismatch with LED chips or low quantum efficiency, which are still far from practical applications. Here, a new efficient FR emitting double-perovskite phosphor BaLaMgTaO R) and FR light absorbing form (PFR), and they can be transformed into each other depending on the wavelength of the irradiation light. PR will convert to be PFR under 660 nm deep-red light irradiation, and this process can be reversed under 730 nm FR light irradiation or nighttime conditions. The concentration of PR and PFR affects the physiological changes of plants, from vegetative to flowering growth. As a result, controlling the wavelength of irradiation light (deep-red or FR light) can change the concentration ratio of PR to PFR so as to regulate the flowering period of plants. Prolonged deep-red light irradiation can increase the concentration of PFR, resulting in early flowering of long-day plants. Raising the exposure time of FR light can increase the concentration of PR, which postpones the flowering time for long-day plants but brings forward the flowering time for short-day plants. In addition, FR light could also prevent bitter taste caused by excessive growth of vegetables and deterioration of food quality, which is very important for planting high-value vegetables. Therefore, by adjusting the spectral composition of light source, the growth and development process of plants could be regulated to improve the quality and yield of crops.Like many other external environment conditions, such as temperature, water and minerals, light is essential for the growth and development of plants. Light provides an energy source for plant growth through photosynthesis and also serves as a light signal that modulates the entire process of plant growth and development ,2,3. PlaTraditional light devices, such as incandescent lamps and fluorescent lamps, have high energy consumption and low light efficiency, and generally they do not match the spectra required for plant growth. As a result, traditional light sources are not optimal for plant growth. Comparatively, light-emitting diodes (LEDs) show the extraordinary advantages of low energy consumption, high light efficiency, long lifetime, small volume and fast response time. Most importantly, the spectral composition of phosphor-converted LEDs (pc-LEDs) is tunable through optimizing the component and proportion of phosphors. Therefore, pc-LEDs have been offered as a promising light source for indoor plant cultivation and crop harvest ,3,4,5,9.4+ ion as a rare-earth-free activator for red-emitting phosphors has attracted considerable attention  for Mn4+ incorporation, which shows excellent photoluminescence (PL) properties  [6] octahedron structure, which is favorable for Mn4+ occupancy to exhibit excellent PL properties due to its ionic radius similar to Ta5+ (r(Mn4+) = 0.53 Å and r(Ta5+) = 0.64 Å when CN = 6). Recently, Zhou and co-workers  and [TaO6] octahedrons alternatively connect to each other by sharing vertexes to form a 3D network structure, while Ba and La atoms randomly occupy the structure interstices which are surrounded by 12 oxygen atoms. In view of the similar ionic radius in the same coordination environment, Mn4+  ions preferentially replace Ta5+  ion sites in BLMT lattice.4+ precursor xerogel in a RM-1400 °C range. There are obviously four exothermic peaks in the DTA profile at <400, 400–650, 800–950 and 1200–1300 °C, respectively. Below 400 °C, the DTA curve presents a broad exothermic peak accompanied by a large drop with a mass loss of about 35% in the TG curve, which is mainly caused by the decomposition of organic matters and the evaporation of residual water  and 1/(kBT), the slope (−ΔEa) can be obtained. As shown in Ea = 0.41 eV for BLMT:Mn4+ phosphor, which is larger than other Mn4+ activated double perovskite phosphors, such as La2ZnTiO6:Mn4+ (0.30 eV) [2LaSbO6:Mn4+ (0.36 eV) [2GdNbO6:Mn4+ (0.36 eV) [2LaTaO6:Mn4+ (0.32 eV) [2LuTaO6:Mn4+ (0.25 eV) [6:Mn4+ (0.34 eV) [The thermal quenching behavior of phosphors is a nonradiative relaxation related to temperature, which can be illustrated by the coordination energy diagram, as shown in the inset of equation :I=I01+Ae0.30 eV) , Ca2LaSb0.36 eV) , Ba2GdNb0.36 eV) , Ca2LaTa0.32 eV) , Ca2LuTa0.25 eV)  and SrLa0.34 eV) .4+ phosphor in plant growth lighting, the LED devices were fabricated by coating BLMT:0.6%Mn4+ phosphor samples on NUV (~365 nm) and blue (~460 nm) chips. 4+ phosphor (see in 4+ FR emitting phosphor has great application potential in plant growth lighting.In order to further evaluate the application potential of BLMT:0.6%Mnr see in a, which r see in a and anor see in b, and thr see in . All res4+ was successfully synthesized by sol-gel method. BLMT:Mn4+ phosphor shows two strong and broad excitation bands in NUV and blue regions, and when excited by 365 nm or 460 nm light, it produces an intense FR emission peaking at 704 nm owing to the 2Eg → 4A2g transition of Mn4+. The FWHM of FR emission is as wide as nearly 40 nm, which overlaps greatly with the absorption spectrum of PFR phytochrome. When the Mn4+ concentration is 0.6 mol%, the PL QE of BLMT:Mn4+ is as high as more than 60%. BLMT:Mn4+ phosphor has a good thermal stability with an activation energy of 0.41 eV. According to crystal field analysis, the parameters Dq, B and C calculated are 1976 cm−1, 690 cm−1 and 3012 cm−1, respectively, and the Dq/B value is 2.86, which suggests that Mn4+ experiences strong crystal field in BLMT crystal. The FR LED devices constructed by combining NUV/blue chips with the as-prepared BLMT:Mn4+ samples demonstrate that BLMT:Mn4+ is a promising FR emitting phosphor for plant growth LEDs.A FR emitting double-perovskite phosphor BLMT:Mn


The molecular characteristics of sporadic inclusion body myositis (sIBM) have been intensively studied, and specific patterns on the cellular, protein and RNA level have emerged. However, these characteristics have not been studied in the context of HIV-associated IBM (HIV-IBM). In this study, we compared clinical, histopathological, and transcriptomic patterns of sIBM and HIV-IBM.In this cross-sectional study, we compared patients with HIV-IBM and sIBM based on clinical and morphological features as well as gene expression levels of specific T-cell markers in skeletal muscle biopsy samples. Non-disease individuals served as controls (NDC). Cell counts for immunohistochemistry and gene expression profiles for quantitative PCR were used as primary outcomes.+ or CD57+ cells, while the number of PD1+ cells did not differ significantly between the two groups. All markers were shown to be significantly upregulated at gene expression level with no significant difference between the IBM subgroups.14 muscle biopsy samples  of patients and 6 biopsy samples from NDC were included. Clinically, HIV-IBM patients showed a significantly lower age of onset and a shorter period between symptom onset and muscle biopsy. Histomorphologically, HIV-IBM patients showed no KLRG1+ cells discriminated sIBM from HIV-IBM. This may be explained by longer disease duration and subsequent T-cell stimulation in sIBM. Thus, the presence of TEMRA cells is characteristic for sIBM, but not a prerequisite for the development of IBM in HIV+ patients.Despite HIV-IBM and sIBM sharing important clinical, histopathological, and transcriptomic signatures, the presence of KLRG1The online version contains supplementary material available at 10.1007/s00415-023-11779-y. A lower proportion of naïve CD4 and CD8 T cells and more effector memory T cells have been described in HIV patients compared to HIV-seronegative patients in the same age group [+ cells were detected in the muscle [+, CD244+, T-bet+, CD57+/CD28−, CD62L−) [HIV-associated myositis has been described as a complication of HIV/AIDS since the beginning of the HIV/AIDS pandemic . Differege group , 8. Simige group , 10. Thige group , 8. In age group , 12. A cge group , and T cge group . Hence tge group . In sIBMe muscle , 14. The CD62L−) . During  CD62L−) . These c CD62L−)  and are  CD62L−) . Additio CD62L−) . In summ CD62L−) . Of noteIn this study, we aimed to characterize the clinical, morphological, and molecular features, including KLRG1 expression, of HIV-IBM to explore putative differences between HIV-associated IBM and sporadic IBM.We identified 19 patients with myositis in the context of HIV infection. Seven of them fulfilled the ENMC criteria of clinicopathological defined IBM  and wereMuscle biopsy specimens from participating partner sites were cryopreserved immediately after removal at − 80 °C before analysis.8 µm cryostat sections were stained according to standard protocols, including H&E, Gomori trichrome, Elastica van Gieson, non-specific esterase, acid phosphatase, Kongo red, SDH and COX-SDH.A Benchmark XT Immunostainer  was used for immunohistochemical staining in a standardized manner. The primary antibodies are listed as follows : mouse anti-human: CD8 , CD20 , CD45 , CD56 , CD68 , C5b-9 , HLA-ABC/MHC cl. I , HLA-DR/MHC cl. II , MHCneo , CD57 , PD1 ; rabbit anti-human: CD4 , CD3 , p62 , KLRG1 , CD27 , PD-L1 ; rat anti-human: PD-L2 .Fluorescence staining with luminescent conjugated oligothiophenes (LCO) and fluorescence double staining was performed in staining chambers after fixation in acetone for 10 min. For LCO staining, the sections were incubated with pFTAA for 30 min and afterwards washed with phosphate-buffered saline (PBS) and distilled water.For double immunostaining, serum of the secondary antibody species was added first for 30 min to avoid non-specific binding. The incubation with the first primary antibody was at 4 °C overnight and was followed by the first fluorescent secondary antibody with an incubation time of 1 h at room temperature.The protocol was repeated in the dark with a second primary antibody and a second fluorochrome-coupled secondary antibody on the same sections, each with an incubation time of 1 h. Each incubation was followed by a washing step with PBS for 2 × 5 min. Finally, the sections were aqueously mounted with Vectashield mounting medium with DAPI and stored at 4 °C.Fluorochromes and other fluorescence staining solutions are listed as follows (dilution and company): goat anti-rabbit: AF488 , Cy3 ; goat anti-mouse: AF488 , Cy3 ; LCO pFTAA .We used irrelevant antibody stains  as negative control, as well as omission of the primary antibodies. For positive controls, we used the respective tissues as mentioned by the companies.To evaluate the expression of calibre variance and atrophy, connective/fat tissue proliferation, necrosis, regeneration, MHC cl.-I/MHC cl.-II expression, rimmed vacuoles, mitochondrial alterations, autophagy and cellular infiltration, a semi-quantitative score was used to differentiate between low, medium, and severe levels.2), 4–20 cells per HPF and > 20 cells per HPF. Capillary pathology, complement activation and amyloid deposits were divided into present or absent. A severity level of 0–3 or 0–1 was assigned for each category, and the sum of these results in an overall severity score of 0–10. The results are illustrated in a heat map. For the quantitative counting of the cells, 10 HPF were averaged. The values are presented as boxplots with 1.5*IQR (Interquartile range) whiskers.Mitochondrial accumulation was categorized into cap-like subsarcolemmal accumulations, subsarcolemmal accumulations surrounding the entire fibre, and “ragged blue” fibres. Autophagy was separated into singular fine-granular sarcoplasmic labelling by p62, presence within vacuoles in a few fibres and presence in vacuoles in many fibres. The stages of cellular infiltration were divided into 1–4 cells per high power field (HPF = 0.096 mmPGK1), quantitative PCR was performed using the 5' nuclease method on an Applied Biosystems™ QuantStudio™ 6 Flex Real‐Time PCR System  with the following conditions: 95 °C 0:20, 95 °C 0:01, 60 °C 0:20, 45 cycles . All genes were run as triplicates. The Ct values of the target genes were related to the endogenous control gene and ΔCt (= Ct—Ct of the endogenous control) was calculated.According to the manufacturer's instructions, RNA was isolated using the triazole/chloroform method . The concentration of RNA and the degree of purity were measured with the Infinite M200 Microplate Reader . Afterwards the complementary DNA was produced by reverse transcription using the High-Capacity cDNA Archive Kit . To measure the expression level of the gene transcripts and an endogenous control gene (B3GAT1 (CD57), Hs01024500_m1; CD244, Hs00175569_m1; CD27, Hs00609654_g1; KLRG1, Hs00195153_m1; PD1, Hs01550088_m1; PDL1, Hs00204257_m1; PDL2, Hs00228839_m1; PGK1, Hs99999906_m1; TBX21, Hs00894392_m1.The following Taq-Man Gene Expression Assays from Life Technologies/ThermoFisher were used: ImageJ Version 1.53 (RRID: SCR_003070) was used for the image processing. GraphPad Prism 9.0.2 software  was used for the graphical illustration. The ∆CT values are shown on the inverse axis. Statistical calculations were performed for the Kruskal–Wallis test with Bonferroni–Dunn correction to analyse the quantitative differences of mRNA transcripts. The level of significance was set at 5% (*) and 1% (**).+ nadir was between 34 and 320 cells/µl, but data were missing for four patients. HIV-associated coinfections were present in two patients with Hepatitis B. Complications of HIV infection included non-Hodgkin lymphoma, CMV pneumonia, and polyneuropathy. The mean time between HIV diagnosis and muscle biopsy was 12 years (SD = 7.5) and the majority (72%) received antiretroviral therapy at the time of biopsy. In one patient, the antiretroviral treatment was interrupted with a CD4+ count of 500 cells/µl at the biopsy, and data were missing from another patient. Some antiretroviral drugs like Zidovudine can cause myopathy with mitochondrial damage too [p < 0.01) and had a shorter period between the onset of symptoms and biopsy than patients with sporadic IBM. Other clinical parameters showed up almost comparable in both groups.The clinical data for all patients are shown in Table mage too , but  serum levels were similar in both groups , autophagy, necrosis, capillary pathology, and inflammation markers Fig. a. The mi+ cells per HPF (7–104 cells) in the HIV-IBM group and 43 (18–83 cells) in the sIBM group  in the HIV-IBM group and eight CD27+ cells per HPF (1–21 cells) in patients with sIBM  but showed a significantly (p < 0.01) higher expression in the sIBM group with a median value of six positive cells per HPF (1–19 cells)  in the HIV-IBM group and 21 cells per HPF (3–28 cells) in the sIBM group Below is the link to the electronic supplementary material.