Skeletal muscle regeneration is a complex process regulated by many cytokines and growth factors. Among the important signaling pathways regulating the myogenic cell identity are these involving SDF-1 and NOTCH. SDF-1 participates in cell mobilization and acts as an important chemoattractant. NOTCH, on the other hand, controls cell activation and myogenic determination of satellite cells. Knowledge about the interaction between SDF-1 and NOTCH signaling is limited.. Naive myogenic potential was assessed in vivo, using subcutaneous engrafts and analysis of cell contribution to regeneration of the skeletal muscles.We analyzed two populations of myogenic cells isolated from mouse skeletal muscle, that is, myoblasts derived from satellite cells (SCs) and muscle interstitial progenitor cells (MIPCs). First, microRNA level changes in response to SDF-1 treatment were analyzed with next-generation sequencing (NGS). Second, myogenic cells, i.e., SC-derived myoblasts and MIPCs were transfected with miRNA mimics, selected on the basis of NGS results, or their inhibitors. Transcriptional changes, as well as proliferation, migration, and differentiation abilities of SC-derived myoblasts and MIPCs, were analyzed in vitroDll1, Jag2, and NICD. Furthermore, miR10a, miR425, and miR5100 down-regulated the expression of factors involved in cell migration: Acta1, MMP12, and FAK, myogenic differentiation: Pax7, Myf5, Myod, Mef2c, Myog, Musk, and Myh3. However, these changes did not significantly affect myogenic cell migration or fusion either in vitro or in vivo, except when miR425 was overexpressed, or miR5100 inhibitor was used. These two molecules increased the fusion of MIPCs and myoblasts, respectively. Furthermore, miR425-transfected MIPC transplantation into injured skeletal muscle resulted in more efficient regeneration, compared to control cell transplantation. However, skeletal muscles that were injected with miR10a transfected myoblasts regenerated less efficiently.SDF-1 treatment led to down-regulation of miR10a, miR151, miR425, and miR5100 in myoblasts. Interestingly, miR10a, miR425, and miR5100 regulated the expression of factors involved in the NOTCH signaling pathway, including SDF-1 down-regulates miR10a, miR425, and miR5100, what could affect NOTCH signaling, differentiation of myogenic cells, and their participation in skeletal muscle regeneration.The online version contains supplementary material available at 10.1186/s13287-023-03429-x. Skeletal muscle regeneration is a multistep process that allows the reconstruction of skeletal muscle tissue after injury. Such damage, caused by exercise, mechanical injury or induced under experimental conditions, e.g., freezing or cardiotoxin (ctx) injection, leads to a cascade of events resulting in tissue reconstruction , 2. MuscReconstruction of skeletal muscle tissue occurs properly due to the presence of SCs. These cells are located between the sarcolemma and the basal lamina of muscle fibers and are characterized by the presence of the PAX7 transcription factor. When activated, SCs proliferate and follow the myogenic program driven by proteins from the myogenic regulatory factors (MRF) family. MRFs include MYF5, MYOD, myogenin, and MRF4, which sequential expression results in terminal myogenic differentiation of SCs, i.e., myofiber formation , 12. SCsOne of the factors that promote cell mobilization, migration, and participation in skeletal regeneration is a stromal cell-derived factor 1 (SDF-1/CXCL12). SDF-1 is a cytokine that binds to the CXCR4 or CXCR7 receptors. The SDF-1/CXCR4 axis leads to activation of the PI3K/AKT, phospholipase C, and ERK1/2 MAPK pathways. Alternatively, SDF-1/CXCR7 acts through β-arrestin and induces the JAK/STAT, ERK1/2 MAPK or AKT pathways. We showed previously that SDF-1 increases MMP activity and expression of adhesion proteins, such as CD9, and altered actin organization via FAK , CDC42 (cell division control protein 42), and RAC-1 (Ras-related C3 Botulinum Toxin Substrate 1) –26. We amiRNAs are short noncoding RNAs that have the ability to post-transcriptionally regulate gene expression by binding to 3 'UTR' of target mRNAs. Such binding leads to destabilization or degradation of the transcript, which results in inhibition of its translation –29. SDF-The aim of present study was to identify microRNA involved in SDF-1 signaling. As a result, we show that SDF-1 treatment of myoblasts led to down-regulation of miR10a, miR151, miR425, and miR5100. Down-regulation of CXCR4, that is, the SDF-1 receptor, on the other hand, caused up-regulation of miR10a, miR151, miR425, and miR5100 in myoblasts. Interestingly, microarray analysis showed that three of these miRNAs, i.e., miR10a, miR425, and miR5100, regulated the level of transcripts involved in NOTCH signaling. Thus, we suggest that these molecules are involved in combing SDF-1 and NOTCH signaling in myogenic cells. For this reason, to follow the impact of these miRNAs on myogenic identity, mouse SC-derived myoblasts and MIPCs were transfected with miRNA mimics encoding abovementioned miRNAs and their inhibitors, and analyzed in vitro and in vivo. We confirmed that miR10a, miR425, and miR5100 regulated the transcripts involved in NOTCH signaling, as well as migration and myogenic differentiation. Thus, SDF-1 acting through miR10a, miR425, and miR5100 affects NOTCH signaling, migration, and differentiation of myogenic cells.Animal studies were approved by the Warsaw Local Ethics Committee No. 1 (permit number 668/2018 and 1186/2021). All details such as animal care and monitoring or experimental procedures were included in the permit. In animal experiments  in each group (control or treated), the experimental unit was one animal. In each case, three independent experiments were performed. Total number of animals was 64. The 2–3-month-old male C57/BL6 mice were used. No additional criteria for including or excluding animals were used. The study was randomized.Gastrocnemius, EDL, and Soleus muscles of 2–3-month-old male C57/BL6 or C57/BL6 mice carrying the LacZ transgene at the ROSA26 locus, according to Rosenblatt et al. [Satellite cells (SCs) were isolated from t et al. . BrieflyGastrocnemius, EDL, and Soleus muscles of 2–3-month-old C57/BL6 or C57/BL6 male mice carrying the LacZ transgene at the ROSA26 locus, as described previously [Muscle interstitial progenitor cells (MIPCs) were obtained from eviously . BrieflymRNA (ID: s64091). Appropriate negative control siRNA was used, according to the manufacturer's recommendation. The siRNA duplexes were diluted in DMEM to a concentration of 100 pmol, and Lipofectamine RNAiMAX (Life Technologies) was added according to the manufacturer's instructions. Mouse recombinant SDF-1 (10 ng/µl) was added to cell culture 24 h after transfection. The cells were then collected and processed 48 h after SDF-1 treatment for RNA isolation and RNA sequencing.MIPCs or SC-derived myoblasts were cultured in growth medium until at least 50% confluence was reached. One hour before transfection, the medium was changed to OptiMem (Thermo Fisher Scientific) supplemented with 15% FBS. SC-derived myoblasts were transfected with predesigned Silencer Select siRNA (Life Technologies) complementary to CXCR4 encoding mirVana miRNA mimics or miRNA inhibitors: miR1 or one of the following: miR10a, miR151, miR425, and miR5100 , according to the manufacturer's protocol. Cells transfected with the miR1 mimic were considered positive controls and cells transfected with the negative control #1 mimic  were considered negative controls. Furthermore, cells were transfected with miRIDIAN miRNA mimic transfection control conjugated with Dy547 fluorescent dye  to evaluate transfection efficiency. The transfection procedure lasted for 48 h, and then cells were washed with PBS and further cultured in medium that supports myogenic differentiation, that is, DMEM supplemented with 2% horse serum  for 5 days. Alternatively, after 48 h of transfection, cells were suspended in Matrigel® and transplanted subcutaneously, or suspended in PBS and transplanted into the injured Gastrocnemius muscle.MIPCs and SC-derived myoblasts were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) and 50 nM SC-derived myoblasts and MIPCs, control or transfected with mimic miRNAs, were incubated in 0.5 µM carboxyfluorescein succinimidyl ester  in PBS at 37 °C for 10 min. Cells were rinsed in PBS and cultured for 24 h or 48 h in growth medium under standard conditions. Cells were then rinsed in PBS, trypsinized, and subjected to flow cytometry analysis  using CytExpert software. Unlabeled cells (negative control) and cells analyzed immediately after CFSE labeling (positive control) were included in each experiment. Three independent experiments were performed.SC-derived myoblasts and MIPCs were cultured and transfected as described above. After 48 h of transfection, cells were cultured in medium that supports myogenic differentiation for another 5 days. Finally, cells were fixed with 3% PFA in PBS for further analysis. The fusion index was calculated as a percentage of cell nuclei present within the myotubes compared to all visible nuclei. Three independent experiments were performed; nuclei were counted from 10 random fields of view.Migration of SC-derived myoblasts and MIPCs, control ones or transfected with mimic miRNAs, was analyzed using a scratch wound healing assay. Cells were scratched off the plate using a plastic tip to create the 'wound'. The wound healing manifested by the ability of cells to fill the created gap was observed. Pictures of the 'wound' area were taken at three time points: 0 h, 6 h, and after 24 h. The scratch area was calculated using ImageJ. Three independent experiments were performed.5 of control or transfected MIPCs or SC-derived myoblasts were suspended in 0.5 ml of Matrigel® GFR High Concentration (HC) basement membrane matrix (Corning). Next, aliquots of approximately 0.4 ml of Matrigel® containing cells were injected into the subcutaneous connective tissue on the back of C57/BL6 male mice. After 21 days, the transplants were isolated for further analysis. Three independent experiments were performed for each of the cell populations examined.5 × 10Gastrocnemius muscles in the hind limb were injected with 50 µl of Naja mossambica cardiotoxin (CTX) , while the collateral leg was injected with 0.9% NaCl. After 24 h from injury, ~ 5 × 105 of control or transfected MIPCs and SC-derived myoblasts carrying the LacZ transgene at the ROSA26 locus were suspended in PBS and transplanted into damaged Gastrocnemius muscle. After 14 days of regeneration, mice were sacrificed by cervical dislocation, muscles were isolated from both legs, from tendon to tendon, and frozen in isopentane cooled with liquid nitrogen. Muscles were sectioned using cryostat  to obtain 7 μm thick cross sections which were then air dried, fixed with 3% PFA in phosphate buffered saline (PBS), and stored for further analyzes. Other samples were ground using mortar and pestle cooled with liquid nitrogen, and the whole protein was isolated with RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitors  and phosphatase inhibitors .Mice were anesthetized with isoflurane . p value ≤ 0.05.RNA was isolated from SC-derived myoblasts transfected with siRNA complementary to mRNA encoding CXCR4 or treated with SDF-1. A total of three independent samples were collected. RNA was extracted in AROS Applied Biotechnology A/S Science Park Skejby (Denmark) with the QIAsymphony RNA extraction kit from QIAGEN (Germany) using the QIAsymphony SP Biorobot from QIAGEN (miRNA CT 400 program). MiRNA was quantified using a Qubit fluorometer, and approximately 150 bp-long barcode libraries were created. The quality of the libraries was validated using the Bioanalyzer 2100 DNA High Sensitivity Kit . Libraries' concentrations were measured with the KAPA Library Quantification Kit during qPCR. Libraries were pooled with a 30% increase in control libraries PhiX (Illumina) and hybridized to a sequencing flow cell. Single read 50 bp sequencing was performed using an Illumina HiSeq sequencer with the use of a TruSeq rapid SBS kit . The quality assessment of the raw reads was performed with the FastQC tool. First, only reads containing adapters were selected for further analysis. Later, adapters and low-quality reads were discarded. Then, reads with identical sequences were grouped/collapsed into one entry and consequently were converted into read-count format. The miRNA expression profile was performed using Partek Flow v6.0 and Partek Genomics Suite v6.6 software . Briefly, reads were aligned with the mouse reference genome (assembly mm10) using Bowtie1. Next, miRBase (release 21) was used to provide annotations for RNA fragments assigned to the mouse genome. Finally, TMM normalized mapped reads were used to infer lists of differentially expressed miRNAs between analyzed groups. Unsupervised hierarchical clustering was performed with the use of the Partek Genomics Suite on normalized reads to visualize differentially expressed miRNAs. The p-value cut-off point for differentially expressed miRNAs between groups was established at the p value < 0.05 was applied. The list of detected differentially expressed transcripts was analyzed using Ingenuity Pathway Analysis  software to identify significant interactions and pathways. All the analysis and the corresponding plots were performed following the software guide, limiting the IPA database information to molecules and relationships where the information was experimentally observed. The data obtained were also analyzed with and Transcriptome Analysis Console , miRDB [Total RNA along with the microRNA fraction was extracted from control and transfected SC-derived myoblasts and MIPCs using the RNAqueous Micro Total Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Furthermore, the integrity of the obtained RNA was analyzed with the 2100 Bioanalyzer (Agilent Technologies) using the RNA 6000 Nano Lab Chip Kit (Agilent Technologies). All RNA samples had an integrity number greater than 8.5. Whole transcriptome analysis was performed using the Clariom S Pico Assay  according to the manufacturing protocol. The prepared samples were hybridized with a single Clariom S mouse array and incubated for 16 h in the Affymetrix GeneChip Hybridization Oven 645 at 45 °C, 60 rpm. The arrays were stained with an Affymetrix GeneChip Fluidics Station 450, according to the specific fluidics protocol, and scanned with an Affymetrix GeneChip Scanner 3000 7G. The raw intensity CEL files generated by GeneChipTM Command ConsoleTM were imported into Transcriptome Analysis Console (TAC) 4.0 (Applied Biosystems). Microarray data were normalized and analyzed using Transcriptome Analysis Console 4.0 following the TAC user guide. Each variance analysis was performed by one-way ANOVA. To determine the significance of differentially expressed genes (DEGs), a cut-off point was applied for the fold change value ± 1.5 and the ), miRDB , and Tar), miRDB .Twf1, Acta1, Ilk, Mmp12, Dll1, Jag1, Jag2, Hes1, Hey1, Pax7, Myf5, Myod, Mef2c, Myog, Musk, Myh3 . The average expression of hypoxanthine phosphoribosyltransferase 1  and glyceraldehyde-3-phosphate dehydrogenase  was used as a reference for further calculations. The reaction was carried out with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) using LightCycler96 (Roche) under the following conditions: preincubation 2 min,50 °C; preincubation 10 min, 95 °C; amplification (40 cycles) 15 s, 95 °C and 1 min, 60 °C. All reactions were performed in duplicates. Expression levels were calculated using 2−(ΔCt) formula based on the relative expression of the average expression of Hprt1 and Gapdh. MiRNA reverse transcription was carried out with the TaqMan MiRNA reverse transcription kit (Thermo Fisher Scientific) and TaqMan miRNA assays (Thermo Fisher Scientific) under the following conditions: 30 min, 16 °C; 30 min 42 °C; 5 min, 85 °C. MiRNA levels were assessed using quantitative real-time PCR analysis (qPCR). RT primers and TaqMan probes were used for specific miRNA: miR1 (002222), miR10a (002288), miR151 (001190), miR5100 (462702_mat), miR425 (001516), U6 (001973). The average expression of noncoding U6 snRNA was used as a reference for further calculations. The reaction was carried out with TaqMan Universal Master Mix II, without UNG (Thermo Fisher Scientific) using LightCycler96 (Roche) under the following conditions: preincubation 2 min, 50 °C; preincubation 10 min, 95 °C; amplification (40 cycles) 15 s, 95 °C and 1 min, 60 °C. All reactions were performed in duplicates. Three independent experiments were performed.Total RNA, including the miRNA fraction, was extracted from the control and mimic miRNA transfected SC-derived myoblasts and MIPCs using the RNAqueous-Micro Total Isolation Kit (Thermo Fisher Scientific), according to the manufacturer's protocol. RNA-based cDNA was synthesized using the RevertAid First-Strand cDNA synthesizer kit (Thermo Fisher Scientific), according to the manufacturer's protocol, under the following conditions: 25 °C for 5 min, 42 °C for 90 min, and 70 °C for 5 min. mRNA levels were evaluated using quantitative real-time PCR analysis (qRT-PCR) with TaqMan assays (Thermo Fisher Scientific) for the following genes: Skeletal muscle frozen sections (7 µm), transplants, or SC-derived control/transfected in vitro cultured myoblasts or MIPCs were fixed with 3% PFA in PBS. Next, the samples were washed in PBS and permeabilized in 0.05% Triton X100 (Sigma-Aldrich) in PBS, washed in PBS and incubated in 0.25% glycine (Sigma-Aldrich) in PBS, followed by incubation in 3% bovine serum albumin (Sigma-Aldrich) with 2% donkey serum albumin (Sigma-Aldrich) in PBS. The samples were then incubated with primary antibodies anti-cleaved NOTCH1 , anti-skeletal myosin , anti-laminin , anti-β-galactosidase  diluted 1:100 in 3% BSA with 2% donkey serum in PBS at 4 °C overnight, followed by incubation in appropriate secondary antibodies conjugated with Alexa Fluor 488 or 594  diluted 1: 500 in 1.5% BSA in PBS at room temperature for 2 h. Negative controls using secondary antibodies were performed. Cell nuclei were visualized by 5-min-long incubation in Hoechst 33342 (Thermo Fisher Scientific) diluted 1:1000 in PBS. The actin cytoskeleton was visualized using phalloidin conjugated with TRITC (tetramethylrhodamine B isothiocyanate). The samples were mounted with fluorescent mounting medium (Dako Cytomation) and analyzed using the LSM 700 confocal microscope (Zeiss) and ZEN software (Zeiss). The proportion of cells expressing the examined proteins was calculated from 10 fields of view on each slide, and each experiment was performed three times.Muscle sections were stained with Gill’s hematoxylin (Sigma-Aldrich) and eosin Y (Sigma-Aldrich) and mounted in permanent aqueous mounting agent for microscopy (Dako). Sections will be analyzed using Axio Scan Z1 (Zeiss). Newly formed myofibers with centrally located nuclei were counted from 10 fields of view on each slide, and each experiment was performed three times.Gastrocnemius muscles, control or injected with cells, using RIPA buffer (Thermo Fisher Scientific). Twenty-five micrograms of total protein lysate were denatured by boiling in Laemmli buffer, separated using SDS-Page electrophoresis, and transferred to PVDF membranes (Roche). The membranes were blocked with 5% milk/Tris buffered saline (TBS) for 1 h and incubated with primary antibodies diluted 1:1000 in 5% milk/TBS, at 4 °C, overnight, followed by secondary antibodies diluted 1:20,000, at room temperature, for 2 h. The protein bands were then visualized with the SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and exposed to a chemiluminescence positive film . The film was developed using a developer and fixer (Fuji). The density of the examined bands was compared with the density of α-tubulin bands. The following primary antibodies were used: mouse monoclonal anti-β-actin , rabbit monoclonal anti-NICD , rabbit polyclonal anti-FAK , rabbit polyclonal anti-phospho FAK , mouse monoclonal anti-MYOD , rabbit polyclonal anti-MCK (SAB4500267 Sigma-Aldrich), and mouse monoclonal anti-α-tubulin . Secondary antibodies used: rabbit anti-mouse IgG conjugated with peroxidase  and goat anti-rabbit IgG conjugated with peroxidase , goat anti-rabbit IgG conjugated with peroxidase . Three independent experiments were performed. Blots were analyzed using Gel Doc XR+ and Image Lab 5.1 (BioRad).Proteins were isolated from control or transfected MIPCs, SC-derived myoblasts, or p < 0.05 . The mean value and standard deviation were shown in each graph presented. All statistical analyses were performed using GraphPad 7 software (Prism).The Gaussian distribution of values was analyzed with the Shapiro–Wilk normality test. The fold change was calculated by comparing the average values of the nontreated samples to those of all samples. Data were analyzed using the t-test or the one-way ANOVA test and post hoc with Dunnett’s multiple comparisons test. All data were compared to results coming from analyzes of control group, i.e., nontreated cells. Differences were considered statistically significant when Cxcr4 siRNA transfected SC-derived myoblasts , decreased significantly in transfected cells -derived myoblast migration and their participation in skeletal muscles regeneration –26, 38. Cxcl12 (encoding SDF1) and Ptk2  involved in cell migration and adhesion, Mef2b/c/d, Myod, Myf6 (MRF4), and Myh4 involved in myogenic differentiation, as well as Jag1 and Dll4 involved in NOTCH signaling , integrin-linked kinase (Ilk), and matrix metalloproteinase 12 (Mmp12) , muscle-specific kinase (Musk), and myosin heavy chain 3 (Myh3). After 48 h, in SC-derived myoblasts transfected with selected mimics the level of transcripts did not change  and cultured for additional 5 days. No significant changes were observed in the level of the analyzed transcripts  Fig. . 48h aftlls Fig. A. Howeveics Fig. B. The lests Fig. A. A signDll1, Jag2, at the mRNA level and NICD at the protein level. Furthermore, miR10a and miR425 led to drop in β-actin as well as total and activated FAK levels, that is, proteins involved in cell migration and adhesion. Furthermore, a significant decrease in MYOD was observed in miR10a, miR425, and miR5100 mimic transfected MIPCs  SC-derived myoblasts analyzed 48 h post transfection; (B) MIPCs analyzed 48 h post transfection; (C) SC-derived myoblasts analyzed 7 days post transfection; (D) MIPCs analyzed 7 days post transfection. Figure S4. Impact of miR151 mimic or inhibitor on migration and fusion of control or transfected SC-derived myoblasts and MIPCs. (A) Cell migration assessed 6 and 24 h after performing a scratch. (B) Fusion index. Three independent analyzes were performed. Figure S5. Full-length images of blots presented in Fig. 4. (A) NICD (~ 110 KDa); (B) FAK (~ 120 KDa); (C) pFAK (~ 120 KDa); (D) β-actin (~ 40 KDa); (E) MYOD (~ 45 KDa); and (F) α-tubulin (~ 50 KDa).Three independent biological analyzes were performed. Figure S6. Full-length images of blots presented in Fig. 8. (A) MYOD (~ 45 KDa); (B) MCK (~ 45 KDa) and (C) α-tubulin (~ 50 KDa) Three independent analyzes were performed.


RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+-dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.Intracellular Ca Conversely, Ca2+ homeostasis disruption occurs under pathological conditions such as senescence and neurodegeneration , the endoplasmic reticulum  Complex (MCUc), comprised of a tetramer of MCUs, the structural stabilizer Essential MCU Regulator (EMRE), and gating and regulatory subunits Mitochondrial Calcium Uptake Protein (MICU)-1, −2 or − 3 , GEO accession number GSE52564; and by http://astrocyternaseq.org/ (last access: 2021-11-01), GEO accession numbers GSE84540 and GSE94010, respectively.RNAseq data were mined from the public databases published by 2.2 |Comissão de Ética em Cuidado e Uso Animal do Instituto de Química da Universidade de São Paulo and by the Bioethics Committee of the University of Salamanca (reference 449), following requirements described by the Sociedade Brasileira de Ciência de Animais de Laboratório, European Union Directive 86/609/EEC and Recommendation 2007/526/EC, regarding the protection of animals used for experimental and other scientific purposes, and enforced under Spanish legislation directive RD1201/2005. Adult mice were maintained in groups of 4–5 animals per cage at the specific pathogen-free Animal Experimentation Facility of the University of Salamanca (Biosafety Level 2 environments). Neonates (0–1 days-old) were obtained from breeding cages  from the specific pathogen-free Animal Care Facility of the Institute of Chemistry and Faculty of Pharmaceutical Sciences at the University of São Paulo and from the Animal Experimentation Facility of the University of Salamanca. All animals were maintained in a light–dark cycle of 12 h, 45–65% humidity, 20–25°C, with open and unlimited access to standard solid diet and water, in a microisolator system. Cages were changed and sanitized 1–2 times/week. All animal manipulation was done during the day (light cycle).Experimental design and animal care standards followed ARRIVE 2.0 guidelines . Animal The number of neonates was determined by the demand of primary astrocyte cultures. Protocols and study design were optimized to yield the maximal cell count using the smallest numbers of animals, in accordance with the 3Rs principle . For in NclxloxP/loxP  mice were designed and produced at Dr. John Elrod’s laboratory, as described by In vitro pharmacological experiments with primary astrocytes were conducted in cells from C57Bl/6NTac mice. 2.3 |2, 37°C, humidified incubator. Cells were grown in a 75 cm2 flask for 7 days and then shaken at 200 rpm in an incubator. The supernatant was discarded, and the remaining astrocyte-enriched culture was re-seeded at 50.103/cm2 and grown for 3–7 days for the experiments.Mouse cortical astrocyte primary cultures were conducted as previously described . Briefly2, 37°C, humidified incubator.The C6 cell line stock  was kindly donated by Dr. Cristoforo Scavone . The C6 cell line is not listed by the International Cell Line Authentication Committee’s Register of Misidentified Cell Lines (version 11), and no further authentication was performed. C6 cells were grown and maintained for up to 10 passages in High Glucose DMEM  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, in a 5% COFor the experiments, unless otherwise stated, all cell media were changed for a respective serum-free version and cells were allowed a 1 h equilibration period, after which 10 μM CGP-37157  or sterile DMSO, as a control, were added. When necessary, BAPTA-AM  or DMSO, as a control, was incubated over the last 30 min of the equilibration period.2.4 |3 or 72.103 cells per well, respectively, on XFe24 Seahorse plates  and experiments were conducted at day in vitro (DIV) 15 ± 1, either for acute pharmacological inhibition of NCLX or for NCLX knock-out . Cells were washed once with experimental medium—DMEM  supplemented with 1 mM pyruvate, 4 mM glutamine, 10 mM HEPES, 1% penicillin/streptomycin, and 5.5 mM glucose (for astrocytes) or 25 mM glucose (for C6 cells)—and pre-incubated at 37°C, room atmosphere, for 1 h in 500 μl experimental medium. Tests were conducted as described below, using pre-titrated inhibitor concentrations, and assessing respective oxygen consumption rates (OCR) and extracellular acidification rates (ECAR).Purified astrocytes or C6 cells were plated at a density of 30.10Astrocyte experiments were normalized by automated cell count, as described previously . Seahors2.4.1 |Total ATP production rates were estimated as previously described . Astrocy2.4..2 |CGP was derived from the average between the last three OCR measurements; OCRproton-leak was calculated from the average between the last two oligomycin-insensitive OCR measurements; OCRATP-linked was calculated from the difference between OCRCGP and OCRproton-leak; ∆ECARoligo was calculated as the difference between the first ECAR measurement right after and the one right before the oligomycin addition.C6 cell metabolic assessment was conducted using a MitoStress test, as previously described . Cell pl2.5 |3H]-glucose (2 μCi/well) and 10 μM CGP-37157 or DMSO for 4 h, under gentle orbital rotation (60 rpm) at 37°C. Reactions were stopped by acidification with 20% perchloric acid, and cell media were collected and allowed to equilibrate with a separated tube containing 500 μl deionized water, enclosed in a sealed glass vial, and maintained in a rotating incubator at 60 rpm, 37°C, room atmosphere, for 72 h. 3H2O produced was indirectly measured from these plastic vials through liquid scintillation counting .Glucose-to-glycolysis metabolism was assessed as described elsewhere . In brie2.6 |+ and 22.5 U/ml lactate dehydrogenase, or using a commercial kit . Glucose concentrations were determined by following NADPH production at λ = 340 nm in a Tris buffer , containing 0.5 mM MgCl2, 2 mM ATP, 1.5 mM NADP+, 2.5 U/ml hexokinase, and 1.25 U/ml glucose-6-phosphate dehydrogenase.In brief, cells were washed and maintained in serum-free culture medium for 1 h for equilibration, collected (baseline measurement), and incubated with 10 μM CGP-37157 or DMSO. Cell medium was collected right after CGP addition and after 1 or 4 h, and both glucose and lactate  levels w2.7 |Cre recombinase expression was induced in vitro through an adenoviral vector  or its respective empty vector as a control . Primary astrocytes were infected 2 days after being re-plated at 15 MOI (multiplicity of infection). The virus suspension was removed 24 h after transduction and experiments were conducted 7 days after beginning the infection.For in vivo experiments , Cre rec2.8 |NclxloxP/loxP mice (11.7 ± 2.5 weeks old) were briefly anesthetized with sevoflurane  in a 30% O2 and 70% N2O atmosphere . Animals were appropriately positioned in the stereotaxic apparatus  coupled with a digital readout , maintained under a heat lamp, and had their temperatures monitored by a rectal thermometer. Injections were controlled by a digitally controlled pump , in which 2 μl containing 1.1010 PFU/μl of AAV/5 vectors  or 2.75.1012 viral genome copies/μl of AAV/rh10 vectors  diluted in sterile PBS with 0.001% Pluronic F-68 were administered bilaterally in two depths (1 μl each) at 500 nl/min. Hippocampi were targeted according to the following coordinates, based on Paxinos and Franklin atlas  and 380 nm (F380) and λem = 510 nm in a Leica DMi-8 microscope equipped with a Fura-2 filter (Leica Microsystems). Cells were followed through additions of CGP (10 μM), ATP (100 μM), as well as ionomycin (20 μM) to allow calibration. Analyses were conducted through FIJI ImageJ 1.52p (2+] variation was estimated as the ratio (R) between F340/F380. Data were calibrated by the maximal ratio induced by ionomycin, controlled for background fluorescence oscillations, and normalized by the initial ratio (R0).Calcium levels were live monitored in attached astrocytes through the ratiometric probe Fura-2-AM  as done by eJ 1.52p , in whic2.11 |t-test; one-sample Wilcoxon test with theoretical mean = 0.0, and paired two-way ANOVA, followed by Holm–Šidak’s post hoc test for parametric analyses; and through Mann–Whitney test for non-parametric analyses. No normality test was conducted. A ROUT outlier test, with 5% sensitivity, was used to search for outliers. Full analysis results are detained in the All raw data were organized and analyzed in Microsoft Excel , and statistical analyses were performed in GraphPad Prism 8 , in which all figures were also plotted. According to the experimental design, as appropriately described in the figure legends, data were analyzed through unpaired, paired, or ratio-paired Student’s 3 |2+ transport in brain function. Interestingly, there is literature evidence that NCLX, the main mitochondrial Ca2+ extrusion pathway  when analyzed as astrocyte versus total input tissue in the hippocampus  in cultured astrocytes also modified ATP-induced Ca2+ signaling  induced a decrease in the total ATP production rate  both und3H2O) production from radiolabeled glucose, which showed a trend toward an increased glycolytic flux in astrocytes with NCLX inhibition , NCLX knockout was achieved over the course of days, which could lead to compensatory mechanisms and dynamic changes in metabolic modulations. Notwithstanding, NCLX knockout induced a significant increase in lactate secretion during 1 h measurements . By regulating the levels of fructose-2,6-bisphosphate, a potent allosteric activator of 6-phosphofructo-1-kinase (PFK1), it acts as a key glycolytic flux regulator . It is aycolysis .2+ signaling and glycolytic flux. Indeed, we observed that astrocyte-specific NCLX deletion in the hippocampus improves aspects of mouse cognitive performance  may be involved. By fine-tuning astrocytic glycolysis and lactate secretion, NCLX may act as a control check point in brain metabolism, impacting on the astrocyte-to-neuron lactate shuttle and cerebral function.We demonstrate that NCLX, which is over-enriched in astrocytes, modulates astrocytic glycolytic flux and lactate secretion secondarily to shaping cytosolic Caignaling . While tSupporting InformationSupplemental Table


Since its effect on the pancreatic islets remains unclear, we evaluated insulin sensitivity and glucose-stimulated insulin secretion (GSIS) in rats consuming YM or tap water (C) for 21 days. Glucose tolerance, glycemia, triglyceridemia, insulinemia, TBARS and FRAP serum levels were evaluated. GSIS and mRNA levels of insulin signaling pathway and inflammatory markers were measured in isolated pancreatic islets from both groups. In C rats, islets were incubated with YM extract or its phenolic components to measure GSIS. YM improved glucose tolerance, enhanced GSIS, increased FRAP plasma levels and islet mRNA levels of IRS-1 and PI3K (p110), and decreased TBARS plasma levels and islet gene expression of TNF-α and PAI-1. Islets from C rats incubated with 100 µg/mL dry YM extract, 1 µM chlorogenic acid, 0.1 and 1 µM rutin, 1 µM caffeic acid or 1 µM quercetin showed an increase in GSIS. Our results suggest that YM enhances glucose tolerance because of its positive effects on GSIS, oxidative stress rate and insulin sensitivity in rat islets, suggesting that long-term dietary supplementation with YM may improve glucose homeostasis in pre-diabetes or type 2 diabetes. Ilex paraguariensis A.St.-Hil. (Aquifoliaceae). This socializing beverage is widely consumed in Argentina, Uruguay, Paraguay, and some regions in Brazil. It has been introduced to the European and North American markets as both an herbal beverage and food supplement or herbal remedy with psycho-stimulant, antioxidant, diuretic, and tonic claims. In fact, Yerba mate has been amply shown to be enriched in various compounds with significant in vitro and in vivo biological activities, including antioxidant and anti-inflammatory properties /22.5. β-cell function was quantified by HOMA-β [insulin (μU/L) × 20/glucose (mmol/L)] − 3.5. Since these indexes have been clinically validated in humans but not in rodents, we here compare the values of treatment groups to the values of the control group instead of using a cut-off threshold value.FRAP levels were determined by a colorimetric method (FRAP assay) and expressed as µmol Fe2+/L. Circulating insulin levels were assayed by RIA (antibody against rat insulin) from Sigma Chemical Co., rat insulin standard from Novo Nordisk Pharma, Argentina, and highly purified porcine insulin 2/O2 (5/95%) with 1% (w/v) BSA and either 3.3 or 16.7 mM glucose. After that, aliquots from the medium of each experimental condition were taken in order to measure insulin levels by RIA as described above. Insulin secreted into the incubation medium was expressed as ng of insulin/islet/hour.To elucidate the effect of dietary intervention with Yerba mate on ex vivo insulin secretion, the whole pancreas from each experimental animal was removed immediately after euthanasia and collagenase digestion was employed to isolate pancreatic islets . Ten gro2/O2 (5/95%) with 1% (w/v) BSA and either 3.3 or 16.7 mM glucose, in the absence (control) or presence of Yerba mate , or the different phenolic compounds mentioned above at 0.1 and 1 µM. As mentioned previously, after 1 h incubation period, samples from the medium were collected for insulin determination by RIA.To study the direct effect of Yerba mate and some of its main phenolic components, namely chlorogenic acid, rutin, caffeic acid and quercetin, 10 groups of five islets from untreated normal rats were incubated for 1 h at 37 °C in 0.6 mL Krebs–Ringer bicarbonate buffer (KRB), pH 7.4, previously gassed with a mixture of COTotal RNA was isolated from islets of Langerhans from both experimental groups using TRIzol Reagent . Integrity of the obtained RNA was verified by agarose–formaldehyde gel electrophoresis. Cross-contamination with protein or phenol was checked by measuring the 260:280 nm absorbance ratio. Samples with a ratio of 1.8 or lower were discarded. On the other hand, samples were treated with DNase I  to avoid DNA contamination. Reverse transcription-PCR was conducted with SuperScript III Reverse Transcriptase (Invitrogen), oligo-dT and 1 μg of total RNA as template. qPCRs were run in triplicate using FastStart SYBR Green Master mix (Roche) in the iCycler 5 (BioRad) employing 40 cycles . Every run was followed by a melting curve from 55 °C to 90 °C for checking the presence of only one amplicon. Sequences of oligonucleotide primers (Invitrogen) used in these experiments are shown in t-test was applied for independent samples with normal distribution, while ANOVA followed by the Tamhane test for similar variance samples were applied to evaluate significance in glucose-stimulated insulin release in the presence of phenolic compounds of Yerba mate. Results are expressed as mean ± SEM. Differences between groups were considered significant when p values were <0.05.For the statistical analysis of experimental data, the SPSS program  was employed. Student’s


Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.(1) Background: Ionic transport in  Trypanosoma cruzi, has a complicated life cycle that alternates between intermediate invertebrates and definitive mammal hosts [The etiological agent of Chagas disease, al hosts , and Chaal hosts ,3.T. cruzi growth and differentiation during its life cycle since it is a necessary micronutrient for all forms of life and a cofactor of many enzymes in a considerable number of metabolic pathways [Iron (Fe) is one nutritional element that controls pathways . Fe is apathways .2 at neutral pH [−9 and 10−18 M, which is lower than the concentration necessary for microbial development [−24 M [Fe is essentially physiologically inaccessible due to the limited solubility of its thermodynamically stable +3 oxidation state in the presence of Outral pH ,7. The celopment , and oxielopment . Furtherelopment ,14,15, rt [−24 M ,14,15. Tt [−24 M .Trypanosoma cruzi requires iron (Fe) for growth, in vitro proliferation of epimastigotes forms (mobilizing heminic or non-heminic Fe), and pathogenicity in mice [Trypanosoma cruzi can hijack Fe-proteins from mammalian hosts. In culture, adding deferoxamine, a Fe chelator, or transferrin-free serum can reduce amastigotes cell multiplication, demonstrating that Fe is an essential nutrient [Trypanosoma cruzi also uses heme as a Fe source; it can boost T. cruzi growth in culture in a dose-dependent way [T. cruzi epimastigotes internalize heme/porphyrin through a process that might be mediated by an ABC transporter protein [T. cruzi genome, the first heminic ring hydrolysis for Fe release is the limiting step for pathogenic trypanosomatids using heme [ in mice . Trypanonutrient . This panutrient . Transfenutrient . Trypanodent way . Further protein . Howevering heme .3+, it must be converted to Fe2+ by the Fe-reductase enzyme [3+ reduction is frequently linked to Fe2+ transport in bacteria, yeast, plant, and animal cells [Leishmania chagasi [L. amazonensis [T. cruzi [2+ transport mechanism in trypanosomatids.Since Fe is present in aerobic conditions as Fee enzyme  to be tral cells . As a re chagasi , L. amazzonensis , and, suT. cruzi  was a st3+ to Fe2+, followed by the absorption of Fe2+ by specialized transporters [L. amazonensis, which belongs to the zinc and iron transporter family (ZIP family), gave early support to this idea. ZIP family members are said to be capable of transporting Zn2+; however, some members of this family can also transport Fe2+ [T. cruzi [2+ uptake in this parasite.The discovery of Fe-reductase activity in trypanosomatids reinforced the hypothesis of a two-step Fe transport mechanism: first, the reduction of Fesporters . In addisporters ) in the ort Fe2+ . RecentlT. cruzi  supportsT. cruzi  and TcITT. cruzi blood trypomastigotes. A putative Fe transporter in the form of a metal ion in the acidocalcisome of T. cruzi supports the hypothesis of Fe storage in this organelle [Due to its low redox potential, Fe is a suitable element for redox catalysis processes ,31, actirganelle . This merganelle .T. cruzi’s redox status and metabolic pathways, as well as the proliferation and differentiation of the parasite. The study revealed molecular mechanisms and intracellular processes modulated by exogenous ionic Fe that had not previously been reported.Although Fe is a critical micronutrient for trypanosomatids, as previously stated ,10,11,12Trypanosoma cruzi (Dm28c strain) epimastigotes were grown in stationary phase at 28 °C in Brain Heart Infusion (BHI) medium supplemented with 10% FBS, 30 µM hemin, and 1% penicillin-streptomycin (P/S) cocktail . Iron-Depleted Media (IDM) was prepared using an iron-free BHI medium, as described by Dick et al. [6 cells/mL) into the BHI medium on the sixth day of growth to test epimastigotes proliferation . Every day, cell proliferation was measured by counting the number of cells in a hemocytometer. Dm28c is a strain that differentiates “in vitro” and was previously used in our previous studies that demonstrated the existence of a Fe-reductase and a Fe-transporter in T. cruzi [k et al. . BrieflyT. cruzi ,29. This8 parasites/mL in triatomine artificial urine (TAU) medium  NaHCO3, and 8 mM phosphate buffer at pH 6.0). After 2 h at 28 °C, the cultures were diluted 100-fold in 10 mL TAU medium supplemented with 10 mM proline and 250 mM glucose (TAU-P) plus 500 g/mL G418  and transferred to T25 flasks—lying at a 45° angle to increase the area in contact with O2—and kept at 28 °C to promote metacyclogenesis. Following 3–5 days, parasites were counted using hemocytometry, and the proportion of metacyclic epimastigotes (Tryp) was determined using their morphology after Giemsa staining.Metacyclogenesis was induced according to Koeller et al. . Epimast8 parasites were obtained from various cultures and washed three times with PBS pretreated with 5 g/100 mL Chelex resin (Sigma-Aldrich). The cells were lysed with 100 µL of 50 mM NaOH, and then 100 µL of 10 mM HCl was added; the release of ionic Fe bound to intracellular structures was induced by adding 100 µL of a mixture containing 1.4 M HCl and 4.5% (w/v) KMnO4 (1:1) to the cell lysate, followed by incubation at 60 °C for 2 h. Then, 30 µL Fe detection reagent was added . The sample’s absorbance at 550 nm was measured after 30 min of incubation at room temperature. A standard curve with known FeCl3 concentrations   from epimastigotes kept at RM, IDM, or IDM + Fe for 5 days (as indicated in the figure legends). The high-capacity cDNA reverse transcription kit was used to reverse-transcribe whole RNA . For RT-PCR, 100 ng/µL cDNA per well  was utilized, coupled with a 5 µM primer mix and a 7 µL PowerUp SYBR green master solution (Thermo Fisher Scientific). Primers were designed using the Primer3 software, with predicted amplicon sizes of 100 pb each [Total  pb each . The pri pb each .v/v) paraformaldehyde in phosphate-buffered saline  for 1 h for flow cytometry and imaging using the fluorescence microscope AxioObserver  after staining with DAPI.Epimastigotes were submitted to endocytosis with 30 µg/mL of transferrin-FITC, hemoglobin-FITC, or BSA-FITC in Roswell Park Memorial Institute (RPMI) medium for 30 min at 28 °C. The parasites were fixed with 4% , counting 10,000 events at the FL2 channel. The data were analyzed by the BD Accuri C6 software. This analysis was performed in three independent experiments.v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature. After a wash in cacodylate buffer, cells were post-fixed using an osmium-thiocarbohydrazide-osmium (OTO) protocol as already described [w/v) uranyl acetate and lead citrate, and observed in an HT7800 transmission electron microscope  operating at 100 kV.Samples were fixed with 2.5% (escribed . Samples7 cells/mL) were washed twice in ice-cold phosphate buffer saline  and lysed in 0.5 mL radioimmunoprecipitation assay buffer (RIPA buffer) for 30 min. PKA activity was assayed in the presence of 4 mM Hepes-Tris (pH 7.0), 0.4 mM MgCl2, 1 mM CaCl2, 1 µM ATP, and 50 µg of lysed cells in a final volume of 50 µL, in the presence or absence of 5 µM 3-isobutyl-1-methylxanthine , in MTS-11C mini tubes . The reaction was triggered by adding 50 μL of the Kinase-Glo luminescent kit, and after 10 min at 37 °C, the samples were placed in a GloMax Multi JR detection system . PKA activity was quantified as the difference between the reading in the presence of IBMX and in the absence of the activator.Epimastigotes cells (5 × 107 parasites/chamber) was measured using an O2k-system high-resolution oxygraph  at 28 °C with continuous stirring. The cells were suspended in a 2 mL respiration solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris–HCl (pH 7.2), and 50 μM digitonin was added to permeabilize the parasites. Following that, 10 mM succinate and 200 μM ADP were added. Uncoupled respiration was induced with 3 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and then blocked with 2.5 μg/mL antimycin A to assess residual O2 consumption [2 consumption were recorded using DatLab software coupled to Oxygraph-2K.Oxygen consumption of intact epimastigotes  was determined by the increase in absorbance due to the reduction of ferricytochrome c at 550 nm ,41. Frozstandard .T. cruzi epimastigotes using the MitoProbe JC-1 assay kit . Epimastigotes (1 × 107 cells/mL) were loaded with 10 µM 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) and incubated for 40 min at room temperature. The fluorescence intensity ratio of red (540 nm excitation and 590 nm emission) to green (490 nm excitation and 540 nm emission) was measured using a multi-well fluorescence reader [Mitochondrial membrane potential was analyzed in the e reader .i) was measured using an ATP bioluminescent somatic cell test kit (Sigma-Aldrich). In brief, epimastigotes (1 × 107 parasites per tube) were incubated in a solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris-HCl in 0.1 mL (pH 7.2 adjusted with HCl). Cellular extracts were prepared by combining them with 0.1 mL of somatic cell ATP-releasing reagent and then chilling the mixture for 1 min. The mixture was transferred to MTS-11C mini tubes containing 0.1 mL ATP assay mix  and swirled for 10 s at room temperature. A GloMax Multi JR detection system (Promega) measured the overall quantity of light emitted. In each experiment, the total intracellular ATP concentration per 107 cells was determined using a standard ATP curve [Intracellular ATP  (Leuconostoc mesenteroides), 0.1% Triton X-100, 1 mM NaF, 5 mM NaN3, 1 mM ATP, and 50–100 μg/mL protein [+, and quantified spectrophotometrically following the reduction of β-NADP+ to β-NADPH (λ = 340 nm) for 10 min. Total NADPH generation was determined using the NADPH standard curve.Cellular extracts of epimastigotes were incubated in a reaction buffer containing 20 mM Tris-HCl (pH 7.4), 5 mM MgCl protein . After 3w/v) Tween 20, probed overnight at 4 °C with the primary mouse anti-GAPDH antibody , and detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody . The loading control was probed with a primary rabbit anti-tubulin antibody  and detected using an HRP-conjugated anti-rabbit IgG secondary antibody . Luminescence was detected using an ImageQuant LAS 4000 digital imaging system  after the reaction with LuminataTM Forte Western HRP Substrate . Densitometric analysis was performed using ImageJ software version 1.50i  with background correction.For western blotting detection, epimastigotes were lysed with 1 mL RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and 5 mM leupeptin, for 30 min at 4 °C. Then, homogenates were centrifuged at 16,000 rpm for 10 min. Aliquots of the supernatants  were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes , which were blocked with 5% milk in PBS plus 0.1%  to the simultaneous oxidation of the non-fluorescent Amplex® Red probe to the fluorescent resorufin [7 parasites/mL were incubated with 5 mM Tris-HCl (pH 7.4), 1.7 μM Amplex® Red , and 6.7 U/mL horseradish peroxidase (Sigma-Aldrich) in a final volume of 100 µL, for 30 min at room temperature. Fluorescence evolution was observed at excitation/emission wavelengths of 563/587 nm. H2O2 concentration was determined using a standard curve.The rate of Hesorufin . Brieflyt-test was used to compare two means. When comparing more than two means, a one-way ANOVA with Tukey’s test was applied, as specified in the text or the figure captions. The normal distribution was assessed before each ANOVA analysis. When results were expressed as a percentage of the RM group, the SEM was obtained from the absolute values. A comparison of the absolute results was carried out using the parametric test. Significance was set at p < 0.05. Except when otherwise indicated, different lowercase letters in superscripts indicate statistical differences among means in the same line of tables. Asterisks  were used in figures and for comparing values from different lines in figures. GraphPad Prism 7.0 was used for statistical analysis and the preparation of figures .The data are provided as mean ± standard error of the mean (SEM). The unpaired Student’s T. cruzi epimastigotes during the exponential growth phase . The proliferative capacity of the cells was recovered with 8 µM Fe citrate added to the Fe-depleted medium : the number of protozoa after 7 days increased from 1.0 × 107 to 5.1 × 107 parasites/mL, a condition similar to the control. In addition, Fe citrate supplementation was enough to restore intracellular Fe concentration compared to RM, while cells maintained at IDM presented low intracellular Fe content  to IDM compared to RM and IDM + Fe (line 1). The O2 consumption in the presence of succinate without further additions matched the succinate-cytochrome c oxidoreductase activity (line 5). However, after additions of ADP (oxidative phosphorylation state-OXPHOS) (line 2) and H+ ionophore FCCP (electron transfer system uncoupled from phosphorylation-ETS) (line 3), there was a significant and similar decrease in O2 consumption in both IDM and IDM + Fe. After adding antimycin A to inhibit the mitochondrial complex III, the residual respiration (ROX) was almost completely abolished under all conditions (line 4). The impact of Fe depletion (even after Fe citrate supplementation) on mitochondrial internal membrane potential (ΔΨm) can be seen in line 6. The ratio between the safranin A fluorescence in the absence and presence of FCCP (F/FFCCP) increased by 600% in parasites grown in IDM and attained an intermediary level ~200% higher in IDM + Fe conditions.As mentioned above, mitochondrial proteins decrease when the HRI→eIF2α pathway is dysfunctional , as encoi) more than twice as high when compared to parasites grown in IDM or IDM + Fe media (oF1-ATP synthase [i content by more than 50% with respect to the RM group without inhibitors (line 8), indicating that, under these conditions, the cellular ATP supply comes from mitochondrial activity. Moreover, the addition of iodoacetamide (line 9), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase (GAPDH): (i) reduced ATPi in parasites grown in RM to levels that are similar to those encountered in IDM and IDM + Fe conditions in the absence or presence of oligomycin; (ii) provoked a further 50% decrease in ATPi from IDM and IDM + Fe parasites when compared to that grown in RM, indicating that the glycolytic pathway is responsible for ATP production in IDM and IDM + Fe conditions.Parasites maintained in the RM medium presented intracellular ATP content  superoxide dismutase (TcSOD), which has Fe as a cofactor in T. cruzi [2O2 to H2O. We measured TcSOD activity by following two strategies. First, by evaluating the formation of nitroblue tetrazolium formazan (NBT formazan) after oxidation of xanthine to uric acid and generation of O2•−, a reaction in which the higher superoxide dismutase activity is, the lower NBT formazan formation will be. Second, measuring the H2O2 formed through different processes, including the premature transfer of electrons to O2 in the T. cruzi mitochondrion. 2•− formed during the oxidation of xanthine increased by approximately 200% in parasites grown in IDM regardless of supplementation with Fe. Moreover, when the total H2O2 production was assayed, a significant decrease of about 40% was found in IDM parasites, which was suppressed in the IDM + Fe group. Finally, the expression profile of TcSOD was the mirror of the activity, whereas that of TcAPX matched the activity, i.e., decreased in the parasites grown in IDM, with recovered levels in the IDM + Fe group.The mutual regulation of hexokinases and redox signaling comprises important events in parasites that cause severe diseases, e.g., T. cruzi . This isT. cruzi , and  were neither added to IDM nor to IDM + Fe, TcFR is upregulated in both cases . While FeSODB has a crucial role in the defense of parasites against O2•− , FeSODA 2•− [2O2 . A downrrentiate .2O2 formation, which stimulate parasite differentiation, thus interfering with parasite virulence. These related mechanisms and processes modulated by exogenous ionic Fe have implications for human health because, by providing energy support for parasite growth and differentiation, they ensure the continuity of the T. cruzi life cycle and the propagation of Chagas disease.Finally,


Microbacterium proteolyticum ustc, a member of the Gram-positive order Micrococcales of the phylum Actinomycetota that is resistant to high concentrations of heavy metals and participates in metal detoxification. The genome consists of one plasmid and one chromosome.A complete genome is presented for  Microbacterium spp. include >110 cultivable strains isolated from anaerobic waste digesters   (16 mg/L) was added to the serum vials containing 50 mL mineral medium  of 0.3. Samples were taken periodically, and the absorbance of supernatants at 540 nm was measured to monitor Cr(VI) levels  , was enriched by adding heavy metal chromium ions 1 mM Cr2O72−)  to the l2−  to thl medium  with 20 ) levels . After 1) levels .N50, 18,951 bp; N90, 2,717 bp) were acquired. AfterQC filters out bad reads , detects and eliminates the sequencer’s bubble effects, trims reads at the front and tail, detects sequencing errors and corrects some of them, and finally outputs clean data; it generated 102,534 reads , which ensured that filtered reads could be assembled using Canu v1.5  following the guidelines provided by the manufacturer. The sequencing libraries were generated using a rapid barcoding kit SQK-LSK109) and sequenced in an R9.4 flow cell with an Oxford Nanopore Technologies system (9 and seqanu v1.5 .Racon v3.4.3 software  was utilGene prediction was performed by using Prodigal v2.63 software , whereasSAMN34004116, BioProject accession number PRJNA950593, and SRA accession number SRR24745629, and the assembly is available under GenBank assembly accession number GCA_029639405.1.The genome sequence has been submitted to GenBank under BioSample accession number


Additionally, the cell was tested toward the detectionof oxalate within a spiked synthetic urine sample, obtaining recoveriesof 104%. This work highlights how, using mixed material composites,excellent electrochemical performance can be obtained at a reducedmaterial cost, while also greatly improving the sustainability ofthe system.Mixing of graphiteand carbon black (CB) alongside recycledpoly(lacticacid) and castor oil to create an electrically conductive additivemanufacturing filament without the use of solvents is reported herein.The additively manufactured electrodes (AMEs) were electrochemicallybenchmarked against a commercial conductive filament and a bespokefilament utilizing only CB. The graphite/CB produced a heterogeneousrate constant,  To improve patient care, real-time measurementsof key analytes are sought after. Electroanalytical sensing platformscan realize this due to their ability to gather highly sensitive,selective, and fast measurements while being easily miniaturized andlow cost.Oxalate , especiallyfused filament fabrication (FFF), is seeing a surge in popularitydue to its many benefits,17 The use of single-shot electrodes is attractivein healthcare monitoring settings due to the necessity to avoid crosscontamination between samples or patients. However, this leads tomassive amounts of waste within the sector, with an estimated 1.6million tons of plastic waste a day being consumed during the COVID-19pandemic.18 The sustainability of practiceshas become increasingly important, with the United Nations outlining17 sustainable goals for development globally.19 Goal 12 of this initiative is to “ensure sustainableconsumption and production patterns”, which is challengingfor areas that require single-use items. To help improve the sustainabilityof AM electrochemistry, Sigley et al.20 produced bespoke conductive FFF filament from recycled coffee machineplastic pod feedstock that later was successfully applied for theelectrochemical detection of caffeine. Further improvements in thesustainability of conductive filament production were made throughthe introduction of castor oil as a biobased plasticizer.21 Additionally, it has recently been shown howused AM electroanalytical sensing platforms can be recycled into anew filament (both conductive and nonconductive) for the productionof sensors.22Most additivelymanufactured electrodes (AMEs) are a single-useitem due to difficulties replenishing the surface of the electrodeand the ingress of solution within the polymer matrix.23 CB (Super P) is produced from thepartial oxidation of petrochemical precursors and costs approximately£300 per 100 g for the specific grade used.20 On the other hand, graphite is naturally occurring andthe most stable form of carbon under standard conditions, with a costroughly 1% of the CB used above. Due to its good thermal stability,corrosion resistance, high specific strength, and electrical conductivity,graphite is commonly used to produce electrodes for electrochemicalapplications, such as within ink formulations to produce screen-printedelectrodes.24 While a conductive graphite/poly(lacticacid) (PLA) filament has been reported in the literature, it had aresistance of 6 kΩ cm–1, showed poor performancewhen compared to nanocarbon and commercial options, and was producedusing multiple steps and dichloromethane.25Within the work above,carbon black , was added asa conductive filler with a loading of ∼25 wt %, increasingthe amount found in the commercial conductive filament by ∼4.5wt %.26 This work highlights how usingmixed carbon material composites can create conductive filament withenhanced electrochemical properties and significantly lower the materialcost while using recycled polymer and biobased plasticizers to improvethe sustainability of the process and the use of additive manufacturingallows for bespoke cell designs to be produced for specific applications.In this work, we propose the development of conductive filamentfor FFF through a composite of CB and graphite in recycled PLA withthe biobased plasticizer castor oil to increase the low-temperatureflexibility of the filament. Utilizing a single mixing process, thepolymer is subjected to minimal thermal steps for filament productionand removes the use of harsh chemicals such as dichloromethane seenin previous reports.20 All solutions were prepared with deionized water of resistivitynot less than 18.2 MΩ cm from a Milli-Q Integral 3 system fromMillipore UK .Hexaamineruthenium(III) chloride (98%), castoroil, potassium ferricyanide (99%), potassium ferrocyanide (98.5–102%),sodium hydroxide (>98%), sodium oxalate (≥99.5%), potassiumchloride (99.0–100.5%), sodium sulfate (≥99.0%), ammoniumchloride (≥99.5%), potassium phosphate monobasic (≥99.0%),calcium chloride (≥96.0%), sodium chloride (≥99.0%),graphite powder (<20 μm), and phosphate-buffered saline (PBS)tablets were purchased from Merck . CB ,urea (98+%), creatinine (98+%), and hydrochloric acid (37% ACS grade)were purchased from Fisher Scientific . RecycledPLA was purchased from Gianeco . Commercial conductivePLA/CB filament  was purchasedfrom Farnell . Recycled nonconductive PLA filament wasproduced in-house as previously reported.Recycled PLAwas dried in an oven at 60 °C for a minimum of 2.5 h before anymixing or filament production. The polymer composition was preparedusing 65 wt % rPLA, 10 wt % castor oil, 15 wt % CB, and 10 wt % graphitepowder. These are mixed (190 °C) with Banbury rotors (70 rpmfor 5 min) using a Thermo Haake Poydrive dynameter fitted with a ThermoHaake Rheomix 600 . This is allowed to coolto room temperature before being granulated to create a finer granulesize using a (Rapid Granulator 1528). This is next processed throughan EX6 extrusion line  using a singlescrew with heat zones of 60, 190, 195, and 195 °C, respectively,which are extruded from a 1.75 mm die head. Then, the filament isready to use for Additive Manufacturing (AM).–1. Details of the physiochemical characterization and electrochemicalexperiments are reported in the Supporting Information.Computer designs are producedusing Fusion 360 . These files were slicedand converted to .GCODE files, which are printed by open-source software,PrusaSlicer . The AMEs were3D-printed using FFF technology on a Prusa i3MK3S+ . All AMEs were printed using a 0.6 mm nozzlewith a nozzle temperature of 215 °C, 100% rectilinear infill,0.15 mm layer height, and print speed of 70 mm s27 where 0.28 g CaCl2, 0.73 g NaCl, 0.35 g KH2PO4, 0.40 g KCl, 0.25g NH4Cl, 0.56 g Na2SO4, 6.25 g urea,and 0.28 g creatinine were dissolved in 250 mL of ultrapure water.The pH was adjusted to 6.0 using a 1.0 M HCl solution. The samplewas first spiked with 500 μmol L–1 sodiumoxalate salt and then diluted (∼20-fold) in the supportingelectrolyte for a standard addition study.A synthetic urine sample was prepared according to Laubeet al.,21 This methodology is a single thermal mixing step, which removesany requirement for additional solvents seen within other reportedmethods.26 The filament produced had excellent room-temperatureflexibility, as shown in 21The productionof additive manufacturing filament from recycled PLA(rPLA), castor oil, CB (Super P), and graphite was achieved in thesame way as reported previously and is presented in 21 and was a significant improvement upon the commercial conductivefilaments reported values of 2–3 kΩ. The significantimprovement in the commercial system is primarily due to the qualityof the conductive filler and increased carbon loading. Thermogravimetricanalysis of the graphite/CB filament, Table S1. It can be seen that the rPLA used throughoutthis study had an average onset of degradation temperature of 304± 4 °C, which showed good agreement with previous work.28 As previously reported, castor oil produced an onset temperatureof 250 ± 3 °C.21 The bespokegraphite/CB filament produced an average onset of degradation temperatureof 283 ± 4 °C, which indicated that the conductive carbonfillers provided a stabilizing effect by acting as a barrier for gasdiffusion out of the polymer, slowing the rate of decomposition.29 The onset temperature seen for the graphite/CBfilament is similar to that seen for the CB only filament publishedpreviously,21 indicating that replacingsome CB with graphite is not detrimental to the thermal stabilityof the filament.The graphite/CB filamentgave a resistance across10 cm of thefilament of 875 ± 38 Ω, which is statistically the sameas the filament using only CB reported previously21 Additionally, in 31 This peak was not required in previously reported work on only CB/castoroil filaments and suggests that the graphite flakes could penetratethe print surface, where the CB was mostly embedded below the rangeprobed by XPS . In the activated AME C 1s spectrum, 31The chemical composition of the printed AMEswas examined usingX-ray photoelectron spectroscopy (XPS) and scanning electron microscopy(SEM), before and after electrochemical activation. The as-printedand activated AME C 1s spectra are shown in Supporting Information bringsthe SEM images of the CB-only filament, where the same aspects areobserved before and after activation , but with the absence of the flakes attributed to the graphiteinserted in the new filament.Overall, these data provideevidence for the electrochemicalactivationprocedure successfully removing surface PLA to reveal increased amountsof conductive graphitic material below, which should improve the electrochemicalperformance of the AME toward inner-sphere molecules. SEM images frombefore and after the electrochemical activation, 32 A summary of thefindings from all of the electrochemical characterization can be foundin Ep) obtained for [Ru(NH3)6]3+ (1 mM in 0.1 M KCl) at 25 mV s–1 scan rate, the calculated k0 and Ae, along with the solution resistance (Rs) and charge-transfer resistance (Rct) from electrochemical impedance spectroscopy in a solutionof ferri/ferrocyanide ([Fe(CN)6]3–/4–) (1 mM in 0.1 M KCl) for the graphite AME and two benchmark filaments.The electrochemicalperformance of the AMEs printed with the graphite/CB filament wastested using electrodes 3D-printed as a simple lollipop-shaped design, 3)6]3+ (1 mM in0.1 M KCl), k0, and the real electrochemicalsurface area of the AME, Ae.34 A comparison of the CV response to [Ru(NH3)6]3+ at 25 mV s–1 from the bespoke graphite/CBfilament, the commercially purchased CB/PLA filament, and a bespoke,previously published,21 CB/rPLA filamentis presented in k0 and Ae values obtained.Initially, the as-printed graphite/CB AMEs were testedusing cyclicvoltammetric scan rate studies against the near-ideal outer-sphereredox probe [Ru(NH6]3–/4– (0.1 M KCl). The response measured at 25mV s–1 represented in 6]3–/4– (0.1 M KCl). Once again, the graphite/CB/rPLAfilament performed well with an Rct of609 ± 166 compared to 2842 ± 458 for the commercial filament.It can be seen from Rs values of 155 ± 15 and 177 ± 23 for the graphite/CB/rPLAand CB/rPLA AMEs, respectively. These values were significantly reducedfrom the commercial filament, which produced an Rs value of 768 ± 96 Ω.Activated AMEs made from graphite/CB/rPLA, CB/rPLA,and commerciallypurchased CB/PLA were tested against 1 mM [Fe(CN)The characterizationof the graphite/CB/rPLA filament indicatesthe electrochemical performance is vastly improved when compared tothe commercially available filament and close to the performance ofthe bespoke lab-made CB/rPLA filament while replacing 40 wt % of theconductive filler with a significantly lower cost material. Basedon the prices from the Super P CB supplier, graphite powder is approximately12 times cheaper than CB, offering a significant improvement on theAME cost when used. Overall, the cost of producing a single electrodeusing the graphite/CB composite was £0.05, reduced from £0.09for an AME produced using only the bespoke CB filament.Figure S2A-C shows the design of the additivemanufactured electrochemical cell used in this work, where the threeelectrodes are inserted into the removable lid, allowing them to bewashed and reused after each analysis, saving cost to the user. TheAME working electrode has been designed to be removable from the system,allowing for a new AME to be used on each analysis, avoiding samplecontamination and any issues with solution ingress.17 With the design of an appropriate cell, the next challengeis applying the AMEs toward detecting oxalate. According to the literature,the electrochemical oxidation of oxalate involves a two-electron mechanismto produce CO2,35 as shown in Figure S3.2SO4 usinga graphite/CB electrode. A very well-defined peak was observed inNa2SO4 (0.1 M). Figures S4 and S5, with the best resultsobtained using astep potential of 3 mV and an amplitude of 60 mV. Then, optimizedDPV was used to produce the analytical curves for the detection ofoxalate using AMEs printed from both commercial conductive filament, Figure S6, and the bespoke graphite/CB filament, I (μA) = 0.0196 COxalate (μM) + 0.0223; R = 0.9996), compared to 80–500μM (I (μA) = 0.0045 COxalate (μM) – 0.1628; R = 0.9947) for the commercial AME. The graphite/CB filament gaveimproved electroanalytical results in all cases, with a sensitivityof 0.0196 μA/μM, limit of detection (LOD) of 5.7 μM,and limit of quantification (LOQ) of 18.8 μM. These resultscompared well with other electrochemical sensors reported within theliterature for oxalate detection, as summarized in Table S2.First, DPV parameterswere optimized, as shownin Figure S7, with a relativestandard deviation of 7.1% obtained over the course of 8 measurements.Furthermore, the selectivity of the electroanalytical platform wasassessed by analyzing oxalate in the presence of twice the concentrationof common interferents, such as ascorbic acid, uric acid, and glucose, The repeatability of oxalate measurements with graphite/CBAMEwas then tested, Figure S8, to mimic the electroanalyticalsensing platform use within a real-world application. Using the celldesigned in Figure S2, a sample could beadded to the container, filling the line. The electrodes were placedwithin the marked slots and the lid was screwed into place to ensurea reproducible experimental setup. A good recovery value of 104% wasobtained, highlighting the applicability of this AM electroanalyticalsensing platform for detecting oxalate in urine.The detection of oxalate was then performed intoa spiked syntheticurine sample, k0 of (1.26 ±0.23) × 10–3 cm s–1 and AMEresistance of only 155 ± 15 Ω, compared to (0.30 ±0.03) × 10–3 cm s–1 and 768± 96 Ω for the commercial AME, respectively.This work has presented the productionand characterization ofa graphite/CB composite filament utilizing recycled PLA and castoroil as a plasticizer. The filament was physiochemically characterizedthrough TGA, XPS, and SEM to provide evidence for successfully incorporatingthese materials into an additive manufacturing filament. The graphite/CBfilament was electrochemically characterized against a previouslyreported filament containing only CB and a commonly used commercialconductive filament. The cost of printing AMEs from the graphite/CBfilament was reduced to £0.05 per electrode, compared to £0.09for the previously reported CB-only filament. The graphite/CB performedwell compared to the CB-only filament and outperformed the commercialfilament, producing a A bespokeAM cell was designed to allow for the simple and quickdetermination of oxalate within urine samples. This was used to testthe electroanalytical performance of the graphite/CB AMEs, where asensitivity of 0.0196 μA/μM, LOD of 5.7 μM, andLOQ of 18.8 μM was obtained. The graphite/CB AMEs showed excellentreproducibility and selectivity against excess concentrations of commoninterferents. Additionally, the cell was tested for the detectionof oxalate within a spiked synthetic urine sample, obtaining recoveriesof 104%. This work highlights how through the use of mixed materialcomposites, excellent electrochemical performance can be obtainedat a reduced material cost while also improving the sustainabilityof the system.


MHC class II molecules are essential for appropriate immune responses against pathogens but are also implicated in pathological responses in autoimmune diseases and transplant rejection. Previous studies have shed light on the systemic contributions of MHC haplotypes to the development and severity of autoimmune diseases. In this study, we addressed the B cell intrinsic MHC haplotype impact on follicular inclusion, germinal center (GC) participation and plasma cell (PC) differentiation in the context of systemic lupus erythematosus (SLE).We leveraged the 564Igi mouse model which harbors a B cell receptor knock-in from an autoreactive B cell clone recognizing ribonuclear components, including double-stranded DNA (dsDNA). This model recapitulates the central hallmarks of the early stages of SLE. We compared 564Igi heterozygous offspring on either H2b/b, H2b/d, or H2d/d background.This revealed significantly higher germinal center (GC) B cell levels in the spleens of H2b/b and H2b/d as compared to H2d/d (p<0.0001) mice. In agreement with this, anti-dsDNA-antibody levels were higher in H2b/b and H2b/d than in H2d/d (p<0.0001), with H2b/b also being higher compared to H2b/d (p<0.01). Specifically, these differences held true both for autoantibodies derived from the knock-in clone and from wild-type (WT) derived clones. In mixed chimeras where 564Igi H2b/b, H2b/d and H2d/d cells competed head-to-head in the same environment, we observed a significantly higher inclusion of H2b/b cells in GC and PC compartments relative to their representation in the B cell repertoire, compared to H2b/d and H2d/d cells. Furthermore, in mixed chimeras in which WT H2b/b and WT H2d/d cells competed for inclusion in GCs associated with an epitope spreading process, H2b/b cells participated to a greater extent and contributed more robustly to the PC compartment. Finally, immature WT H2b/b cells had a higher baseline of BCRs with an autoreactive idiotype and were subject to more stringent negative selection at the transitional stage.Taken together, our findings demonstrate that B cell intrinsic MHC haplotype governs their capacity for participation in the autoreactive response at multiple levels: follicular inclusion, GC participation, and PC output. These findings pinpoint B cells as central contributors to precipitation of autoimmunity. Similarly, there are three pairs of classical class II alpha and beta chain genes, encoding HLA-DP, -DQ and -DR, which are expressed on the surface of professional antigen-presenting cells and function to present peptides to T-helper cells. The class I locus is mirrored in mice by H2-K, H-2D and H-2L, each encoding a classical class I molecule in combination with β2-microglobulin. A number of strains, including C57BL/6 mice, however, lack the H2-L allele. The class II locus is mirrored by I-A and I-E, which encode two isoforms of classical class II alpha and beta chain genes. In several commonly used inbred strains of mice, including C57BL/6, 129 and BXSB, the I-E alpha-chain is a pseudogene, leaving only a single functional isoform of MHC-II (The major histocompatibility complex (MHC) locus is well-established as the main genetic determinant of susceptibility to infections and autoimmune diseases , 2. In hf MHC-II .in vivo in the context of a physiological response has historically been challenging as contributions from other antigen-presenting subsets such as dendritic cells and macrophages could not be excluded. Here we leverage a well-characterized B cell receptor (BCR) knock-in model with specificity for nucleic acid autoantigens, the 564Igi model , less is known about the isolated role of specific subsets of APCs. Among APC subsets, B cells are particularly interesting because they are subject to elaborate diversification and selection processes which depend on their capacity to present peptides to T cells, and because their antigen-presentation is intimately linked with their adaptive receptor specificity. Thus, B cells have a powerful capacity to focus the immune response to particular antigenic targets . This isgi model , whereby22.1d/H2bByJ, JAX stock #000359) were obtained from Jackson Laboratories. 564Igi mice (B6.129S4(Cg)-tm1(Igk564)Tik Ightm1(Igh564)Tik/IgkJ) Tik/J)  were kin2.2564Igi Heavy chain: A ddPCR assay was set up for 564Igi heavy chain using 564Igi Heavy probe (TGA CAT CTG AGG ACT CTG CGG T) with 5’-FAM and 3’-MGBEQ, 564Igi Heavy Forward primer (TCC AGC ACA GTC TAC ATG CAA) and 564Igi Heavy Reverse primer (CCC CCG ATC TTG CAC AGT AAT), using a HEX-probe based housekeeping assay (Bio-Rad 10042962).564Igi Kappa chain: a ddPCR assay was set up for 564Igi Kappa chain using 564 Kappa probe (ACC CAC TCA CGT TCG GTG CTG) with 5’-HEX and 3’-MGBEQ, 564Igi Kappa Forward primer (AGG CTG AAG ATG CTG CCA TT), and 564Igi Kappa Reverse primer (TTC AGC TCC AGC TTG GTC C), using a FAM-probe based housekeeping assay (Bio-Rad 10042959).Both reactions were run with the following conditions: 95°C 5 min, then 40 cycles of 94°C 30 s, 62°C 45 s, and 72°C 45 s, followed by 96°C 5 min, and 12°C hold.2.3Assay 1: a ddPCR assay for H2b vs. H2d was established based on JAX Protocol 18706, using primers 22205 (CAC AGG AGA GAG GAT GTT CTG) as forward primer and 22206 (AGG GTT GCA GAT CCA TAA TTG) as reverse primer, with MGB probes 21943 (ATG CGA TGC GTG ATG AAG) carrying a FAM (channel 1) for H2b detection and 21944 (AAG GAT GCG TGA TGA AGG AT) carrying a HEX (channel 2) for H2d detection.Assay 2: a ddPCR assay for H2b vs. H2d was established based on JAX Protocol 21973, using primers 22202 (CCA AGA ACC TCC TCT CTC ATG T) as forward primer and 22203 (GCA GAT GTC CCA CCC TAT TC) as reverse primer, with MGB probes 21926 (ATG GCT GGG CAG AAG AGA G) carrying a HEX (channel 2) for H2b detection and 21928 (ATG GCT GGG CTG AAG AGA G) carrying a FAM (channel 1) for H2d detection.Both reactions were run with the following conditions: 95°C 5 min, then 40 cycles of 94°C 30 s, 60°C 45 s, and 72°C 45 s, followed by 96°C 5 min, and 12°C hold.2.4All animal experiments were conducted in accordance with the guidelines of the European Community and were approved by the Danish Animal Experiments Inspectorate (protocol numbers 2017-15-0201-01348 and 2017-15-0201-01319).2.5g, 10 min, 4°C) and resuspended to 1*108 cells/mL. The donor cell mixtures were used to reconstitute the recipient mice by retroorbital injection of 200 µL  into each recipient mouse.Recipient mice were irradiated with 9 Gy  in a MultiRad 350 (Faxitron), with 350 kV, 11.4 mA, a Thoraeus filter , and with a beam-distance of 37 cm, as described . Irradia2.6g for 5 minutes at 4°C, lysed in RBC lysis buffer , incubated at RT for 3 minutes, centrifuged, and finally resuspended in FACS buffer. For histology, spleen slices were placed directly into cryo-molds containing Tissue-Tek O.C.T. media  and frozen at -20°C. Collected blood samples were allowed to coagulate for 1 hr, then centrifuged at 3,000 g for 10 minutes at room temperature, the supernatant was collected, and centrifuged again at 20,000 g for 3 minutes at 4°C. Serum samples were stored at -80°C.Mice were anesthetized with isoflurane , decapitated, and the blood was collected in 1.5 ml tubes. Spleen, IngLN and MesLN were harvested, and the spleen was divided using surgical scissors. Tissues were either placed into ice-cold FACS buffer for flow cytometry analysis or prepared for histology. For flow cytometry, spleens, and lymph nodes in ice-cold FACS buffer were mechanically dissociated using pestles and samples were filtered through 70 µm cell strainers. Spleen samples were centrifuged at 200 2.7g for 5 minutes at 4°C, supernatant was removed, and cells were fixed for 30 minutes in PBS, 0.9% formaldehyde  at RT. Following fixation, the plates were centrifuged at 200 g for 5 minutes at RT, the supernatant discarded, and the samples resuspended in FACS buffer. Flow cytometry evaluation was performed the following day using a 4-laser  BD LSRFortessa analyzer with FACSDiva software version 8.0.2 (BD Biosciences). Data were analyzed in FlowJo version 10.8.1. The hybridoma producing 9D11 anti-idiotypic antibody reactive toward the 564Igi clone was kindly provided by Elisabeth Alicot, Boston Children’s Hospital, and the antibody was produced, purified, and labeled in-house with iFluor-647. All other antibodies were from commercial sources.Twenty µL Fc-block  diluted 1:50 in PBS and 100 µL of each sample was added onto a 96-well plate then incubated for 5-10 minutes on ice. Antibodies and fixable viability dye  were diluted 1/500 and 1/2,000, respectively, in FACS buffer. One hundred µL antibody mix was added to each sample well and incubated for 30 minutes on ice. The plate was centrifuged at 200 2.83+-labeled streptavidin  diluted 1:1,000 in TBS/Tw containing 25 µM EDTA was subsequently added to the wells and incubated at RT for 1 hour. Finally, the wells were washed thrice with TBS/Tw, and 200 µL enhancement buffer  was added. The plate was shaken for 5 minutes and counts were read in a time-resolved fluorometry plate reader .FluoroNunc Maxisorp 96-well plates were coated with 100 µg/mL salmon sperm dsDNA  in PBS and incubated overnight at 4°C. Wells were blocked with 200 µL TBS containing 1% bovine serum albumin (BSA)  for 1 hour at RT and washed thrice with TBS/Tw ). Samples, standards, and quality controls were diluted in TBS/Tw containing 5 mM EDTA and 0.1% w/v BSA, and subsequently added in duplicates. The plates were incubated at 37°C for 1 hour. Wells were washed thrice in TBS/Tw and incubated with biotinylated goat-anti-mouse Ig , 1 µg/ml TBS/Tw, at 37°C for 1 hour. Wells were washed 3 times in TBS/Tw, and Eu2.9S. cerevisiae and kindly provided by Steffen Thiel, Aarhus University) in coating buffer ; 10 µg/ml acetylated bovine serum albumin (AcBSA) (Sigma B2518) in coating buffer; 1 µg/ml mouse IgG from serum (Sigma I5381) in coating buffer; or 20 µg/ml Zymosan (Sigma Z4250); and incubated overnight at 4°C. Wells were blocked with 200 µL TBS containing 1 mg/ml human serum albumin (HSA) for 1 hour at RT and washed thrice with TBS/Tw. Serum samples were diluted 1/100 and 1/200 (mannan), or 1/200 and 1/400 (AcBSA and mIgG) in assay buffer , or 1/40 and 1/80 (Zymosan) in alternative buffer , pH 7.4). Dilutions and setup were performed on ice, and 100 µl was added to each well. A standard curve was included based on serial dilution of 8 H2b/b sera arbitrarily assigned the value 1,000 mU. Following incubation for 1.5 hrs at 37°C, the wells were washed thrice with TBS/Tw/Ca2+  or TBS/Tw (Zymosan). For mannan, AcBSA and mIgG coats, biotinylated anti-mouse C4 Ab (Cedarlane CL7504) was added at 0.5 μg/ml TBS/Tw/Ca2+. For Zymosan coat, anti-mouse C3 Ab (Cedarlane CL7503) was added at 0.5 µg/ml TBS/Tw/Ca2+. Wells were incubated 1.5 hrs at room temperature with anti-C4 or anti-C3 Ab. The wells were then washed thrice with TBS/Tw/Ca2+  or TBS/Tw (Zymosan), and europium-labeled streptavidin 0.1 μg/ml TBS/Tw containing 25 μM EDTA was added. After 1 h incubation and wash, again thrice with TBS/Tw/Ca2+, enhancement solution (PerkinElmer) was added, followed by reading of time-resolved fluorescence in a plate reader .FluoroNunc Maxisorp 96-well plates were coated with 100 µl of: 1 µg/ml mannan  were performed for each data set. If two or more tests confirmed normality, it was accepted. If one test confirmed normality, QQ-plots were made to evaluate normality. Data that were neither normally distributed based on normality tests nor QQ-plots were log-transformed and re-tested for normality following the same procedure. Data in 33.1d/H2bByJ line (B6.H2d). The B6.H2d line is in essence a B6 mouse, in which the H2d locus of the BALB/c strain has been introgressed, so the only locus that differs between regular B6 and B6.H2d is exactly the MHC locus. We established ddPCR assays for genotyping with copy-number determination of 564Igi heavy and light chains . We also established two ddPCR assays to monitor the H2 locus during the backcrossing of 564Igi heavy and light chains from H2b onto H2d background . Assay 1 targeted the 3’ UTR of H2-Q4 in the Qa locus of the murine H2 complex on Chromosome 17. Assay 2 targeted the 5’ end of H2-Eb2 in the I locus of the murine H2 complex on Chromosome 17. The two assays are spaced ~1 megabase apart, proximal to each their end of the H2 complex, providing a check on potential cross-over events during back-crossing of 564Igi heavy and light chains to H2d background  and mesenteric lymph nodes (MesLN) for analysis by flow cytometry J is of the Igha allotype, whereas the C57BL/6J-derived WT Igh locus of B6 and B6.H2d mice is of the Ighb allotype. Hence in heterozygous 564Igi offspring on either of the three H2 haplotype combination backgrounds, B cells that express the knock-in receptor also carry the linked Igha constant region, and B cells that escape the knock-in and express a WT receptor carry the linked Ighb constant region. For several of the Ig isotypes, these allotypes can be distinguished by allotype-specific antibodies. In the case of the mature repertoire of B cells characterized by expression of IgM, we can thus distinguish 564Igi B cells as IgMa vs. WT B cells as IgMb, within individual mice. Consequently, we asked whether there were any differences in IgMa vs. IgMb levels in mice of the three H2 haplotype combinations. However, we observed only very minor variations in IgMa and IgMb levels between the groups of offspring, with no systematic variation across tissues , hence aTaken together, our findings indicated that overall, the primary repertoire of B cells in 564Igi heterozygotes on either of the three H2 haplotype backgrounds was comparable. Yet H2b/b seemed to be more permissive for output of idiotype positive B cells, and H2d/d presented an attenuated spontaneous GC formation.3.2To unravel the potential functional implications of the observed decreased GC output in H2d/d mice compared to H2b/b and H2b/d groups, we analyzed the levels of autoantibodies in their serum. To this end, we again leveraged the linkage of knock-in vs. WT BCRs with Igha vs. Ighb respectively, which allowed us to discriminate the origin of the major Ig isotype involved in anti-dsDNA responses via IgG2a vs. IgG2c detection, respectively , 17, 18.These observations were overall congruent with the observed GC B cell differences presented in 3.3The H2 locus also harbors class III genes, which importantly include many complement genes . Complem3.4Having substantiated that the differences observed between H2 haplotype backgrounds in the 564Igi model with regards to autoreactive B cell activation were likely not determined by differences in complement activity, but rather classical MHC genes, most likely MHCII, we sought to corroborate these differences further. One issue with our initial experiments was that the diverse breeding setups employed, although random, could potentially affect phenotypes in the offspring. For example, we have previously shown in another autoimmune model, that offspring can receive quite robust levels of autoantibodies from autoimmune mothers , and henAccordingly, we devised a single cross, in which we could generate H2b/b, H2b/d and H2d/d littermates with 564Igi heterozygous status  displays epitope spreading , and fouAt 8 weeks post reconstitution, the bone marrow chimeras were euthanized, and spleens were harvested and analyzed. We first confirmed the expected haplotype distribution within the B cell compartment , and we To understand if there were any differences in the WT precursor repertoires, we used CD93 (AA4.1) staining to define progressive maturation stages of splenic B cells from transitional to mature, as CD93 high, intermediate, and low. Segregating these by H2 status, it was evident that whereas H2b and H2d cells were relatively equally represented across CD93hi, int and lo, 564Igi H2b/d cells were overrepresented among CD93hi and severely underrepresented among CD93lo cell subsets , 23, andin vivo.All in all, our findings demonstrated an intrinsic higher capacity of H2b B cells to enter or persist in autoreactive GCs and contribute to their output, compared to their H2d counterparts. Importantly, these findings were obtained within a B cell driven autoreactive environment, in which diverse WT-derived repertoires of H2b and H2d cells competed on equal terms, within the exact same environment 4The MHCII locus is well-known to play a substantial role in the development of autoimmune disease in general and in SLE specifically. However, and though much work has been done to investigate this, the pathways linking MHCII haplotypes to autoimmune phenotypes remain not fully elucidated. To investigate the B cell intrinsic contribution to this complex phenomenon, we utilized a well-described mouse model of SLE, the 564Igi mouse, and investigated not only the role of different MHCII haplotypes in the model itself, but also in two bone marrow chimera set-ups derived from this model.A priori, a possible explanation for this could be that the surface density of each MHCII molecule is reciprocally connected to the number of different MHCII molecules, meaning that the density of each MHCII-peptide-complex in a H2b/d mouse would be lower than in an H2b/b or d/d mouse. Indeed, this was previously suggested by the observation of similar MHCII expression levels on cortical epithelial cells across various strains  and NZW (H2z) mice . Followi strains . However1 setting referenced above, Nishimura et al. suggested that the exacerbation of autoimmunity could be caused by the formation of mixed haplotype AαdAβz heterodimers at low levels, which due to their scarcity would not adequately tolerize autoreactive T cells in the thymus (βz transgenic (NZB x NZW.H2d)F1 animals (In the (NZB x NZW)Fe thymus . Clearlye thymus –31. Spece thymus  and in F1 mice and reconstituted these with equal amounts of B6/lpr.Igha and B6/lpr.I-Eαd bone marrow F1 transgenic lines and bone marrow chimeras (α peptides prevents activation of would-be autoreactive T and B cells, by virtue of their high affinity to the I-A molecules.To determine whether autoantibody was preferentially made by I-E-negative B cells, Eisenberg and colleagues irradiated (B6/lpr.Igha x B6/lpr.I-Ee marrow . They suchimeras . Taken tThus, there is a preexisting mechanistic framework for understanding the observations in the present study, which we believe adds novel insights on the relative contributions in the context of GC and PB/PC responses that were not previously investigated. Specifically, most prior studies were performed in complex genetic models that could not truly isolate the contribution of B cells, and the dominant negative effect of I-E was largely observed in transgenic models with vastly unphysiological expression levels , 29, 33.Compared to H2d/d, H2b/d are slightly more represented in GC B and PC compartments, whereas H2d/d is slightly more represented among PBs, potentially indicating a preference for extrafollicular responses, which could be either T-dependent or T-independent.Finally, we also investigated the B cell intrinsic role of H2 in the context of selection at the transitional stage and subsequent epitope spreading. This was done in three-donor chimeras using 564Igi H2b/d driver cells, WT H2b/b, and WT H2d/d donors. Here, the 564Igi cells should be equally able to communicate with the two WT compartments, and preferential selection of one over the other would reflect differential ability to partake in the autoimmune response. The analyses revealed that H2b/b were again vastly superior to H2d/d in terms of ability to contribute to the GC compartment Figure 7Beyond MHCII molecules, additional factors associated with the MHC locus are known to play a role in autoimmunity, such as numerous complement factors  and seveTo summarize, while previous studies have confirmed the importance of MHCII in autoimmunity generally, our study underlines the vital role of the MHCII haplotype in B cells in autoimmunity and show that the MHCII haplotypes themselves are intrinsically different in their autoimmune potential. This is of immense significance, as B cells are key players in any autoimmune response, and specifically in diseases such as SLE driven largely by autoantibody production and thereby differentiation of B cells. Dissecting the molecular pathways for this effect thus provides necessary steppingstones in the direction of understanding the pathology and etiology of autoimmune diseases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by The Danish Animal Experiments Inspectorate. The study was conducted in accordance with the local legislation and institutional requirements.CW: Formal Analysis, Writing – original draft, Writing – review & editing, Data curation, Investigation, Methodology, Visualization. TW: Data curation, Investigation, Methodology, Writing – review & editing, Supervision. LV: Data curation, Investigation, Methodology, Writing – review & editing. GW: Data curation, Investigation, Methodology, Writing – review & editing. LJ: Investigation, Methodology, Writing – review & editing. AF: Investigation, Methodology, Writing – review & editing. SF: Investigation, Methodology, Writing – review & editing. CF-H: Investigation, Methodology, Writing – review & editing, Data curation, Visualization. SD: Writing – review & editing, Conceptualization, Formal Analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft.