Radioprotection was previously considered as a function of hematopoietic stem cells (HSCs). However, recent studies have reported its activity in hematopoietic progenitor cells (HPCs). To address this issue, we compared the radioprotection activity in 2 subsets of HSCs (nHSC1 and 2 populations) and 4 subsets of HPCs (nHPC1–4 populations) of the mouse bone marrow, in relation to their in vitro and in vivo colony-forming activity. Significant radioprotection activity was detected in the nHSC2 population enriched in lymphoid-biased HSCs. Moderate radioprotection activity was detected in nHPC1 and 2 populations enriched in myeloid-biased HPCs. Low radioprotection activity was detected in the nHSC1 enriched in myeloid-biased HSCs. No radioprotection activity was detected in the nHPC3 and 4 populations that included MPP4 (LMPP). Single-cell colony assay combined with flow cytometry analysis showed that the nHSC1, nHSC2, nHPC1, and nHPC2 populations had the neutrophils/macrophages/erythroblasts/megakaryocytes (nmEMk) differentiation potential whereas the nHPC3 and 4 populations had only the nm differentiation potential. Varying day 12 spleen colony-forming units (day 12 CFU-S) were detected in the nHSC1, nHSC2, and nHPC1–3 populations, but very few in the nHPC4 population. These data suggested that nmEMk differentiation potential and day 12 CFU-S activity are partially associated with radioprotection activity. Reconstitution analysis showed that sufficient myeloid reconstitution around 12 to 14 days after transplantation was critical for radioprotection. This study implied that radioprotection is specific to neither HSC nor HPC populations, and that lymphoid-biased HSCs and myeloid-biased HPCs as populations play a major role in radioprotection. Recently, we have suggested that HSCs are enforced to differentiate at an early time after transplantation.7 This has raised the possibility that HSCs also contribute to radioprotection. In this study, we asked whether both HSCs and HPCs have radioprotection activity.Radioprotection can be defined as the condition of being protected from the lethal effect of irradiation. For instance, it is observed when lethally irradiated mice are rescued by bone marrow transplantation. Thus, bone marrow cells have radioprotection activity. Radioprotection has been considered as one of the functions of hematopoietic stem cells (HSCs).8 Day 12 spleen colonies appeared to develop depending on the SCF/c-Kit signal.9 Day 12 CFU-S activity and radioprotection were suggested to be associated.11The number of day 12 CFU-S and radioprotection activity in the bone marrow of W mutant mice were found to be significantly reduced.12: CD201+CD150+CD48−CD41−CD34−Kit+Sca-1+Lin− cells (nHSC1), CD201+CD150−CD48−CD41−CD34−Kit+Sca-1+Lin− cells (nHSC2), CD201+CD150+CD48−CD41+CD34−Kit+Sca-1+Lin− cells (nHPC1), CD150+Flt3−CD34+KSL cells (nHPC2), CD150−Flt3−CD34+KSL cells (nHPC3), and CD150−Flt3+CD34+KSL (nHPC4) cells. In this study, we compared the radioprotection activity, in vitro colony-forming activity, and in vivo CFU-S activity among these populations. Varying degrees of these activities were detected among HSCs and HPCs. Neither high in vitro nor in vivo colony-forming activity was a sufficient condition of radioprotection. This study suggested that appropriate timing, degree, and context of myelopoiesis are required for radioprotection.We have recently characterized 2 HSC and 4 HPC populations22.1+Sca-1+Lin− (KSL) cells in mouse bone marrow are significantly enriched in HSC and HPC activities.2 CD34 expression levels can be used to separate HSCs from HPCs.4 Furthermore, CD150 expression level can be used to separate lymphoid-biased HSCs from myeloid-biased HSCs.17 Accordingly, the HSC1, HSC2, HSC3, HPC1, HPC2, HPC3, HPC4, and HPC5 populations were defined as shown in Figure Kitn = 20). After photographs were taken, individual colonies were stained with antibodies and analyzed by flow cytometry. After 7 and 14 days of culture, the numbers of live and dead cells and the proportions of cells of each lineage per colony were determined.Single-cell colony assays were used to compare the proliferation and differentiation potentials of the HSC1, 2, and 3 populations and the HPC1, 2, 3, 4, and 5 populations, which accounted for 2.2 ± 1.5%, 5.0 ± 1.8%, 3.6 ± 2.3%, 3.4 ± 1.6%, 3.8 ± 1.6%, 10.5 ± 4.5%, 34.7 ± 12.7%, and 31.2 ± 12.5% of KSL cells, respectively , macrophages (m), megakaryocytes (Mk), erythrocytes (E), and Mk/E progenitors (P) were identified by flow cytometry. Fluorospheres were simultaneously used to count the cells in each colony. The numbers of n, m, Mk, E, and P produced by single cells from 8 populations after 7 and 14 days of culture are shown in Figure Figure 2.2The HSC1 and HPC1 populations showed a variety of differentiation potentials during the in vitro culture. Figure In contrast to these results, a skewed distribution of differentiation potentials among the HPC4/5 populations was observed. Almost all cells in the HPC4/5 populations gave rise to nm colonies . The nHSC1, nHSC2, nHPC1, nHPC2, nHPC3, and nHPC4 populations accounted for 4.9 ± 1.5%, 15.3 ± 4.0%, 1.2 ± 0.8%, 6.1 ± 1.5%, 21.5 ± 3.2%, and 53.7 ± 3.6% of KSL cells, respectively .To study B and T cell differentiation potentials, we next performed a transplantation assay. To see more significant differences among populations, we redefined the HSC and HPC populations with gaps between the populations. We additionally used CD48 and CD201 to increase the purity of the cells. We also replaced CD41 with Flt-3 for the detection of HPC populations. The new HSC1 and 2 populations were designated the nHSC1/2 populations, while the new HPC1–4 populations were designated the nHPC1–4 populations. Their sorting gates and the relationship between the previously defined and redefined populations are shown in Supplemental Fig. 1 and Supplemental Table 1. CD482.4Hundred cells each from the nHSC1, nHSC2, nHPC1, nHPC2, nHPC3, and nHPC4 populations were transplanted into 10 lethally irradiated mice. Mice transplanted with no cells, nHPC3 cells, and nHPC4 cells similarly died by day 21 after transplantation Fig. . In cont2.5−KSL populations. nHPC2/3 cells formed significantly more day 12 colonies than did nHPC4 cells .Next, 100 cells each from the nHSC1, nHSC2, nHPC1, nHPC2, nHPC3, and nHPC4 populations were transplanted into lethally irradiated mice. Day 12 CFU-S were detected in all populations, consistent with a previous study.ion Fig. B, consisWe also analyzed the peripheral blood (PB) and bone marrow (BM) on days 7 and 12 after the transplantation of 100 cells each from the nHSC1, nHSC2, nHPC1, nHPC2, nHPC3, and nHPC4 populations Fig. C and D. 2.6To analyze longer reconstitution, the PB of the surviving recipients after transplantation with 100 nHSC1/2 and nHPC1–4 cells but without competitor cells was retrospectively analyzed. As representatively shown in Figure 320 Of importance was that the old populations  covered all new populations  so that data from the old populations were interpretable. The nHSC1 population was enriched in myeloid-biased HSCs while the nHSC2 population was enriched in lymphoid-biased HSCs  without gaps between its subpopulations . The nmEMk differentiation potential was detected in the nHSC1/2 and nHPC1/2 populations which all exhibited radioprotection activity. In contrast, consistent with previous studies,19 the nHPC3/4 populations lacked the EMk differentiation potential, and did not exhibit the radioprotection activity. Since nHSC1 cells need more time to produce platelets and neutrophils after transplantation than do nHSC2 cells,14 the radioprotection activity in the nHSC1 population appeared to be lower than that in the nHSC2 population  mice were purchased from Beijing HFK Bioscience Co.  and Sankyo Lab Service, Co. . B6-CD45.1 mice were bred and maintained at State Key Laboratory of Experimental Hematology. Only female mice were used. All animals were kept in certificated environment. The procedures for care and use of animals were approved by the Ethics Committee of Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, with approval number IHCAMS-DWLL-2017-KT001-1, date April-6, 2017. All applicable institutional and governmental regulations concerning the ethical use of animals were followed.4.2+Sca1+lineage− (KSL) cells were divided into 8 populations as shown in Figure 14For in vitro assays, bone marrow cells were collected from the femora, tibiae, and iliac crests of 8 to 10 week-old B6 mice and stained with antibodies. For flow cytometry sorting, c-Kit12 KSL cells were divided into new HSC1–2, and HPC1–4 populations . The relationships between the HSC1–3 and nHSC1,2 populations and between the HPC1–5 and the nHPC1–4 populations are demonstrated in Supplemental Fig. 1 and Supplemental Table 1.For in vivo assays, bone marrow cells were collected from B6-CD45.1 mice. c-Kit-positive cells were enriched and stained as described.4.3Unlike HSCs, HPCs have been poorly defined. In this study, we operationally defined HPCs as progenitors detected in the KSL population.4.4l-glutamine, 50 u/mL streptomycin/penicillin, 20 ng/mL recombinant murine stem cell factor, 20 ng/mL recombinant human thrombopoietin, 10 ng/mL murine interleukin-3, and 1 U/mL recombinant murine erythropoietin was prepared. Single cells were sorted to each well and cultured at 37°C and 5% CO2 in an Aztec air jacket incubator.A 96-well U-bottom tissue culture plate  in which each well had 200 μL of minimum essential medium  containing 10% fetal calf serum, 200 μM +CD115−, Gr1−CD115+, Gr1−CD115−CD41+, and Gr1−CD115−Ter119+ fractions were found to contain neutrophils (n), monocytes and macrophages (m), megakaryocytes (Mk), and erythroblasts (E), respectively. Cells in the CD41+Ter119+ fraction were operationally defined as progenitors (P). Cells with none of these lineage markers were shown as negative cells (Neg). Colony sizes were calculated using the following equation: P = C(T/B), where P is the population of the colony, C is the cell count from flow cytometric data, T is the total number of flow count fluorophores added to the sample, and B is the flow count fluorophore count from flow cytometric data. Colonies were defined as >200 live cells of any 1 lineage, as identified by flow cytometry. Clusters containing <200 live cells were excluded from the study.On days 7 and 14 of culture, the colony cells in each well were stained with antibodies. Flow-Count Fluorospheres (Beckman Coulter) were added to each well to estimate the cell number, and PI was added to exclude dead cells from analysis and sorting. Cells were then analyzed with a MACSQuant Analyzer (Miltineyi Biotec). The Gr14.5+ donor-derived Gr-1/Mac-1+ myeloid cells, B220+ B cells, and CD4/8+ T cells at the indicated time points.nHSC1–2 cells; and nHPC1–4 cells isolated from B6-CD45.1 mice were transplanted via the tail vein into B6-CD45.2 mice irradiated at 8.5 Gy in split doses, 3 to 4 hours apart. After transplantation, the peripheral blood of the recipients was stained with antibodies, and analyzed with a FACSCanto using Summit software (Beckman Coulter) to detect CD45.14.6If lethally mice transplanted with test donor cells survived >30 days after transplantation, test donor cells were considered to have radioprotection activity.4.7The mice were sacrificed on days 7 and 12 after transplantation. Spleens were removed from the mice and fixed for 24 hours with Bouin solution made of 5 mL of 5% acetic acid, 75 mL of 0.9% picric acid, and 25 mL of 9% formaldehyde.4.8t test, ANOVA, and long-rank test were performed using Prism 6 software. Data were shown as mean ± SD.Student's two-tailed


CO2 is a potent inhibitor of cellular proinflammatory responses caused by H2O2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5–8% CO2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways.Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are COThe online version contains supplementary material available at 10.1007/s00018-021-04005-3. However, the influence of this common gas on the functioning and components of cells is poorly understood, even though all cells in which organic substances are oxidised in the processes of oxidative cellular respiration produce CO2. While CO2, as a small molecule, can be efficiently transported through membranes by diffusion, CO2 is spontaneously hydrated to HCO3− at the pH of the cytoplasm. Carbonic anhydrases can convert HCO3− to CO2 and vice versa. The widespread presence of carbonic anhydrases in bacteria, archaea and eukaryotes and the existence of additional CO2 and HCO3− transporters indicate the benefits of rapid regulation of CO2 levels in cells, further suggesting the importance of CO2 for cell functions. It is, therefore, expected that there are mechanisms of CO2 sensing that are common to different groups of organisms. However, conserved CO2 receptors have not yet been found, and the currently identified eukaryotic CO2 sensors are specific to only particular groups of organisms, e.g. insect chemoreceptors. In mammals, several families of proteins are considered HCO3− receptors, including protein tyrosine phosphatases, which are inactivated by HCO3−-assisted oxidation using H2O2 [The physiological importance of carbon dioxide  have been found in all eukaryotes. They regulate many aspects of cell survival, proliferation, differentiation, migration, apoptosis, neurodegeneration and oncogenesis. Typically, MAPKs transduce signals from receptors to a wide variety of effector proteins, including transcription factors. Several human MAPKs, including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38, are involved in hundreds of documented signalling pathways in all organs, tissues, and cell types. JNK, ERK1/2 and p38 are activated through phosphorylation at regulatory tyrosine and threonine residues in the activation loop by upstream MAPK kinases (MEKs) and dephosphorylated and inactivated by MAPK phosphatases (MKPs). MAPKs are key players in the regulation of inflammation. In response to a wide variety of chemical and biological agents, such as reactive oxygen species (ROS), they regulate the production of ROS and proinflammatory cytokines, including interferon-gamma (IFNγ), interleukin-1β, interleukin-6 (IL-6) and tumour necrosis factor-α (TNFα) , 5.. Therefore, the inhibition of active MAPKs could be a strategy to prevent acute severe coronavirus disease 2019 (COVID-19).Angiotensin-converting enzyme 2 (ACE2) is a potent inhibitor of MAPK signalling and thus efficiently prevents both activation of MAPKs and pneumonia caused by exposure to cigarette smoke, particulate matter 2.5 (PM2.5), lipopolysaccharides (LPS) and bleomycin , 7. Howe2 binding, and active MAPKs are inactivated by CO2 [2 in vitro and in human cells.Since inactive plant MAPKs are activated upon COd by CO2 , we invel-glutamine and 1% penicillin/streptomycin. The EA.hy926 cell culture medium was additionally supplemented with 2% hypoxanthine-aminopterin-thymidine (HAT). The cells were cultured in a humidified 5% CO2 atmosphere at 37 °C until they reached approximately 80% (BEAS-2B) or 95% (EA.hy926) confluence, and the medium was exchanged every 3 days. The cells were treated with the indicated concentrations of the receptor-binding domain (RBD) of SARS-CoV-2 spike protein  and dexamethasone (dex) , 1 mg/ml LPS , 10 ng/ml TNFα , 7.5 ng/ml IFNγ , 5 mM (E/Z)-BCI hydrochloride , 2.5 µM U0126 , 25 µM PD98059  and phosphate-buffered saline (PBS) and/or dimethyl sulfoxide (DMSO) as controls 2 days after the cells had reached confluence and 12 h (for treatments shorter than 1 h) or 2 h (for longer treatments) after medium exchange.The stable human umbilical vein endothelial cell (EC) line EA.hy926  and normal human bronchial epithelial cell line BEAS-2B  were cultured in Dulbecco’s modified Eagle’s medium  or DMEM/nutrient mixture F-12 , respectively, supplemented with heat-inactivated 10% foetal bovine serum (FBS), 1% 2 bovine plasma fibronectin . All experiments were conducted at passages 2–3 using confluent HUVECs and HPMECs at 70–80% confluency after exchange of EGM-2 or EGM-2 MV with heparin- and hydrocortisone-free media. A number of administered spike-pseudotyped lentivirus particles corresponded to the doubled tissue culture infectious dose 50% (TCID50) in a standard assay (2 days at 37 °C with 5% CO2) on HEK293 cells expressing ACE2.Primary cells, i.e. human umbilical vein endothelial cells  and human pulmonary microvascular endothelial cells , were cultured in endothelial cell growth medium-2  or microvascular endothelial cell growth medium-2 , respectively, according to the manufacturers’ protocols. The HPMECs were grown on vessels coated with 2 μg/cmThe following reagents were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, heat inactivated (NR-52286) and SARS-Related Coronavirus 2, Isolate USA/CA_CDC_5574/2020, heat inactivated, NR-55245. The following reagents were obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Wuhan-Hu-1 spike-pseudotyped lentivirus, Luc2/ZsGreen, NR-53818, SARS-Related Coronavirus 2, Wuhan-Hu-1 spike D614G-pseudotyped lentivirus, Luc2/ZsGreen, NR-53819, and Spike glycoprotein (stabilised) from SARS-Related Coronavirus 2, B.1.1.7 lineage with C-terminal histidine and Avi tags, recombinant from HEK293 cells, NR-55311.2 concentrations (6.5–10%) used for ERK1/2 regulation are in the physiological range based on the following assumptions. (i) In central venous blood, the normal partial pressure of CO2 (pCO2), 45 mm Hg [2 dissolved from an atmosphere containing 6% CO2. (ii) Typical therapeutic breathing of 5% CO2 (see “”) leads to an increase in the blood pCO2 by 10–13 mm Hg. After summing (58 mm Hg), the pCO2 corresponds to dissolved CO2 from air containing 8% CO2. (iii) The intracellular pCO2 is higher than that in blood. CO2/HCO3− calculations were made using Aqion software (www.aqion.de).The COions 6.5–% used foCO2 see “” leads tImmunoblotting was carried out as described previously . The folg, 1 min, 4 °C). The pellet containing 2 × 108 ECs was lysed (5 min on ice) in 2 ml of buffer containing 100 mM Tris–HCl pH 7.5, 600 mM NaCl, 75 mM NaF, 6 mM EDTA, 0.1% NP40, 4 mM DTT, 1 mM PMSF and protease (Roche) and phosphatase (Thermo Scientific) inhibitor cocktails. Then, 5 ml of ice-cold deionised water was added, and the lysates were centrifuged . The supernatants were incubated for 1 h at 4 °C with 250 μl of Dynabeads® Protein G  for preclearing. Then, the Dynabeads® Protein G was replaced with 40 ml of anti-ERK1/2 antibody coupled with Dynabeads. The suspensions were incubated overnight at 4 °C with slow agitation and then washed six times with PBST and two times with deionised water.ECs were rinsed twice with ice-cold PBS, scraped off and centrifuged . After FastAP inactivation with 50 mM EDTA followed by six washes with PBST and two washes with deionised water, ERK1/2 were used in in vitro phosphorylation reactions.Immunoprecipitated ERK1/2 were dephosphorylated at 37 °C for 1 h in a reaction mixture containing 10 mM Tris–HCl pH 8.0, 5 mM MgCl2, 200 μM ATP and EDTA-Free Protease and Phosphatase Inhibitor Tablets  with the indicated concentration of freshly dissolved NaHCO3. Then, the proteins were denatured in SDS-PAGE sample loading buffer and subjected to immunoblotting with an anti-phospho-MBP antibody.Immunoprecipitated ERK1/2 were incubated  with 5 μg of dephosphorylated MBP  in buffer containing 100 mM MOPS (pH 6.7 or 7.4), 0.5 mM EGTA, 1 mM DTT, 20 mM MgClMeasurement of the level of IL-6 in cell culture supernatants was carried out using an enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.® Green Master Mix  and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) were used to perform qPCR with primers specific to ACE2 (ACCAGTGGATGAAAAAGTGGTG and AGAAACATGGAACAGAGATGCG), GAPDH (GTCTCCTCTGACTTCAACAGCG and ACCACCCTGTTGCTGTAGCCAA) and ACTB (CACCATTGGCAATGAGCGGTTC and AGGTCTTTGCGGATGTCCACGT). The 2−ΔΔCT method [ACE2 mRNA.RNA extraction with TRI reagent (Sigma–Aldrich) and reverse transcription using a RevertAid H Minus First-Strand cDNA Synthesis Kit  were conducted according to the manufacturers’ protocols and described previous particulars . PowerUpT method  was applT-test  or one-way or factorial ANOVA followed by Tukey’s post hoc test . The means ± SDs of three independent experiments are presented. *, ** and *** indicate significant differences in ERK1/2 activity .ImageJ software was employed for densitometric analysis of the immunoblotting bands. The expression levels of total kinases and GAPDH and Ponceau S staining were used for data normalisation. For relative quantification, the value of the mock treatment group was considered 1. The means ± standard deviations (SD) are shown. The significance of differences was calculated using a two-tailed 2 in regulating ERK1/2 activity, because ECs in the lungs, heart and brain are strongly affected in COVID-19, and COVID-19 is considered a form of inflammatory endothelialitis [We focused on ECs (cell line EA.hy926) to test hypotheses regarding the potential role of COlialitis , 15.2 , whereas activation was weak at pH 7.4 (8.7% CO2 and 91.2% HCO3−). This result indicates that dissolved CO2, but not HCO3−, activates ERK1/2. The decrease in ERK1/2 activity in the presence of 30 mM HCO3−, which is close to the concentration in plasma, suggests that ERK1/2 can be activated by both increasing and decreasing the cellular CO2 concentration. Then, we found that transient activation of ERK1/2 in ECs occurred at all tested CO2 concentrations  and neutralised (pH 7.4) CO2 . To verify that CO2 directly activated ERK1/2 in ECs, we showed that ERK1/2 were still activated by CO2 in the presence of MEK1/2 inhibitors (U0126 and PD98059), while activation of ERK1/2 by TNFα was abolished under these conditions , H2O2 at a concentration of 100 μM permanently activates ERK1/2. Activated ERK1/2 were successfully inhibited by 30 mM HCO3− over a time range of 2 to 4 h . More stable ERK1/2 inactivation can be achieved by repeated treatment with buffered (pH 7.4) HCO3−  or CO2 . Transient hypercapnia, or an increased pCO2, is a physiological condition affecting the blood vessels, and further experiments were conducted using a transient increase in the CO2 concentration to avoid the introduction of additional compounds  to the cell culture medium.Since p38 and JNK are inactivated by CO2 Fig. f and actin vitro , we aske2 concentration for 11 min .Efficient inhibition of ERK1/2 below baseline levels was induced by an increase in the CO2 concentrations. Therefore, we looked for other factors affecting ERK1/2 activity induced by CO2. After stimulation with H2O2 alone or in combination with 10% CO2 , ERK1/2 were initially activated by CO2 before inactivation, and the time required to achieve baseline ERK1/2 activity was much shorter in H2O2-treated ECs than in control ECs  CO2 for different duration at 3-h intervals . Since ERK1/2 inactivation by dex depends on increased MKP-1 expression [2. Indeed, ERK1/2 were not inactivated in the presence of BCI, a specific MKP-1 inhibitor . Fifteen hours after RBD application, ERK1/2 were activated in a concentration-dependent manner up to 400 ng/ml RBD, and ERK1/2 activation was higher in ECs exposed to 25 mM glucose than to those exposed to 5.55 mM glucose. This is consistent with the glucose-mediated induction of ACE2 expression, SARS-CoV-2 load, and cytokine and ROS production , 21. The2O2, RBD-induced ERK1/2 activation was less effectively inhibited, and exposure to 10% CO2 three times at an interval of 3 h was necessary to retain baseline ERK1/2 activity .Although activation of ERK1/2 in response to RBD was weaker than that in response to H2-induced inactivation of ERK1/2. In contrast to studies showing that ACE2 is not expressed at the mRNA level in BEAS-2B cells [Then, we aimed to show the versatility of CO2B cells , we conf2B cells  demonstr2B cells . Therefo2B cells  and acti2B cells , 7.2, ERK1/2 activity was effectively blocked under these conditions in both cell lines . Therefore, we applied RBD in together with TNFα or RBD together with TNFα and IFNγ to induce ERK1/2 activity in ECs and BEAS-2B cells Fig. d–f. Follnes Fig. d–f, whilnes Fig. e. Neithenes Fig. f. ImportECs Fig. d–f.Notably, aspirin is known to induce aspirin-exacerbated respiratory disease (AERD), in which IFNγ plays a crucial role . Moreove2O2  and intercellular adhesion molecule 1 (ICAM-1) and phosphorylation of NFκB p65 and mitogen- and stress-activated protein kinase 1 (MSK1) were induced by H2O2 Fig. a or RBD 2O2 Fig. b or RBD 2O2 Fig. c and couCO2 Fig. d–f.Fig. 2 in a more natural context, we employed primary ECs from the umbilical vein and from the pulmonary microvasculature, trimeric spike glycoprotein, spike-pseudotyped lentivirus and heat-inactivated SARS-CoV-2. At a low glucose concentration (5.55 mM), ERK1/2 activation by spike, spike-pseudotyped lentivirus or heat-inactivated SARS-CoV-2 was clearly stronger in HUVECs  efficiently inhibited not only ERK1/2 activity but also the increase in IL-6, ICAM-1 and HIF-1α expression in primary ECs  can be partially repressed by two treatments with CO2 after TNFα and IFNγ administration  increases pulmonary artery pressure. However, inhaled 5% CO2 reduces existing pulmonary hypertension [2 activates inactive ERK1/2 and inactivates active ERK1/2.Hundreds of clinical trials have confirmed that short-term inhalation of 5% COup to 8% . Inhalattoo weak . On the rtension . Corresp2 is effective for the treatment of febrile seizures [2 enables oxygen to be released from haemoglobin [2, the oxygenation of the arterial blood increases, which results in better oxidation and susceptibility of tumours to radiotherapy [2 increases blood oxygen saturation and the supply of oxygen to tissues [2 is not only increasingly being used for carboxytherapy in aesthetic medicine but also increasingly being administered via inhalation and baths for the treatment of kidney, cardiovascular and neurological diseases.Inhaled COseizures , centralseizures , and seiseizures  and counseizures . CO2 enamoglobin . As a reotherapy . Similar tissues . CO2 is 2 sensing seems to be a universal process among eukaryotes, currently recognised HCO3− sensors, namely, GCs and ACs, are absent in many eukaryotes. Moreover, CO2 signalling pathways, which are crucial for vertebrates, are independent of GCs and ACs, including the pathways responsible for the regulation of breathing in response to changes in CO2 concentrations [2 sensing by olfactory sensory neurons [3−-dependent regulation of spontaneous heart rate and cardiac force development. Interestingly, the activity of AC and GC in Dictyostelium discoideum depends on DdERK2, the activation of which is independent of MEKs [2 signalling are functionally closely associated with MAPKs; Sch9 kinase [Although COtrations , CO2 sen of MEKs . Yeast p9 kinase  is regul9 kinase . Intrigu9 kinase .2 at the level of MAPK activity. However, activation of MAPKs in response to many factors is transient. Regarding this type of MAPK activation, the effectiveness of both MAPK inhibitors [2 [In this work, we focused on long-term activation of ERK1/2 to clearly demonstrate the effectiveness of COhibitors  and a siitors [2  in vivo 2-induced activation of inactive ERK1/2 and inactivation of active ERK1/2 have been demonstrated in pheochromocytoma cells [2 on ERK1/2 activity. More recently, Xu et al. [2 in rat skeletal muscle.The conclusions from the present work are consistent with previous studies; COma cells . Howeveru et al.  showed t2 on ERK1/2 is as marked as that of a high dose of dex. Moreover, CO2 treatment is the most effective method for inactivating ERK1/2 in epithelial cells in the presence of increased IFNγ and TNFα levels, which is characteristic of severe COVID-19.Although ERK1/2, JNK and p38 have been shown to facilitate the replication of many viruses and nuclear export of viral ribonucleoprotein complexes, the MAPK pathway that is currently attracting the most attention is the pathway involved in the regulation of proinflammatory cytokine expression. Dex is widely used in the treatment of inflammation in severe COVID-19. However, the serious conditions of patients often result from comorbidities, and contraindications to the use of dex coincide with factors that increase the risk of a severe COVID-19. In addition, among other factors, chronic obstructive pulmonary disease (COPD), cigarette smoke extract, bleomycin‐induced acute lung injury and oxidative stress have been shown to cause resistance to corticosteroids in bronchial and alveolar epithelial cells. We have shown that the inhibitory effect of lower doses of dex in combination with CO2 treatment and inhibition of MAPKs [2 clearly inhibits ROS generation in many human and mouse tissues and stimulates the production of antioxidants [2 and MEK inhibitors. MAPK inhibitors [2 inhalation for 10 min [2 [2 [2 concentration is lower in deceased patients than in patients who have recovered from COVID-19 [In addition to a cytokine storm, infection with SARS-CoV-2 induces ROS generation . In seveof MAPKs . CO2 cleoxidants . There ahibitors  or 5% COr 10 min  is prote0 min [2  and MEK 0 min [2  attenuat0 min [2  and CO2 in [2 [2  counteraCOVID-19 . In addiCOVID-19 .2 is a potent inhibitor of ERK1/2 activity that acts independently of cell type and proinflammatory cytokines, which is in contrast to cytokine-susceptible aspirin and dex. Unlike dex and aspirin, CO2, which readily penetrates tissues and cells and acts independently of appropriate transporters, receptors and signalling pathways, inactivates mainly overactivated ERK1/2. Thus, our study supports previous report [2 as a potential therapeutic agent for COVID-19.We have shown that COs report . We encoSupplementary file1 (PDF 973 KB)Below is the link to the electronic supplementary material.


The Fe3O4 supports were decoratedwith Pd and Pt active sites and served as the local heat generatorswhen subjected to an alternating magnetic field. For hydrogenationover Pd/Fe3O4, both heating methods gave similarliquid product selectivities, but thermogravimetric analysis–differentialscanning calorimetry measurements showed no coke accumulation forthe RF-heated catalyst versus 6.5 wt % for the conventionallyheated catalyst. A different trend emerged when hydrogenation overPt/Fe3O4 was performed. Compared to conventionalheating, the RF increased the selectivity to stearic acid by an additional15%. Based on these results, RF heating acting upon a magneticallysusceptible nanoparticle catalyst would also be expected to positivelyimpact systems with high coking rates, for example, nonoxidative dehydrogenations.Radio frequency (RF)induction heating was compared to conventionalthermal heating for the hydrogenation of oleic acid to stearic acid.The RF reaction demonstrated decreased coke accumulation and increasedproduct selectivity at comparable temperatures over mesoporous Fe Typical temperatures and pressuresfor this application are 300–360 °C and 15–27 bar(0–5% H2), respectively. These catalysts are preferredwhen diesel-fuel-like products are desired.9Industrialprocessing of the longer chain fatty acids, such aslauric, palmitic, and oleic acid, traditionally focuses on decreasingthe degree of unsaturation while maintaining the carboxylic acid groupand avoiding isomerization.1 While these metals are generally selective forC=C hydrogenation, minimizing defect sites and adding an electropositivepromoter such as Co, Ge, Fe, Ga, Ni, or Sn both increase the metals’electronegativity and decrease their ensemble size, increasing theselectivity to unsaturated alcohols as a result.6 Othershave explored various supports such as SiO2, Al2O3, and metal oxides such as TiO2, ZnO, andCeO2 that can also increase selectivity to the unsaturatedalcohol by either a strong metal–support interaction or theoxygen-exchange ability of the support with reactants.11 Both approaches have been shownto have significant effects on selectivity.11Due tothe high interest in the hydrogenation of fatty acids, thereare numerous papers focused on supported Pt, Pd, Rh, and Ru.13 Radio frequency(RF) induction heating allows increased efficiency due to the highpenetration capability of RF waves and their ability to induce magneticdipoles in a magnetic material.15 As a result, RF-heatedcatalysts can be 90% energy-efficient in an industrial environmentcompared to 50% for a conventional reactor.16 One approach uses sub-millimeter metallic beads to act as a RF absorberwhich then heats the process stream and catalyst bed.17 Another approach is to incorporate ferrimagnetic or ferromagneticnanoparticles into the catalyst itself and utilize the material’smagnetic hysteresis. By the latter method, the RF field localizesheat generation at the catalyst surface through hysteresis lossesand minimizes the temperature gradient between the catalyst and substrateinterface, which is expected to increase the overall rate of reaction.18 Furthermore, the increased temperature uniformity due to the alternatingmagnetic field and the Curie temperature (Tc) of the material can prevent hot-spot generation leading to unwantedside reactions.19 While improving overall heat transfer, the constraints imposed bythe magnetic material increase the difficulty of designing an effectivecatalyst. For example, the active metal or metal oxide crystallitesize and morphology can influence hysteresis losses and their controlin optimal catalyst design can conflict with the demands of inductionheating.18 Currently,there are no known industrial applications of catalytic inductionheating, but smaller-scale experiments have been published.21 For example, in methane steam reforming, induction-heated catalysiscould achieve 90% methane conversion over a single catalyst at temperaturesof 700–800 °C.21 A similarapproach has been attempted with microwave heating in dry reformingcatalysis, where a reduction of coke formation has been observed.22Much fatty acid hydrogenation researchexplores the effects oftemperature, pressure, and catalyst composition while still relyingon thermal conduction and convection to supply the necessary energyto drive the reaction. While these methods of energy transfer arewell understood in the reactor design, they are also inefficient andslow compared to nonionizing radiation.3O4 nanoparticles.The Fe3O4 support, composed of ∼21 nmnanoparticles, demonstrated the expected hysteresis heating basedon specific loss power (SLP) measurements, while the two noble metalshave distinct selectivity characteristics for oleic acid hydrogenation.24 The catalysts were characterized by thermogravimetric analysis (TGA)–differentialscanning calorimetry (DSC), chemisorption, X-ray diffraction (XRD),and X-ray photoelectron spectroscopy (XPS).This project aims to evaluate the effectsmagnetic nanoparticleRF induction heating has on the activity and selectivity in the hydrogenationof a typical fatty acid, oleic acid. The catalytic performances ofRF and conventional thermal heating are compared for Pd and Pt supportedon the microspheres of ferrimagnetic Fe22.16H8O7, Fisher Chemicals,99.7%) and sodium bicarbonate were used for the synthesisof the trisodium citrate dehydrate (C6H9Na3O9). Ferric chloride hexahydrate , sodium acetate , and ethylene glycol  were used for theFe3O4 solvothermal synthesis. Palladium(II)chloride and hydrochloric acid  were used for the Pd deposition, and hydrogenhexachloroplatinate(IV)hydrate (H2PtCl6·xH2O) was used for the Pt impregnation. Hydrochloricacid  and nitric acid  were used for the inductivelycoupled plasma optical emission spectrometry (ICP-OES) digestion process.Dodecane  andoleic acid  were used as the reactor solvent and reactant, respectively.Citric acid  was created by dissolving 1.0g of PdCl2 in a 70:100 mixture of 37 wt % HCl and DI water.The solutionwas then diluted to 15 mM with DI water, and 1.28 g of iron oxidesupport was suspended in 8.1 mL of this solution for 6 h to give anominal 1.0 wt % Pd catalyst (1Pd–Fe3O4). The pH was brought to 10 with 1 M sodium carbonate, and then thesolution was heated to 70 °C overnight to reduce the volume by15–25%. The precipitate was then washed four times with DIwater, centrifuged, and heated at 100 °C in flowing N2 for 12 h. The powder was then reduced at 250 °C in flowingHe for 3 h.The Pd deposition method was adapted from Freundet al. 1b.26 Her2PtCl6·6H2O was dissolved in 60 mL of DIwater to givenominal 3 wt % Pt (3Pt–Fe3O4) for 1.20g of iron oxide support  using temperature-programmed oxidation (TPO). In flowingN2, the sample was equilibrated at 250 °C, rampedat 10 °C min–1 to 325 °C, held for 60min, and then ramped at 10 °C min–1 to 400°C, with a 30 min final hold. This treatment prior to oxidationwas designed to remove any residual water and organics that may remainafter synthesis. The gas was then switched to air for 60 min and thenramped to 600 °C at 10 °C min–1.The Fe2 (assumed 1 H/Pt). The morphology and size of the Fe3O4 microspheres were measured by high-resolution transmissionelectron microscopy (HRTEM) using a JEOL JEM-1400 operating at 120kV and using an Orius Camera SC1000A 1, with a 0.20 nm lattice imageresolution and a 0.38 nm point image resolution. The powder samplewas dispersed in ethanol and drop-cast on a 300 mesh, lacey carbongrid prior to imaging. The elemental composition of the catalystswas measured using a Perkin Elmer Optima 8000 inductively coupledplasma optical emission spectrometer equipped with an autosampler.Samples for ICP-OES analyses were prepared by digesting 2.1 mg of1 wt % Pd/Fe3O4 and 30.0 mg of 3Pt–Fe3O4 in a HNO3 (68–70%) and HCl(36.5–38%) solution, which is heated to ∼70 °C.1Pd–Fe3O4 is diluted to 45 ppm Fe and0.63 ppm Pd, respectively, and 3Pt–Fe3O4 is diluted to 16 ppm for both Fe and Pt using 2% HNO3. Powder XRD data were obtained using the Cu Kα1 (λ =1.54 Å) emission on a PANalytical XRD with a step size of 0.04°and a dwell time of 60 s. XPS results were collected using a ScientaOmicron ESCA 2SR XPS equipped with a monochromatic Al Kα (hν = 1486.6 eV) X-ray source and a hemispherical analyzerwith a 128-channel detector. The inherent Gaussian width of the photonsource was 0.2 eV. The pressure inside the chamber was 1.5 ×10–9 Torr. The XPS spectra were calibrated to theadventitious C 1s peak at 284.8 eV, and peak deconvolution was performed(using the CasaXPS software) using Gaussians after Shirley backgroundsubtraction.Surface area and pore volume measurements were performed on a MicrometricsASAP 2020 utilizing the Brunauer–Emmett–Teller method.Pd and Pt dispersion was quantified by pulse chemisorption on a MicromeriticsPulse Chemisorb 2700, Pd with CO (assumed 1 CO/Pd) and Pt with H2.42 for 30 min and then H2 at 1 atm for 30 min before heating. The induction heating was drivenusing an Ambrell EASYHEAT 8130LI 10 kW induction heater  with a 3-turn, 0.035 m diameter Cu coil. The bulk temperaturewas measured using a Neoptix T1 PTFE-coated fiber optic cable. Thecurrent was adjusted based on the desired bulk temperature  for 4 h. A conventional thermal reaction (oil bath)was used as control with identical temperatures and times.Batch reactions were performed with 500 mg of oleic acid, 5.00gof dodecane solvent, and 50.0 mg of the catalyst in a glass vial.The vial was purged with NN,O-bis(trimethylsilyl)acetamide (BSA)at a molar ratio of 4:1 BSA to oleic acid. Pyridine was added, at1/10th the volume of BSA, to catalyze the derivatization, which createda nonpolar trimethylsilyl ester  orether . Product identification was performed bymass spectrometry using an Agilent GC 6890N/Agilent MSD 5975B. A WassonKC066  dimethylsiloxane column was usedat a 15:1 split ratio at 13.3 mL min–1 total flow.The temperature was ramped at 15.0 °C min–1 from 100 to 230 °C with a 92 min hold time. Product quantificationwas performed on an HP 5890 Series II GC with an FID and an HP 7673autosampler fitted with a Supelco Equity-1  column. A split ratio of 50:1 was used at a total flow of 64.8mL min–1. The temperature was ramped at 7.0 °Cmin–1 from 100 to 230 °C with a hold time of11.5 min. TPO (TA SDT Q600) was used to quantify the coke and coke-likeproducts on the catalyst surface. Before running the TPO, the catalystswere vacuum-dried at 110 °C for 12 h. The sample was equilibratedand held for 30 min at 100 °C in N2, switched to air,ramped at 10 °C min–1 to 650 °C, and heldfor 50 min.Forchromatographic analysis, each sample was derivatized with 33.13O4 component. The support diffraction peaks areconsistent with the cubic magnetite Fe3O4 structure(ICDD: 005-4319). An average crystallite size of 21 nm was extractedfrom the (311) reflection using the Debye–Scherrer equation.In comparison, TEM imaging  and a small shoulder at 336.2 eV that are characteristicof PdO  catalystswas probed by XPS. The spectrum of the fresh 1Pd–Fecof PdO 3a.28,29 336.8 eV 3a.28,29,ectively 3b.32 A m3O4 showed similar characteristicsto that of 1Pd–Fe3O4. For the fresh catalyst,the major peak is at71.7 eV with a small shoulder at 72.6 eV  with a reduction of Fe2O3 consistent with the disappearance of the satellitepeak  Pd size of 5.8 nm usingD is the dispersion(%), M is the atomic weight, ρ is the densityof the metal, Na is Avogadro’snumber, Sa is the area of each surfaceatom, and d is the diameter (nm).38 The surfacearea and pore volume of the Pt catalyst both decreased as expectedfor incipient wetness impregnation. No measurable CO or H2 uptake was found on the Pt catalyst at 23, 50, or 100 °C; therefore,dispersion was calculated approximately from the XPS measurement byquantifying the Fe 2p and Pt 4f peaks, relating the peak areas tomoles of each element, and dividing the calculated exposed Pt fromXPS by the total number of Pt. This lack of adsorption under theseconditions has been observed previously and is attributed to Pt-FeOx SMSI,39 as observedin the XPS spectra. ICP-OES performed on 1Pd–Fe3O4 and 3Pt–Fe3O4 gave 1.6wt % Pd and 3.0 wt % Pt, respectively, suggesting that an additionalPd precursor was deposited, but the target impregnation of Pt wasachieved. These values were then used in the calculation of dispersionand average particle diameter.To quantify the dispersion of themetals and the substrate surfacearea, chemisorption and physisorption measurements were performedon the pure and decorated substrates. These measurements are shownin 2O3, TPO was performed on the uncoatedsubstrates. This compositional analysis is important as the high temperaturesrequired for catalytic reactions require a Fe3O4/γ-Fe2O3 (magnetic moment per formulaunit: 4.0 μβ/2.5 μβ)ratio greater than 0.6.43 A high-temperature N2 treatment was usedto remove any residual organics while minimizing support oxidation.44 The TPO was compared to that of commercial Fe3O4  powders. Both were heated to 400 °Cwith N2 to desorb residual hydrocarbons from the syntheses,and then, at 400 °C, the gas was switched to air. By switchingto air, the percentage of Fe3O4 was estimatedby utilizing the weight change due to oxidation and the theoreticalweight change to Fe2O3. It was estimated thatthe synthesized FeOx support containsat least 70% Fe3O4. In contrast, a typical commercialFe3O4 powder contained only 57% Fe3O4. A temperature-field strength calibration was performedby dispersing 50 mg of Fe3O4 in dodecane. Bulktemperatures were measured by submerging an IR fiber optic probe inthe solution. These solvent and catalyst loadings were typical forthe hydrogenation of oleic acid to stearic acid, oleyl alcohol, oroctadecanol as were the three characteristic temperatures .47 The solutions rapidly reached the desired temperature(∼5 min) upon exposure to the magnetic fields (Table S1). The solvent could be heated to thedesired temperatures with RF fields <20.0 mT under these conditions.The maximum temperature is normally sufficient for more than 80% oleicacid conversion in 4–6 h.48 SLPmeasurements were performed on the Pd and Pt catalysts to characterizethe heat generated between the range of 10.8 and 21.2 mT .44 At 21.2mT, 130 ± 13 and 93 ± 9.3 W/g were measured for 1Pd–Fe3O4 and 3Pt–Fe3O4,respectively, and are comparable to the previously reported SLP values.To confirmthe above XRD results, specifically the relative lackof Fe3.246 a saturatedalcohol (octadecanol), and more fully hydrogenated products such asalkanes , with no oleic acid conversiondetected for either approach.The hydrogenationof oleic acid can produce a saturated acid (stearic acid), an unsaturatedalcohol ,alkanes 4a.Acidiic acids 4b.7,49 T3O4 and 3Pt–Fe3O4 catalysts areshown in 3O4 catalystunder RF heating converted ∼60% of the oleic acid, 28% higherthan that with conventional heating  was heated thermally at 7.9 bar H2 pressure, 150 °C.50 The increase in pressure(coverage) showed no effect on conversion but changed the selectivityto 1-octadecanol from 2 to 14%  to stearic acidwith minimal production of side products  at all three bulk temperatures.The catalytic results for the 1Pd–Fe heating 5a. Howev50 For 1Pd–Fe3O4,the increase of the H2 partial pressure increased the productselectivity to the saturated alcohol. Since the catalyst activitydid not change, the increased ratio of hydrogen to other adsorbedspecies on the catalyst must be responsible for the change in selectivity.52 It is known that monometallic Pd prefers to hydrogenate the C=Cbond in aliphatic and aromatic carboxylic acids and aldehydes.53 However, Neri et al. have shown that for Pd/Fe2O3 (even at 1 bar H2), strong metal–supportinteraction can result in electron donation to the metal, favoringthe adsorption of the C=O group and increasing selectivityto alcohols.55 Since neither RF nor thermal heating modes showed selectivity toalcohols at 1 bar, there is likely no Pd–Fe interaction here.It has been previouslyreported that hydrogen partial pressurecan affect the selectivity and activity depending on the metal/support.3O4 catalyst was significantlyless active and produced more cracked/heavy acids and heavy productsthan the 1 wt %Pd-Fe3O4 catalyst . Selectivityto stearic acid increased proportionally with the reactor temperature,ultimately reaching 45% for RF heating versus 30%for conventional heating. Therefore, RF was able to decrease the yieldof unwanted products and increase selectivity to stearic acid forat least one catalyst.The selectivities of thePt catalyst were also affected by theheating method. At 70 °C, less than 2% stearic acid was foundfor both conventional and RF heating, but at 110 °C, the stearicacid selectivity increased to 34% for RF 2O3 catalystscan selectively hydrogenate the C=O bonds of carboxylic acids.58 However, this requires a Pt–Fe alloy to be formed duringthe H2 reduction of the catalyst.39 Studies have shown that the rate and degree of reduction dependheavily on temperature, reducing gas partial pressure, and concentrationof additional elements.61 The reason why Fe2O3 can form the Pt–Fe alloy is that when it isreduced , both Fe3O4 and Fe0 are produced.39 The Fe0 formed then alloys with Pt.57 The slight shift in binding energies for the used catalyst and thedisappearance of the Fe2O3 satellite peak maybe an artifact of this Pt-Fe interaction  used here were insufficient to reduce to FeO/Fe0. For the reduced Pt/Fe3O4, no FeO orFe diffraction peaks were observed.The literature suggests that Pt/Feeraction 3c,d. In 3O4 and3Pt–Fe3O4 catalysts. The weight derivatives while thatoxidized at the higher temperatures is more graphitic.65 The exotherm seen at 570 °C and above is due to the exothermicdecomposition of γ-Fe2O3 to α-Fe2O3.66 The weight lossand endothermic behavior at these temperatures are due to the reductionof Fe2O3 to Fe3O4 andFe3O4 to FeO.68 For RF heating,no coke was found on the spent Pd catalyst, whereas conventional heatingresulted in 6.5 wt % of coke relative to the catalyst weight  on the RF-heated sample (75 and have been attributed to betterheating control preventing unwanted side reactions.76TPO measurements were performedto quantify coke deposition onthe used (150 °C) 1Pd–Feivatives6a and thivatives6b of thet weight 2. When hd sample 2. The hi77 For similar bulk temperatures, RF has demonstrated that it can improveconversions from 5 to 25%, depending on the catalyst, compared tothose of conventional thermal heating. Such improvements were alsoobserved here for both catalysts. The nature of RF heating allowsheat to be generated on the surface of the magnetic catalysts insteadof being heated by external sources and transporting heat to the catalystthrough traditional means.18 What is likely occurring here for the Pt catalyst  is that the surface of the catalysts is ata significantly higher temperature than the liquid bulk (∼20–30°C).77 Since the reaction is kinetically limited,any temperature gradient from the catalyst surface to the bulk liquidwould increase the observed activity. However, this does not explainthe positive effects on selectivity  we have observed. We offer theseresults as proof that RF heating can do more than just enhance catalystactivities through enhanced heat transfer. Moving forward, hierarchicalcore–shell nanostructures could be used to isolate transitionmetal-supported catalysts from Fe3O4 while stillmaintaining the beneficial heating aspects of the RF system to furthercontrol the selectivity of these reactions.Higher catalytic conversions usingRF heating have been shown forreactions such as steam reforming and Fischer–Tropsch syntheses,likely due to the nature of localized heating.43O4 mesoporous microspheres composed of 28–32nm spherical nanoparticles. These two magnetically susceptible catalystswere synthesized to study the effect RF induction heating has on thehydrogenation catalysis of a typical fatty acid. This work revealedthat induction heating paired with a catalyst that produces heat locallythrough magnetic hysteresis losses increased both activity and selectivityfor the hydrogenation of oleic acid compared to conventional heatingmethods. For the Pt catalyst, RF provided a minor increase in activityand larger increases in product (stearic acid) yield. At the two higherbulk temperatures, the selectivity increased by a factor of 1.8. Forthe Pd catalyst, the RF input roughly doubled the stearic acid yieldat the lowest bulk temperature, but there were no effects at highertemperatures due to kinetic limitations. The most significant resultwas that there were no coke-like heavy products on the RF-heated catalyst,in contrast to conventional thermal heating. This result suggeststhat RF paired with a ferrimagnetic catalyst can greatly limit therate of coke formation and therefore extend the catalyst lifetime.The ability of RF induction heating to reduce coke formation willbe even more beneficial in reactions with traditionally high ratesof coking, such as nonoxidative dehydrogenation.In summary, 1 wt % Pd and 3 wt % Pt catalystswere prepared onFe


The rat model of IgA nephropathy was successfully established and verified by biochemical tests and histological staining. Meanwhile, the clinical 18F-AlF-NOTA-PRGD2 probe molecule was utilized to visualize and further confirmed the IgA nephropathy in vivo via positron emission computed tomography. Subsequently, the Fe3O4 NPs were conjugated with arginine–glycine–aspartic acid (RGD) molecules (Fe3O4-RGD), and their integrin αvβ3-targeted T2-weighted imaging (T2WI) potential has been carefully evaluated. The Fe3O4-RGD demonstrated great relaxation in vivo. The T2WI signal of renal layers in the targeted group at 3 h after intravenous injection of Fe3O4-RGD was distinctly lower than baseline, indicating MRI signal decreased in the established IgA nephropathy rat model. Moreover, the TEM characterization and Prussian blue staining confirmed that the Fe3O4-RGD was located at the region of glomerulus and tubular interstitium. Moreover, no obvious signal decreased was detected in the untargeted Fe3O4 treated and normal groups. Collectively, our results establish the possibility of Fe3O4-RGD serving as a feasible MRI agent for the noninvasive diagnosis of IgA nephropathy.IgA nephropathy is the most common glomerular disease in the world and has become a serious threat to human health. Accurate and non-invasive molecular imaging to detect and recognize the IgA nephropathy is critical for the subsequent timely treatment; otherwise, it may progress to end-stage renal disease and lead to glomerular dysfunction. In this study, we have developed a sensitive, specific, and biocompatible integrin αvβ3-targeted superparamagnetic Fe IgA nephropathy is the most common glomerular disease in the world, and approximately 50% of IgA nephropathy will progress to end-stage renal disease within 30 years regardless of treatment . The golβ , as a common examination for clinical disease diagnosis, possesses high spatial resolution and good tissue contrast and is not radioactive . Neverthtibility . At prestibility  and CR2-tibility . Howeverin vivo  and low positron energy (0.64 MeV)  possesses superior sensitivity and can be used to observe the distribution of specific tracers as well as quantitatively measure the transport rates in vivo  18F is a.64 MeV) . The appbaseline . Moreoveiagnosis . Particurin αvβ3 . However3O4 nanoparticles (NPs). Subsequently, these paramagnetic NPs were functionalized with RGD molecules (RGD-Fe3O4) as an integrin αvβ3-targeting MRI nanoprobe to specifically recognize the glomerular mesangial region of IgA nephropathy  RGD-Fe3O4 NPs have been carefully evaluated in the IgA nephropathy rat model. Therefore, our study provided a foundation into the development of safe and effective T2WI for molecular imaging of detecting IgA nephropathy noninvasively.In our work, a facile method was used to prepare the uniform BSA-modified Fehropathy . At firs3O4 NPs were firstly synthesized by a simple wet chemistry method. Briefly, 200 ml of 0.18 mol/L Fe (NO3)3 solution and 200 ml of 0.12 mol/L FeSO4 were mixed homogeneously. The above mixed solution was kept stirred at 40oC under the protection of N2 atmosphere. Subsequently, 48 ml of NH3.H2O solution was added and stirred for 15 min and then aged at 40oC for 5 min. Finally, Fe3O4 magnetic NPs were obtained by centrifugation and washing process. Fe3O4 magnetic NPs were then modified with BSA molecules. Specifically, an appropriate amount of Fe3O4 NP solution was added to 30 ml of BSA (10 mg/ml) solution and stirred for 24 h, mechanically. The Fe3O4-BSA NPs were obtained by separating and washing processes.Fe3O4-BSA NP solution (2 mg/ml) was uniformly dispersed in 28 ml of ultra-pure water, and then followed by adding 2 ml of PBS (pH 7.4) solution. The above solution was subjected to further ultrasonic dispersion for 10 min. Subsequently, 230 mg of EDC was added to the above mixed solution and stirred for 0.5 h in the dark. One hundred fifty milligrams of NHS was further added and vigorously stirred for 1 h under dark conditions. Fifteen milliliters of RGD (1 mg/ml) was then added and stirred for 24 h under dark conditions, continually. Finally, the above solution was centrifuged through an ultrafiltration tube at a speed of 5,500 RPM and washed three times with ultra-pure water. Fe3O4-BSA-RGD NPs were further dispersed in 20 ml of ultra-pure water for further use.Twenty milliliters of FeoC (room temperature) and 59.6% relative humidity in the SPF degree animal laboratory of the First Affiliated Hospital, School of Medicine, Zhejiang University. All rats were divided into two groups randomly (model group and control group). The rats of the model group were administrated with 100 g/L BSA solution every other day in a dose of 800 mg/kg by gavage. Besides, the model group also received 0.4 ml mixed solution of CCl4 and castor oil weekly by subcutaneous injection, and 0.05 mg of LPS every other week by tail vein injection. Simultaneously, isodose saline was administered to the control group in the same way.For animal model construction about IgA nephropathy, 40 male SD rats (6 to 8 weeks old) were purchased from Zhejiang Academy of Medical Sciences and fed at 22.8After 6 weeks’ feeding, biochemical and pathological examinations were conducted to verify histopathological changes of IgA nephropathy and investigate renal functions in the above two groups. The samples were harvested repeatedly every 2 weeks until the model group got a significant difference in histology compared with the control group.To detect renal functions, blood samples were taken from the tail vein and 24-h urine was collected to determine creatinine (CRE), blood urea nitrogen (BUN), and albumin (ALB) levels by automatic biochemical analyzers .To compare the changes of mesangial cells and matrix in two groups, two rats from the model and control group were sacrificed, and one part of sagittal renal tissue slices was fixed in formalin, embedded in paraffin, sliced, and dewaxed to water for HE, PAS, and MASSON staining. Subsequently, staining images were acquired from the light microscope .18F-AlF-NOTA-PRGD2 probe molecule for micro-PET technology was used. The difference in renal radioactivity uptake between IgA nephropathy rat and the normal group was compared after intravenous application of 18F-ALF-NOTA-PRGD2 in vivo. Rats (n = 1/group) were weighed and anesthetized with 4% chloral hydrate by intraperitoneal injection. Micro-PET was performed at 30, 40, 50, 60, 70, 80, and 90 min after injection of 18F-AlF-NOTA-PRGD2 (about 37 mBq) via tail vein. The images were reconstructed according to a two-dimensional Ordered Subsets Expectation Maximum (OSEM) algorithm and then were processed by the Inveon Research Workspace (IRW). The regions of interest (ROIs) of the entire renal parenchyma were drawn manually, and maximum percent injected dose per gram of body weight (%ID/g) was obtained on the workstation directly.In order to further confirm successful establishment of the IgA nephropathy rat model, the 2O2 solution for 10–20 min. Subsequently, renal sections were treated with citric acid buffer for 15–20 min and sealed with 10% normal serum for 30 min at 37oC. After that, the diluted primary antibody  was added and incubated at 37oC for 60 min. The fluorescent second antibody was added and incubated at 37oC for 30 min. DAPI was added for core staining and section sealing. In the end, the fluorescence images were shot under a fluorescence microscope . It is worth mentioning that quantitative analysis of immunofluorescence intensity was performed by Image-Pro Plus 6.0 . Briefly, the mean density, which was the ratio of integrated option density to area, was acquired to estimate the fluorescence intensity.To confirm the increased expression of integrin avβ3 in model rat kidney, integrin avβ3 fluorescence staining was performed. Firstly, renal tissue was fixed, embedded, sliced, and dewaxed to water conventionally, and then soaked into 3% H3O4-RGD in vitro, a series of Fe3O4-RGD solutions with different Fe concentrations  were prepared. Then, T2-weighted imaging and T2*mapping were performed on the clinical GE 3.0-T MRI . Subsequently, the value of R2 was measured by READ Y View  based on the T2* mapping. Finally, the relative curve between R2 value and Fe3O4-RGD gradient concentration was fitted by GraphPad Prism 8 .For the purpose of evaluating relativity r2 of Fe3O4-RGD solution at a dose of 15 mg Fe/kg. Group 2 (untargeted group) received intravenous tail injection of Fe3O4-BSA solution at a dose of 15 mg Fe/kg. Group 3  group received intravenous tail injection of Fe3O4-RGD solution at a dose of 15 mg Fe/kg.In order to explore the feasibility for the early noninvasive diagnosis of IgA nephropathy through identifying the increased expression of integrin avβ3 early, the relative rat MRI experiment was performed. The rats were divided into three groups: Group 1 (targeted group) received intravenous tail injection of Fe3O4-RGD or Fe3O4-BSA solution through the tail vein on the clinical GE 3.0-T MRI . T2-weighted imaging was acquired with the following parameters: repetition time (TR), 3,000 ms; echo time (TE), 68 ms; flip angle, 142°; field of view (FOV), 8 cm × 8 cm; matrix size, 128 × 128; number of sections, 16; slice thickness, 1.0 mm; total acquisition time, 4 min. The parameters of T2* mapping were as follows: repetition time (TR), 110 ms; echo time (TE), 2.0 to 16.1 ms; flip angle, 20°; field of view (FOV), 9 cm × 9 cm; matrix size, 64 × 64; number of sections, eight; slice thickness, 2.2 mm; total acquisition time, 30 s; the R2 values of renal different layers between groups were measured and statistical analysis were conducted by SPSS 26 .Subsequently, the above three group’s rats were weighted and anesthetized with 4% chloral hydrate at a dose of 0.8 ml/kg; T2-weighted imaging and T2* mapping were performed at baseline and 3 h after administering FeAfter MRI scan, those rats were sacrificed through cervical dislocation. Kidney samples were harvested rapidly and fixed in formalin for Prussian blue staining, as well fixed in 2.5% glutaraldehyde for TEM tests, respectively.3O4 magnetic NPs were initially prepared and then modified with BSA molecule (Fe3O4-BSA) for further RGD grafting process. The morphology and structure were investigated by transmission electron microscopy (TEM). 3O4-BSA NPs exhibited a regularly spherical morphology with the uniform diameters in the range ∼20 nm. Meanwhile, the insert selected area electron diffraction (SAED) pattern demonstrated the defined diffraction rings, implying the polycrystalline nature of Fe3O4-BSA. Furthermore, the HRTEM images of Fe3O4-BSA NPs revealed that the distance between the adjacent lattice fringes is ∼0.1972 nm (3O4-BSA matrix (3O4-BSA NPs were further determined by x-ray diffraction (XRD). As presented in , the characteristic peaks of Fe3O4-BSA are related to the crystal planes of Fe3O4 crystal (JCPDS Card No. 19–0629) . As displayed, the XPS spectrum of Fe3O4-BSA contains C1s, O1s, N 1s, S 2p and Fe 2p peaks , which were determined by TEM and EDS mapping observed results. As demonstrated in 3O4-RGD NPs possess a well-defined spherical morphology with a diameter of approximately 20 nm and contains Fe, O, S, and N elements. A more interesting phenomenon is that the N element signal intensity was enhanced compared with the Fe3O4-BSA, which may be due to the successful grafting of the RGD molecules. The hydrodynamic dimensions of Fe3O4-RGD NPs increased from ∼20 to ∼50 nm . Furthermore, the cytotoxicity of Fe3O4-RGD on normal cells (293T cells) was assessed by CCK-8 test in vitro. The result showed that the cells’ viability slightly decreased with the increase in concentrations after 24-h incubation. All groups demonstrated low cytotoxicity compared with the control group (p > 0.05). About 74% cell viability was still observed at the highest concentration (80 μg/ml) of Fe3O4-RGD  , indicat2p peaks . In the 2p peaks , the mai2p peaks . One notspersion , indicato ∼50 nm . Meanwhio ∼50 nm . Moreovee3O4-RGD . This re control , which ip > 0.05) among two groups (p < 0.05)  . This reTo determine the histopathological changes of IgA nephropathy in the model group, various histological tests were conducted. H&E staining revealed that lots of mesangial cells were observed among expanded mesangial matrix with slightly compressed capillary loop in the model group, which was not obvious in the normal group . Meanwhip < 0.05)  . This in < 0.05) . Therefo18F-AlF-NOTA-PRGD2 probe was then used to confirm the successful establishment of the IgA nephropathy model. The chemical structure of 18F-ALF-NOTA-PRGD2 is shown in 18F-flouride. The radiochemical purity of the sample could reach 98%   . The for < 0.05) ; therefo3O4-RGD for IgA nephropathy detection  were added into tubes, respectively. Subsequently, the above tubes were performed on the clinical GE 3.0-T MRI for T2-weighted imaging. With the Fe3O4-RGD concentration increased, the T2WI signal decreased more  model rats received intravenous tail injection of Fe3O4-BSA solution at a dose of 15 mg Fe/kg. Group 3  received intravenous tail injection of Fe3O4-RGD solution at a dose of 15 mg Fe/kg. The results indicated that the T2WI signal of renal parenchyma in the targeted group was lower than baseline after injection of Fe3O4-RGD for 3 h   and in the normal group (p < 0.001). All of these indicated that the targeted binding of Fe3O4-RGD with integrin αvβ3 achieves T2WI signal reduction. One notable fact is that compared with baseline, the mean value of untargeted group and normal group showed a higher signal ratio after the injection of Fe3O4-RGD NPs; the reason for this is not clear. Because the main metabolism of Fe3O4-RGD NPs is through liver, spleen, and kidney  . These rperiment , and anaperiment , we can rin α3β1 . The sigd kidney , we infe3O4-RGD NPs can be well combined with renal integrin αvβ3 in the targeted group. Thus, no iron element was observed in the untargeted group and normal group for Prussian blue staining. Moreover, the deposition of black iron oxide NPs was observed in the foot process of the targeted group by TEM, while no black iron oxide NPs were detected in the untargeted group and normal group. Finally, we have used the ICP to quantify iron content. This result exhibited that the ratio of Fe content to kidney was 0.240 ± 0.002 mg/g for the administration group after injection of Fe3O4-RGD for 3 h, and the ratio was 0.130 ± 0.010 mg/g for the normal group. These results indicated that the deposition of Fe3O4-RGD in kidney for the administration group was higher than that for the normal group (p < 0.05). Therefore, the above data further illustrated the dominant location of Fe3O4-RGD in the glomerular podocytes’ reduced kidney T2WI signal of the rats with IgA nephropathy.After the MRI detection process, Prussian blue staining and TEM investigation of renal tissue were further performed to explore the targeted location of iron element in the renal tissue. Prussian blue staining demonstrated that the blue dots were scattered in the glomerulus and tubular interstitium in the targeted group. However, no apparent blue dots were found in the untargeted group and normal group . The renal group . Besides3O4 NPs for the noninvasive MR imaging of integrin αvβ3 in IgA nephropathy disease. The rat model was successfully established and verified by biochemical tests and histological staining. Furthermore, the micro-PET 18F-AlF-NOTA-PRGD2 probe molecule was utilized to confirm the successful establishment of the IgA nephropathy in the rat model. Subsequently, the synthesized RGD-Fe3O4 NPs were injected intravenously into model rats and the integrin αvβ3-targeted T2WI RGD-Fe3O4 NPs proved the accurate detection of the IgA nephropathy disease. Therefore, our study demonstrated the clinical possibility of utilizing the safe and effective Fe3O4-RGD for accurate MR imaging to diagnose IgA nephropathy noninvasively.In this study, we have developed a sensitive, specific, and biocompatible integrin αvβ3-targeted superparamagnetic Fe


LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes.  LST1 splice variants of various length (transmembrane and soluble isoforms) were described in human mRNA. However, only one of these variants LST1/A has been detected at the protein level (LST1 genes only as LST1.Leukocyte-specific transcript 1 protein (LST1) is a small, 97 amino acid long, transmembrane adaptor protein. It is composed of a very short extracellular segment with a dimerization cysteine, a single transmembrane domain, immediately followed by a palmitoylation site, and a larger cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs (ITIM). Despite of its small size, at least 16 in level . The rolin level . Interesin level . In thisOur previous analysis of LST1 expression pattern by in-house generated monoclonal antibody LST1/02 recognizing human but not murine LST1 revealed its expression exclusively in leukocytes of the myeloid lineage  and in related cell lines U-937, THP-1) 7, THP-1  and in tLST1 gene  localized in MHCIII locus. This genomic site harbors many immunologically important genes, such as genes coding for Lymphotoxin-β, Tumor Necrosis Factor α, several complement proteins and others. High LST1 expression in leukocytes together with localization of its gene in one of the immunologically most important loci, raises a question about the function of LST1 in the immune system -induced colitis, a mouse model of IBD. We show that the LST1 deficiency results in alterations in innate leukocyte subset composition and in milder progress of DSS-induced colitis, demonstrating LST1 involvement in the regulation of leukocyte homeostasis and inflammation.In HeLa cells, overexpression of LST1 induced formation of tunneling nanotubes he cells –12. In aza virus . This waza virus . Increastm1(KOMP)Vlcg on C57Bl/6J genetic background (abbreviated as -/-Lst1) was obtained from International Knockout Mouse Consortium. These animals were crossed to C57Bl/6J mice from Animal facility of the Institute of Molecular Genetics to obtain heterozygotes -/-Lst1 x C57Bl/6J. Their homozygote offspring were used as littermates for comparative experiments at the age of 6 - 10 weeks. Animal experiments were approved by the Animal Care and Use Committee of the Institute of Molecular Genetics and were in agreement with local legal requirements and ethical guidelines.LST1-deficient mouse strain LST14Cl, 0.1 mM EDTA , 1 mM KHCO3). Colon lamina propria cells were isolated from entire colon. The colon was opened longitudinally, washed with cold PBS and then rocked for 1 hour in 10 ml solution of 5mM EDTA/2% FCS/HBSS without Ca2+ and Mg2+ at 4°C followed by washing with 10 ml HBSS/2% FCS. Colon was then cut to 3 mm pieces and digested with the solution containing collagenase II  and DNase I  in DMEM/2% FCS with shaking . After each digestion round, tissues were poured onto petri dish and triturated gently with plastic Pasteur pipette in order to obtain the lamina propria cells. Cell suspension was filtered through 100 µm Sysmex filters, centrifuged, resuspended in 5 ml of PBS/2% FCS and kept on ice. After the second round of digestion, cells were pooled together, centrifuged, resuspended in PBS/2% FCS and filtered again through 50 µm Sysmex filters to obtain single cell suspension of colon lamina propria cells. For murine colonic epithelial cell isolation, colon was opened longitudinally and washed vigorously in PBS on ice. Then it was cut to shorter fragments and incubated in 20 ml pre-heated HBSS, 3% FBS, 2 mM EDTA twice at 37°C. The suspension with released cells was then collected and filtered through 100 µm filter. Filtered cells were resuspended in 1 ml  TrypLE™ Express Enzyme , resuspended by pipetting and filtered again through 40 µm filter into 15 ml tubes. TrypLE™ enzymes were neutralized by washing filter with 3 ml of HBSS (without Ca2+ and Mg2+), 3% FBS, 2 mM EDTA . Cells were centrifuged for 10 min , stained with CD45 and EpCAM fluorescent antibodies and sorted on BD FACSAria™ cell sorter as CD45- EpCAM++. Bone marrow derived dendritic cells (BMDC) and bone marrow derived macrophages (BMDM) were generated from isolated mouse bone marrow cells by culturing in IMDM culture media supplemented with 10% FCS and 3% supernatant from J558 cells containing GM-CSF for 10 days (BMDC) or 5% supernatant from CMG 14-12 cells containing M-CSF for 7 days (BMDM) as described in detail here  - 0.5 µg/ml and 1 µg/ml for 24 hours followed by lysis (300 µl 2× concentrated SDS-PAGE sample buffer). Lysed samples were sonicated, heated to 95°C for 10 minutes and analyzed by immunoblotting under non-reducing conditions.BMDM were plated in a 6-well plate at 2 × 106 cells per well) and then incubated for indicated time-points with IFNγ (Peprotech) – 50 ng/ml, TNFα (Peprotech) – 20 ng/ml, LPS (Sigma) – 100 ng/ml and PolyI:C  – 20 µg/ml, GM-CSF – 3% supernatant from J558 cells. Subsequently, cells were lysed in 2 × concentrated SDS-PAGE sample buffer  and subjected to immunoblotting with indicated antibodies.BMDM and BMDC were cultured overnight in DMEM without M-CSF/GM-CSF in a 12-well tissue culture plate (10-/-Lst1 mice with recombinant intracellular domain of murine LST1 (starting at Cys40) produced in Escherichia coli. After immunization, splenocytes were fused with Sp2/0 myeloma cells and antibody producing hybridomas were cloned by limiting dilution. Antibodies used for Western blot detection: SHP-1 , SHP-2 , GAPDH , IκBα . Western blot ECL signals were detected with Azure c300 imaging system (Azure Biosystems) and quantified using AIDA image analyzer software .Flow cytometry antibodies are listed in + CD4+; CD8 T cells – CD3+ CD8+; B cells – CD19+ CD3-; NK cells – NK1.1+ CD3-; NK1.1+CD3+; Macrophages – CD11blow F4/80+; Monocytes – CD11b+ Ly6Chi Ly6G-; Neutrophils – CD11b+ Ly6Cmed Ly6G+; Dendritic cells – CD11b+ CD11c+. Colon leukocytes were measured on BD™ FACSymphony flow cytometer (Becton Dickinson) according to International Mouse Phenotyping Consortium standards+ CD4+; CD8 T cells – CD5+ CD8+; B cells – CD19+ MHCII+; NK cells – CD5- CD161+; NKT cells – CD5+ CD161+; Monocytes – CD11b+ Ly6C+ Ly6G-; Neutrophils – CD11b+ Ly6Cmed Ly6G+; Macrophages – CD11b+ F4/80+; Dendritic cells – CD11b+/- MHCII+ CD11c+. Data were analyzed in FlowJo™ software.Cells of bone marrow, spleen and lymph nodes were incubated with fluorescently labeled antibodies and Fc-receptor blocking antibody (2.4G2) and analyzed on BD™ LSRII flow cytometer (Becton Dickinson). Staining was divided into two sets – A , B . Cell populations were gated as: CD4 T cells – CD3Blood from tail vein was collected into EDTA tubes  and centrifuged . Supernatant (plasma) was then transfered to fresh tubes and frozen in -80°C. Colons were homogenized in 400 µl RIPA lysis buffer  containing 5 mM iodoacetamide (Sigma-Aldrich) and diluted Protease Inhibitor Cocktail Set III  using AvansAHM1 Homogenizer on ice . The homogenates were cleared by centrifugation and frozen in -80°C. Concentrations of cytokines in blood plasma or colonic lysates were determined using IL-6 and TNF alpha Mouse Uncoated ELISA Kit with Plates  according to the manufacturer´s protocols.in-vivo under 20% zoletile anesthesia in SkyScan 1176  at the resolution of 8.67 µm with 0.5 mm aluminum filter  with 180° rotation, and reconstructed in NRecon 1.6.10.4  with parameters of smoothing = 4, ring artifact correction = 7, beam hardening correction = 23%, and spread of intensities from 0.007 to 0.11 AU. Only reconstructions without artifacts underwent analysis resulting in 42 femurs analyzed. The femurs were segmented in CT Analyzer 1.16.4.1  and reoriented in DataViewer 1.5.2 . CT Analyzer was also used for semiautomatic selection of regions of interest  and subsequent 2D and 3D analysis. Parameters describing bone volume, porosity, and structural complexity were recorded. Bone mineral density (BMD) and tissue mineral density (TMD) were recorded for trabeculae and corticals, respectively, based on the correlation with calibrated hydroxyapatite phantoms.Femurs of 28 mice were scanned -/-Lst1 and WT mice  were fed with 2,5% DSS dissolved in drinking water for 6 days to induce acute colitis. Starting on day 7, DSS solution was changed for plain water. Parameters defining disease activity index, i.e. stool consistency, occult bleeding  and body weight on days 0, 2, 4, 5, 6, 8 were measured during the experiment. Mice in control group were administered only plain water. On day 8, mice were anesthetized with isoflurane (0.3L/min) for collection of blood sample from the eye vein and sacrificed. Colon length was recorded, organs were collected , processed into single cell suspensions and measured with BD™ FACSymphony flow cytometer. Disease Activity Index was evaluated as average of the three measured parameters  on a particular day. Scoring values were normalized to day 0, which has an arbitrary value 0. Scoring system for body weight: 1, loss of 1% –5% weight; 2, loss of 5% –10% weight; 3, loss of 10% –20% weight; 4, 20% and higher body weight loss. Stool consistency: 0, well-formed stool pellets; 1, loose stool pellets; 2, pasty and semi-formed stools that did not adhere to the anus (mild diarrhea); 3, slimy stool (moderate diarrhea); 4, severe watery diarrhea that adhered to the anus and contained with blood. Occult bleeding: 0, no blood; 2, positive Hemoccult test, and 4, gross rectal bleeding.For LPS challenge, mice were intraperitoneally injected with LPS . After 4 and 16 hours blood samples were collected and then mice were sacrificed by cervical dislocation and splenocyte subsets analyzed. To induce colitis with dextran sodium sulphate (DSS), littermates of 2 .The entire colon was opened longitudinally, flushed with PBS and fixed in 4% neutral buffered formaldehyde for 48 hours as swiss roll. Samples were dehydrated, embedded in paraffin and sectioned at 5 μm and stained with hematoxylin-eosin. For histological evaluation, areas of inflammatory lesions were microscopically evaluated and quantified as described . Area of5 macrophages were added into the insert with migration media and 600 µl of migration media with CXCL12 (100 ng/ml) into the bottom well. After 2 hours at 37°C, 5% CO2 migrated cells were collected (attached cells were released with 0.2% EDTA in PBS). The collected cells were mixed with 10 µl of Flow Cytometry Absolute Count Standard™ (Bangs Laboratories) and counted in BD™ FACSymphony flow cytometer (Becton Dickinson).Corning 6.5 mm diameter Transwell Permeable Supports with 8.0 µm pores were used. Inserts (upper well) with pores were coated with 100 µl fibronectin (50 µg/ml) for 2 hours at room temperature and washed two times with migration media . 10The total cellular RNA was isolated using Quick-RNA Miniprep Plus kit and transcribed into cDNA by RevertAid RT Reverse Transcription Kit, both used according to manufacturer’s instructions. The cDNA was then used as a template for real-time quantitative PCR performed on LightCycler 480 using SYBR Green I Master mix (Roche). Primers used in cytokine mRNA detection are listed in Student´s t-test  and One-way ANOVA with Tukey posttest for P-values and two-sided Grubb´s test for outlier recognition was performed in Graphpad Prism Software. Bars in figures represent mean ± SD, if not stated otherwise. P values lower than 0.05 are marked with asterisks as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.0001.via cysteine bridges , neutrophils (Neu), monocytes (Mon), but not in lymphoid lineage cells  (in vitro in murine BMDM and BMDC after overnight (16 hours) stimulation with IFNγ, LPS, TNFα and PolyI:C  did not confirm any differences in the bone mineral density (not shown). On the other hand, it revealed reduced numbers of trabeculae in the trabecular bone tissue of -/-Lst1 mice , other populations show reduced numbers. These include mainly bone marrow and splenic NK cells and splenic NKT cells  reduction in neutrophils (-/-Lst1 mice were observed (not shown). TNFα and IL-6 levels in the blood at 4 hours after LPS challenge were also not affected by LST1 deficiency . Mice were monitored for Lst1 mRNA expression and for number and area of inflammatory lesions, length of the colon and overall disease activity defined by Disease Activity Index – DAI . Lst1 mRNA expression in the colon tissue did not significantly change during the course of the experiment and it was absent from isolated colonic epithelial cells. . Intriguingly, -/-Lst1 BMDM showed enhanced migration towards CXCL12. While this observation is interesting, it does not show any attenuation of migratory capacity and, as a result, cannot explain the reduced percentages of macrophages and other cell types in the lymphoid organs analyzed in this study. Reduced percentages were frequently observed also for LST1 non-expressing NK and NKT cells  and by institutional funding from Institute of Molecular Genetics, CAS (RVO68378050). The results were obtained using the research infrastructure of the Czech Center for Phenogenomics supported by the projects of the Ministry of Education, Youth and Sports (MEYS) LM2018126 and OP RDI CZ.1.05/2.1.00/19.0395, and CZ.1.05/1.1.00/02.0109 provided by the MEYS and ERDF, and OP RDE CZ.02.1.01/0.0/0.0/16_013/0001789 by MEYS and ESIF. Centre d’Immunophénomique is supported by the Investissement d’Avenir program PHENOMIN .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pulmonary delivery of chitosan nanoparticles is met with nanoparticle agglomeration and exhalation. Admixing lactose-based microparticles (surface area-weighted diameter~5 μm) with nanoparticles mutually reduces particle agglomeration through surface adsorption phenomenon. Lactose-polyethylene glycol (PEG) microparticles with different sizes, morphologies and crystallinities were prepared by a spray drying method using varying PEG molecular weights and ethanol contents. The chitosan nanoparticles were similarly prepared. In vitro inhalation performance and peripheral lung deposition of chitosan nanoparticles were enhanced through co-blending with larger lactose-PEG microparticles with reduced specific surface area. These microparticles had reduced inter-microparticle interaction, thereby promoting microparticle–nanoparticle interaction and facilitating nanoparticles flow into peripheral lung. Lactose is widely used in inhalation dosage form development with reference to dry powder aerosol ,2,3,4. IThe United States Food and Drug Administration indicates that a nanoproduct has at least one nanoscale dimension (about 1 nm to 100 nm), or exhibits physicochemical or biological properties attributable to its nanogeometry, up to one µm . The nanChitosan is widely explored as a pulmonary therapeutic carrier in lung cancer therapy ,14,15,16Chitosan nano- and micro-carriers have been developed as pulmonary drug vehicles ,17,18. TA direct chitosan nanoparticles-lactose-polyethylene glycol 3000 microparticle blend enhances the powder flow, similar to the microparticles-large carriers mix ,24,25. MUnlike co-spray drying, direct chitosan nanoparticles-lactose based microparticle blending reduces the likelihood of changes in nanoparticulate properties ,27,28. TThe size and shape, as well as specific surface area, of nanoparticles, admixed with lactose-based microparticles, affect their inhalation efficiency . Small aLactose monohydrate  was used as the matrix material of microparticles. Polyethylene glycol 400, 1000 and 3000  were used as the stabilizers with ethanol absolute  as the additive. Chitosan  was used as the model matrix material that was transformable into nanoparticles, with glacial acetic acid as a solvent . Lithium acetate anhydrous , ninhydrin and hydrindantin  were the reagents used for the quantitative assay of chitosan.Lactose microparticles L1-6 were prepared by a spray drying technique (The spray-dried powder was collected by a precipitating electrode. It was kept in a 30-mL glass jar. The powder was stored in a desiccator at 25 ± 1 °C for 5 days prior to characterization.Chitosan (0.1 %w/w) was dissolved in 0.5 %v/v acetic acid under continuous stirring for 5 h at 25 ± 1 °C. The solution was subjected to nanospray-drying  with inlet temperature = 70 °C, outlet temperature = 24.8 °C, solution feed rate = 3 mL/min via bifeeding tubes, and atomizing air pressure = 6 bar. The spray-dried powder was collected at the precipitating electrode. It was retrieved into a 10 mL clear glass powder jar by a rubber spatula. The powder was stored in a desiccator at 25 ± 1 °C prior to characterization.ρb). The cylinder was subjected to 200 taps, where there was no further powder bed volume reduction thereafter, at 4 taps/s. The tapped density (ρt) was recorded as the quotient of powder weight to its tapped volume. Both bulk density and tapped density values of powder were used to derive Carr’s index and Hausner ratio by means of the following equations:A known weight of powder was added in a 5-mL measuring cylinder. The quotient of powder weight to bulk volume was recorded as the bulk density , surface area weighted mean diameter , specific surface area, span, volume median diameter d10, d50 and d90 were assessed by means of dry powder dispersion mode (Scirocco 2000) at 1 bar pressure. Triplicates were run for each type of microparticles with the results averaged.The size distribution of microparticles was determined using the laser diffraction analyzer . The volume weighted mean diameter (Da) and circularity (Circ) of microparticles were analyzed by ImageJ software . A minimum of nine measurements of three images were characterized.The surface morphology of microparticles was evaluated by scanning electron microscope (SEM) . The microparticles were adhered onto a carbon tape, and sputter-coated with 5 nm-thick platinum with an auto fine coater . The surface roughness (Rθ) of 3° to 60° at 5°/min scanning speed. Cu-Kα (40 kV and 30 mA) was used as the X-ray resource. Triplicates were conducted and the results were averaged.The crystallinity of microparticles was examined by the X-ray powder diffractometer (XRPD) , over diffraction angles  was calculated as:n at 16° ,30.The size and polydispersity index were determined using the Zetasizer  at 25 ± 1 °C and 90° scattering angle. The nanoparticles (5 mg) were dispersed in deionized water (30 mL) with the aid of magnetic stirring. Triplicates were determined with results averaged.The zeta potential was determined using the Zetasizer  at 25 ± 1 °C. The nanoparticles (5 mg) were dispersed in deionized water (30 mL) with the aid of magnetic stirring. Three measurements were determined with results averaged.® . The impactor was equipped with a vacuum source  and a critical flow controller .Andersen Cascade Impactor  was used to assess the aerosolization and inhalation profiles of nanoparticles as a function of physicochemical characteristics of lactose-PEG microparticles. The stage F-0 were assembled with silicone rubber O-rings to avoid leakage from gaps between stages. A glass fiber filter  was placed beneath stage 7 to capture small particles from upper stages. A pre-separator was attached to stage 0 with a connecting induction port assembled with mouthpiece adapter and Handihaler®. The powder in the inhaler was actuated to the impactor through an air current flow of 48 L/min for 5 s which corresponded to a 4 kPa pressure drop as in human lung. The cut-off diameter of each stage was calculated using Equation (4): Stage 0, 6.91 µm; Stage 1, 4.45 µm; Stage 2, 3.61 µm; Stage 3, 2.53 µm; Stage 4, 1.61 µm; Stage 5, 0.84 µm; Stage 6, 0.54 µm; and Stage 7, 0.31 µm.50,48 and D50,28.3 are the cut-off diameters at 48 and 28.3 L/min respectively.The chitosan nanoparticles and lactose-PEG microparticles (weight ratio 1:9) were blended with 40 Hz vortex for 30 min . Thirty mg of powder mixture were inserted into a size-2 capsule . The capsule was inserted into the chamber of HandihalerThe contents of 5 capsules (15 mg chitosan nanoparticles) were released into the impactor sequentially in a cumulative fashion. Triplicates were conducted, and with impactor cleaned before another 5 capsules were tested. The emitted powders on mouthpiece, induction port, pre-separator, collection plate and filter were collected using 0.5 %v/v acetic acid solution. The samples were stored in individual glass vials, oscillated in a shaker bath  at 25 ± 1 °C for 5 h, and had chitosan content determined by ninhydrin assay method. The chitosan content was assayed using UV-VIS spectrophotometer  at 570 nm wavelength. The detection and quantification limits were 0.048 mg/mL and 0.160 mg/mL respectively. The linearity ranged between 0.025 and 1.000 mg/mL.<4.5µm, FPD<3.6µm, and 0.5µm<FPD<3.6µm, were doses on stages 2 to F, stages 3 to F, and stages 3 to 7 respectively. FPD<4.5µm reflected deep lung-deposited particles. FPD<3.6µm reflected peripheral lung-deposited particles. 0.5µm<FPD<3.6µm represented FPD<3.6µm excluding particles prone to exhalation (<500 nm). The fine particle fraction (FPF) was defined as percentage FPD to ED. The respirable fraction (RF) was denoted by percentage of FPD to DD.The emitted dose (ED) referred to the collective chitosan mass on mouthpiece, induction port, pre-separator and all stages. Deposited dose (DD) referred to chitosan mass on stages 0 to F. Percent dispersed (PD) was defined as percentage ED in relation to total dose (TD). Percent inhaled (PI) was defined as percentage DD in relation to TD. The fine particle doses (FPD), FPDThe cumulative particle size distribution pattern was plotted on a log probability graph. The particle size at 50th percentile of distribution was denoted as mass median aerodynamic diameter (MMAD). The square root of the ratio of particle size at 84.13th percentile to 15.87th percentile was denoted as a geometric standard deviation (GSD). Triplicates were run for each experiment with the results averaged.−1 and at 4 cm−1 resolution. Chitosan nanoparticles, lactose-PEG microparticles and the mixture of both (weight ratio 1:9) were used as the references. The correlation of FTIR spectra between impactor powder samples and references was analyzed using Perkin Elmer Spectrum Version 10.3.6 software  by means of the following equation [iA = spectra absorbance value of test powder at frequency i, iB = spectra absorbance value of reference powder at frequency i, and iw = a statistical weighting factor. Triplicates of experiment were conducted with results averaged.The segregation pattern of chitosan nanoparticles from lactose-PEG microparticles upon inhalation was characterized. L3 and L6 admixed with chitosan nanoparticles were collected from stages 0 to 7. The powder mixture was blended with potassium bromide at a 1:99 weight ratio and was subjected to grinding. The ground mixture was compressed by an axial load  to a maximum pressure of 10 tons for 2 min. The FTIR spectrum of the formed disc was obtained by an IR spectrometer  through scanning over 400 to 4000 cmequation :(5)CorreThe physicochemical characteristics of six lactose-PEG microparticle variants are summarized in d50 ranging from 5.43 ± 0.10 µm (L3) to 13.32 ± 0.15 µm (L5) (Lactose-PEG microparticles were characterized by  µm (L5) . PEG 300Lactose is soluble in water (0.22 g/mL at 25 °C). It is practically ethanol-insoluble. PEG is soluble in ethanol, with a larger molecular weight PEG having a higher solubility . In the All batches of lactose-PEG microparticles were irregular in shape with some variations ; Table 2The spray-dried lactose microparticles were hygroscopic and experienced a marked aggregation during handling. PEG could transform the spray-dried lactose with a higher crystallinity level. It could form hydrogen bonds with water in the course of spray drying. The PEG could delay the lactose drying and provide more time for lactose molecules to rearrange into a crystalline structure ,33.θ=19.98°2 = 1640.67 ± 75.13) and higher than PEG-free spray-dried lactose (peak areaθ=19.98°2 = 15.85 ± 7.14)  . In the The ethanol-free formulations demonstrated a decrease in matrix crystallinity with increasing PEG molecular weight . The wataR of 0.38 ± 0.00 nm and a crystallinity index of 19.40 ± 2.23 %  with smaller specific surface areas brought about a higher respirable fraction of chitosan at the peripheral lung levels . The inhalation performance is improved when the chitosan nanoparticles were blended with lactose-PEG 3000 microparticles . Larger g levels . A reducd50 of lactose-PEG microparticles exerted the largest effect on deposition manner of the chitosan nanoparticles (d50 values, whereas the opposite was applicable for L6. L3 and L6 were adopted to examine the attachment/detachment behavior of chitosan nanoparticles in an inhaled powder blend. FTIR spectra of the test powder blend collected at each stage were compared against the reference spectra, which constituted of lactose-PEG microparticles, chitosan nanoparticles, and their blend at a weight ratio of 9:1.The specific surface area and articles . L3 had The changes in the correlation between the FTIR spectra of the test powder blend and the reference powder reflected the changes in the component ratio in the test powder blend. They did not necessarily reflect the changes in the amount of its components.With reference to L3 and L6, marked correlation changes between spectra took place after stage 2 to 3 . The speAn inhalable microparticulate carrier can be used successfully to deliver nanoparticles in a form of coarse carrier-free dry powder inhaler. The specific surface area of the small microparticle carriers significantly affects the nanoparticle deposition on the airway. Larger microparticle carriers enhance the peripheral lung deposition of nanoparticles. They are characterized by a reduced specific surface area, which in turn facilitates the detachment of nanoparticles from the microparticle carrier and into the lower lung regions during inhalation. Unlike the blend of large carriers and nanoparticles , the inh