EGFR) disrupts the entire complex and causes a rapid, global arrest of the cell cycle. Normal epithelial cells do not recruit these additional receptors to the adhesion mechanism and are not arrested by SSTNEGFR. Although EGFR docking with Sdc4 in the tumor cells is required, cell cycle progression does not depend on EGFR kinase. Instead, progression depends on RON kinase, activated by its incorporation into the complex. RON activates ABL1, which suppresses p38 mitogen-activated protein kinase and prevents a p38-mediated signal that would otherwise arrest the cell cycle. These findings add to the growing list of receptor tyrosine kinases that support tumorigenesis when activated by their association with syndecans at sites of matrix adhesion and identify new potential targets for cancer therapy.Epidermal growth factor receptor (EGFR) is a causal factor in carcinoma, yet many carcinoma patients are resistant to EGFR inhibitors. Potential insight into this resistance stems from prior work that showed EGFR in normal epithelial cells docks to the extracellular domain of the plasma membrane proteoglycan syndecan-4 (Sdc4) engaged with α3β1 and α6β4 integrins. We now report that this receptor complex is modified by the recruitment of syndecan-2 (Sdc2), the Recepteur d’Origine Nantais (RON) tyrosine kinase, and the cellular signaling mediator Abelson murine leukemia viral oncogene homolog 1 (ABL1) in triple-negative breast carcinoma and head and neck squamous cell carcinoma, where it contributes to EGFR kinase–independent proliferation. Treatment with a peptide mimetic of the EGFR docking site in the extracellular domain of Sdc4 (called SSTN It is well known that cooperative signaling between adhesion receptors and receptor tyrosine kinases regulates mechanosensing, cell migration, proliferation, and survival. This cooperative signaling often emanates from adhesion sites that incorporate receptor tyrosine kinases along with integrins, cadherins, or other adhesion receptors, leading to clustering and both ligand-dependent and ligand-independent activation of the kinases , 2, 3, 4Syndecans contain docking sites in their extracellular domains that assemble partner receptors into signaling complexes , 6, 7. IEGFR) (Syndecan-4 (Sdc4) mediates the assembly of the epidermal growth factor receptor (EGFR) with the laminin-332 (LN332)–binding α6β4 and α3β1 integrins , 13. WheEGFR) , 13.e.g., EGF, transforming growth factor-alpha) are overexpressed in up to 90% of head and neck cancer patients    occurs, presumably constitutively activated by metabolic, oncogenic, and/or genotoxic stress in the tumor cells but held in abeyance by signaling from the Sdc4 receptor complex. This suggests that an epithelial cell migration mechanism that relies on Sdc4, EGFR, and the α3β1 and α6β4 integrins adopts a dual role by incorporating Sdc2, RON, and ABL1 to sustain S-phase progression at times of cellular stress.EGFR has been implicated in multiple human cancers, including carcinomas of the head and neck and breast , 15, 16.patients , 18, 19,patients , 21, 22,patients , 24. EGFpatients , 26. Nonpatients , 27, sug (MST1R) , 29, 30) (MST1R) , which, EGFR was compared to nontumorigenic breast, oral, and epidermal epithelial cells. Whereas the proliferation of nontumorigenic, immortalized normal oral keratinocytes (NOKs), human tonsillar epithelial cells (HTEs), mammary epithelial cells (MCF10A), and epidermal keratinocytes (HaCaT) are not affected by SSTNEGFR (A), the peptide causes reduced proliferation of HNSCC (B) and TNBC cells (C) with an IC50 that falls with the range of 3 to 10 μM. This coincides with the approximate concentration required to displace 90% of EGFR and the α3β1 integrin from Sdc4 , to analyze DNA synthesis by flow cytometry. As expected, treated NOKs showed no significant change in EdU incorporation or in the distribution of cells throughout the cell cycle when compared to untreated cells (A). In contrast, an S-phase block after only 3 h treatment of UM-SCC47 cells is indicated by a complete lack of EdU incorporation (A), and the failure of the cells to show any major change in their distribution in the G1-, S-, G2-, and M-phases of the cell cycle suggests the peptide may induce a global block to cell cycle progression. Shorter treatment times utilizing in situ staining of fixed cells show that treatment with SSTNEGFR for as little as 1 h reduces EdU incorporation by well over 90% in HNSCC and TNBC cells, whereas NOKs and MCF10A cells show no reduction (B). To verify the arrest in S-phase, UM-SCC47 cells were synchronized in early S-phase using a double thymidine block, then released for 2 h to recover (C). Cells released for 2 h and chased for an additional 12 h with the vehicle alone progress into late S-phase/G2 or pass through G2/M and reenter G1 (C). In contrast, cells released for 2 h then chased for 12 h in SSTNEGFR remain entirely in S-phase and show little or no EdU incorporation above background (C).For the sake of simplicity, we elected to use a single cell line (UWSCC47 cells) to conduct exploratory studies into this growth mechanism and then extended our findings to the other transformed cells. UM-SCC47 cells and normal NOKs as a control were treated with SSTNed cells A. In conporation A, and theduction B. To ver recover C. Cells enter G1 C. In conckground C.Figure e.g., Chk1 or Chk2) downstream of the DNA damage sensors  that phosphorylate cell cycle regulatory factors, among them the histone variant H2AX (EGFR fail to activate either Chk1 or Chk2 or cause phosphorylation of H2AX (γH2AX)  . This co (γH2AX) .EGFR-arrested cells to accumulate in any one phase of the cell cycle suggests that the block is global rather than focused on any one phase. To test this, we compared the behavior of UM-SCC47 cells subjected to either a double thymidine block or a double SSTNEGFR block. The UM-SCC47 cells have a 24 h cell cycle, with an approximate 8 h S-phase, 2 h G2/M, and 14 h G1. Thus, EdU labels approximately one-third of the cells if they are not synchronized (D). A 24 h thymidine block arrests the one-third of the cells that are spread throughout S-phase, whereas the rest progress to the G1/S start before arresting. A subsequent 16 h release allows these and the cells arrested throughout S-phase to progress through and exit S-phase and enter G1. Reimposition of a second 24 h thymidine block at this point prevents these cells from entering S-phase and they arrest as a cohort at the G1/S interface. Cells labeled at this point show no EdU incorporation (0 h), but cells released for 4 or 6 h transit partially through S-phase, as shown by extensive (greater than 80%) EdU labeling (D). This contrasts with the result seen if the cells are subjected to a 24 h double SSTNEGFR block (D). The cells show no labeling for 1 h after SSTNEGFR removal, but at 2, 4, or 6 h after release, approximately one-third of the cells incorporate EdU, the same as cells treated with vehicle alone, indicating that the number of cells in S-phase at any of these time points remains the same (D). There is no increase in cells entering S-phase, suggesting that cells have not piled up at the G1/S-phase boundary despite the ability of SSTNEGFR to arrest cells in S-phase (C) nor have cells entering S-phase been delayed by a block elsewhere. Instead, the cells appear to globally arrest wherever they are in the cell cycle when SSTNEGFR is applied and then recover and resume their transit when SSTNEGFR is removed.The apparent failure of the SSTNhronized D. A 24 hlabeling D. This cFR block D. The cethe same D. There  S-phase C nor havEGFR, or α3β1 integrin blocking antibody (A). In contrast, the proliferation of NOKs or UM-SCC47 cells is independent of EGFR kinase activity, shown by the failure of gefitinib and erlotinib to block EdU incorporation even when used at 100-fold over their IC50 (B). But, consistent with the proliferation inhibition observed in EGFR and the addition of EGF cannot reverse the block to EdU incorporation by SSTNEGFR in the UM-SCC47 tumor cells (B).We have shown previously that EGFR-stimulated migration of HaCaT and MCF10A epithelial cells on LN332 depends on its coupling with the α3β1 and α6β4 integrins by Sdc4 and requires EGF and active EGFR kinase , 13. Simantibody A. In coneir IC50 B. But, cor cells B.Figure EGFR by significantly reducing EdU incorporation in HNSCC and TNBC cells, but not in NOKs or MCF10A cells (C). Focusing on the UM-SCC47 cells, we find that RON and ABL1 are active in the tumor cells, as evidenced by phosphorylation of Y1238/1239 and Y412 in their respective kinase domains, as well as Y245 in the SH2-kinase linker of ABL1 (D). This phosphorylation is blocked by SSTNEGFR, and ABL1 no longer associates with RON (D). This suggests that by interacting with Sdc4, either directly or indirectly, RON is activated via transphosphorylation when clustered, engages the ABL1 SH2 domain, and activates ABL1 by phosphorylation.These findings prompted us to question the potential role of other kinases in the arrest mechanism. Like EGFR , 13, 36,0A cells C. Focusi of ABL1 , 41 , along with EGFR, the α3β1 integrin, phosphorylated α6β4 integrin, the CD151 tetraspanin known to associate with the α3β1 and α6β4 integrins . This recapitulates prior findings that phosphorylation of the α6β4 integrin depends on kinases in the receptor complex that are activated by syndecan clustering (A), strongly suggesting that they are all in a single receptor complex. SSTNEGFR displaces all but RON from Sdc2 (A), although RON is no longer phosphorylated, explaining the loss of ABL1 as well. This suggests that RON relies on Sdc2 for its linkage to the receptor complex, potentially via an interaction involving the Sdc2 extracellular domain. To confirm this, the receptors that assemble together by docking, either directly or indirectly, with the Sdc4 extracellular domain  were captured using GST-S4ED, a recombinant Sdc4 extracellular domain fusion protein (B); performing this capture in the presence of recombinant His-tagged Sdc2 extracellular domain as a competitor prevents capture of Sdc2 and RON (B).Probing Sdc4 immunoprecipitates from UM-SCC47 cells confirms that active RON and active ABL1 coprecipitate with Sdc4 A, along ntegrins , 47, 48,ntegrins . All arec domain  but is nc domain A. This rustering , 36, 50.ustering A, strongrom Sdc2 A, althou protein B; perfor and RON B.Figure C). These findings suggest that in tumor cells, Sdc2 becomes incorporated into the receptor complex involved in cell adhesion and migration and extends its activity to a mechanism that sustains cell proliferation as well .The incorporation of Sdc2, RON, and ABL1 into the receptor complex in tumor cells contrasts with the makeup of the complex in nontumorigenic cells; originally defined in HaCaT cells, Sdc4 immunoprecipitates contain EGFR and α6β4 and α3β1 integrins that the HaCaT cells use for migration on LN332 , 13. TheFig. 4via its cytoplasmic domain and CD151 that is known to associate with the integrin (E and S2B), revealing a major structural role for EGFR. Silencing α3β1 integrin expression does not affect the assembly of this core set of receptors, but it does prevent the capture of Sdc2 and Sdc2-associated RON kinase (E and S2B). Furthermore, silencing either CD151 or α6β4 integrin has no effect on EGFR engaging Sdc4 but does block the assembly of the adhesion receptor complex consisting of α3β1, α6β4, and CD151 with EGFR (E and S2B). Control siRNA has no effect on the assembly of the receptor complex (B) or expression of the complex’s component receptors (A). Moreover, each receptor siRNA is target-specific with no off-target effects on expression of other components within the complex (A). These findings provide a general order of assembly as shown in Figure 4D, with EGFR providing a link between Sdc4 and a complex of CD151 and the integrins and Sdc2 linking RON to this complex via an as-yet undefined interaction of the Sdc2 extracellular domain. Each member of the receptor complex appears to play an essential role either in assembly of the receptor complex or in signaling because silencing expression of any one of the receptors or kinases  results in cessation of DNA synthesis by well over 90% in UM-SCC47 cells (F).The lack of a requirement for EGFR kinase activity in the cell proliferation mechanism raises the question of what role, if any, EGFR has in this receptor complex. Silencing EGFR expression blocks assembly of the entire complex with Sdc4 except for the α6β4 integrin that remains bound integrin E and S2BN kinase E and S2Bith EGFR E and S2B complex B or expreceptors A. Moreov complex A. These 47 cells F.A). This contrasts with nontransformed epithelial cells. HaCaT cells express cell surface Sdc4, EGFR, α3β1 integrin, and α6β4 integrin but have undetectable levels of cell surface RON and Sdc2 despite the presence of these receptors in HaCaT cell lysates (cf.C). NOK and HTE cells express EGFR and the integrins but have undetectable levels of the Sdc2 and RON, as well as Sdc4, at the cell surface (B).Analysis of receptor expression on representative carcinoma cell lines demonstrates that all express the critical signaling and adhesion receptors of the Sdc4-organized complex at the cell surface A. This cates cf.C. NOK an surface B.Figure EGFR-treated tumor cells but not normal epithelial cells. This revealed an upregulation of stress-activated p38MAPK (A) in the tumor cells treated with SSTNEGFR for 24 h. A time course of SSTNEGFR treatment from 15 min to 16 h confirms that HNSCC and TNBC cells all activate p38MAPK within 15 min of peptide addition and that activation persists throughout the entire time course examined, whereas NOKs and MCF10A cells fail to activate the kinase (B and C). To test if activated p38MAPK is responsible for the S-phase arrest observed when SSTNEGFR causes inactivation of RON and ABL1, UM-SCC47 cells were treated with either SSTNEGFR, RON kinase inhibitor (BMS-777607 or CAS 913376-84-8), or ABL1 kinase inhibitor (PPY-A or GNF-5) for 3 h in the presence or absence of p38MAPK inhibitor BIRB-796 (doramapimod). Treatment with either RON or ABL1 kinase inhibitor alone activates p38MAPK  and blocks EdU incorporation , which is fully rescued by the addition of BIRB-796 (A and D). Arrest caused by activating the DDR using HU is not reversed by BIRB-796 . This reversal extends to each of the representative HNSCC and TNBC cell lines treated with SSTNEGFR, all of which are rescued  either with BIRB-796 or another p38MAPK inhibitor, losmapimod .In the course of these studies, we probed an antibody array looking for signaling markers that change in SSTN p38MAPK A in the e kinase , B and CEGFR peptide leads to a rapid and global arrest of the carcinoma cells throughout the cell cycle. The signaling mechanism builds on a previously described receptor complex found on migrating epithelial cells comprised of the α3β1 integrin and EGFR docked to a juxtamembrane site in the Sdc4 ectodomain and the α6β4 integrin engaged by the Sdc4 cytoplasmic domain . The α6β4 integrin binds a motif in the C-terminus of the Sdc4 cytoplasmic domain and there is no evidence to date for an extracellular interaction between this integrin and the syndecan , most likely in a specialized, tetraspanin-enriched membrane microdomain . There are likely to be other stabilizing interactions as well. Sdc2 and Sdc4 are known to heterodimerize via interactions within their transmembrane domains  provided by Dr Guido David (University of Leuven) and integrin α6β4 (3E1) from Memorial Sloan-Kettering. Anti-Sdc4 (AF2918), EGFR (AF231), RON (AF691 and FAB6647F), pY1238/1239 RON (AF1947), ITGB4 (mAb 422325) Sdc2 (305515) were from R&D Systems . Anti-ITGA3 (NBP2-48514) was from Novus Biologicals. Rabbit mAbs 73E5 (pY245-ABL1), 247C7 (pY412-ABL1), D13E1 (p38MAPK), 11H10 (tubulin), 133D3 (pS345-CHK1), 2661S (pT68-CHK2), D3H8P (PCNA), 1B1B2 (histone H3), 28B10 (pT183/Y185-p38MAPK), and E4I9J (CD151) were from Cell Signaling Technology. Anti-MCM2 (A300-191A) was from Bethyl Laboratories. 8E9 (ABL1), TU-01 (tubulin), and 36-6200 (Sdc2) were from Invitrogen/ThermoFisher Scientific. DO-1 (p53) and H-7 (Sdc2) were from Santa Cruz Biotechnology. JBW301 (pS139-γH2AX), AC-74 (β-actin), and P1B5 (integrin α3β1) were from MilliporeSigma. Antibodies to Sdc2 were produced against GST-S2ED in rabbits and affinity-purified as described , and Losmapimod were from Selleck Chemicals. Cetuximab was provided by Dr Paul Harari (University of Wisconsin-Madison). CAS 913376-84-8, actinomycin D, HU, and propidium iodide were from MilliporeSigma; GNF5 and PPY-A are from R&D Systems. EdU and Click-IT EdU-labeling reagents were from Click Chemistry Tools and MilliporeSigma. CellTiter-GLO was from Promega.2. NOKs and HTEs were cultured in complete Keratinocyte Serum-Free medium containing 100 units/ml penicillin and 100 μg/ml streptomycin (Life Technologies). All other cell lines were cultured as previously described .Parental telomerase reverse transcriptase–immortalized human NOKs and HTEs were described previously , 65. HaCescribed , 8, 9. N5 cells, washed, counterstained with Alexa-488-conjugated goat secondary antibodies, and scanned on a Thermo Fisher Scientific’s Attune NxT bench top cytometer. Cell scatter and PI staining profiles were used to gate live, single-cell events. For cell cycle analysis, asynchronous or synchronous , followed by an additional 24 h with 4 mM thymidine) were released from the double thymidine block and treated with SSTNEGFR after cells recovered from the block and entered S-phase. Cells were labeled with 100 μM EdU for 1 h prior to being suspended and fixed in ice-cold 70% ethanol. Cells were first stained using a Click-IT EdU-labeling reaction for 1 h  followed by DAPI (D1306) staining for 4 h (5 μg/ml in the presence of 1 μg/ml DNAse-free RNase A (Thermo Fisher Scientific) in 0.1% Triton X-100). Cells were then analyzed by flow cytometry to assess levels of AF488-EdU and DAPI staining on a ThermoFisher Attune NxT bench top cytometer.To measure cell surface receptor expression, suspended cells were incubated for 1 h on ice with 1 μg of primary antibody per 5 × 10EGFR peptide was carried out using antibodies to Sdc4 or Sdc2 mAb (or mouse IgG1 as a negative control), GammaBind PLUS Sepharose , and 1 mg of precleared whole cell lysate (WCL)/sample in the presence of protease and phosphatase inhibitors as previously described  and target-specific siRNA oligos directed against human Sdc4 GGAATCTGATGACTTTGAG217 and siRNA ID# s12638, Target Sequence: 536CTACTGCTCATGTACCGTA(tt)554; GenBank Accession number NM_002999.4), Sdc2 1014 and siRNA ID# 12636 868GACAGTCTGTTTAAACGGA(tt)886; GenBank Accession Number NM_002998.4), ITGB4 676 and siRNA ID# s7585, Target Sequence: 580CCAACTCCATGTCCGATGA(tt)598; GenBank Accession Number NM_001005731.3), ITGA3 1044 and siRNA ID# s7541, Target Sequence: 2629GTAAATCACCGGCTACAAA(tt)2647;GenBank Accession Number NM_002204.4), EGFR  and human EGFR-specific siRNA oligo (sc-29301) acquired from Santa Cruz Biotechnology, MST1R 3594 and siRNA ID# s8998, Target Sequence: 3065GCGTAGATGGTGAATGTCA(tt)3084; GenBank Accession Number, NM_002447.4), ABL1 2048 and siRNA ID# s864, Target Sequence: 1836GAAGGGAGGGTGTACCATT(tt)1854; GenBank Accession Number NM_007313.3), and CD151 428 and siRNA ID# s194332, 3′UTR Target Sequence: 935CCCAACTACTGAGCTGAGA(tt)953; GenBank Accession Number NM_004357.5)are from Life Technologies. UM-SCC47 cells (0.35 × 106 per 35 mm well) were transfected with 100 nM siRNA using Lipofectamine RNAiMAX and Opti-MEM I transfection medium (from Life Technologies) at 1:1 ratio (μg siRNA:μl RNAiMAX). At 6 h post-transfection, the wells were supplemented with 10% fetal bovine serum and 3 ml of complete culture medium. At 24 h post-transfection, the cells were suspended and plated on either acid-etched 18mm-#1 glass coverslips in 12-well plates or in 60 mm tissue culture plates. Cells were then harvested at 72 h post-transfection and analyzed by Western blot  or AF488-EdU staining (for active DNA synthesis).Two different siRNAs were used for all treatments with equivalent results, paired with a scrambled siRNA control: EGFR peptide in the presence or absence of the indicated inhibitors for the times indicated. For EdU labeling, cells were incubated with 100 μM EdU 45 min prior to fixation. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 in 1× CMF-PBS (pH 7.4), and blocked for 1 h at RT in a 3% bovine serum albumin (BSA)/CMF-PBS solution. The cells were then stained using a Click-IT EdU-labeling reaction for 30 min at RT  prior to washing with PBT solution (CMF-PBS containing 1% BSA and 0.2% Tween-20) and mounting in ProLong Diamond Antifade Mountant with DAPI (Life Technologies). For PCNA staining, cells were first hypotonically lysed in a buffer containing 10 mM Tris (pH 7.4), 2.5 mM MgCl2, 0.5% NP-40, HALT protease and phosphatase inhibitor cocktail, and 1 mM DTT for 15 min under constant agitation. The cells were then washed with 1× CMF-PBS (pH 7.4), then blocked in a 3% BSA/CMF-PBS solution for 1 h prior to Click-IT EdU AF488-labeling, as described previously. Cells were then stained mAb D3H8P (1:800) for 1.5 h followed by Alexa546-conjugated secondary antibody prior to mounting. Fluorescent images (six representative fields from duplicate wells/experiment) were acquired using either a Zeiss PlanAPOCHROMAT 10× (0.45 NA) or 20× objective (0.8 NA) and a Zeiss AxioCam Mrm CCD camera on a Zeiss Axio Imager.M2 microscopy system. Stained cells were quantified using ImageJ (https://imagej.nih.gov/ij/) using threshold limits based on controls.Cells were plated on glass coverslips overnight and then treated with or without SSTN3/well) were plated in 96-well plates in complete culture medium in the presence or absence of SSTNEGFR peptide or EGFR kinase inhibitors for 72 h. Cell numbers were measured using CellTiter-GLO against a standard curve in accordance with the manufacturer’s instructions.Cells  control cells was conducted as previously described (Screening for stress markers activated in cells treated with SSTNescribed  using tht test were performed using Excel (Microsoft Office 365). Data that satisfy confidence levels of p ≤ 0.05, 0.01, or 0.001 are noted. Data are presented as means ± SEM, unless otherwise noted.Statistical analyses using a two-tailed Student’s All data described in the article are contained herein.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Endoplasmic reticulum stress (ERS) is associated with breast cancer progression. However, the potential role of ribosomal protein L5 (RPL5) on ERS in breast cancer remains unclear. This study aimed to determine the role of RPL5/E2F transcription factor 1 (E2F1) in breast cancer. It was found that RPL5 was downregulated in breast cancer cells and tissues. Additionally, overexpression of RPL5 inhibited cell proliferation. Moreover, the levels of ERS and autophagy markers were estimated using western blotting. Overexpression of RPL5 induced ERS and suppressed autophagy. Additionally, RPL5 downregulated E2F1, which was overexpressed in breast cancer cells. However, E2F1 knockdown promoted the transcriptional activation of glucose regulated protein 78 (GRP78), suppressed ERS response, and promoted autophagy. Rescue assays indicated that the effects of RPL5 on ERS and autophagy were abolished by E2F1. Taken together, RPL5 inhibited the growth of breast cancer cells by modulating ERS and autophagy via the regulation of E2F1. These findings suggest that RPL5 has a tumor-suppressive effect in breast cancer.  The mortality rate of breast cancer has decreased since the 1990s ,3. HowevThe endoplasmic reticulum (ER) is a crucial organelle for the synthesis and folding of new transmembrane and secreted proteins. ER homeostasis is vital for natural cellular activity . ER streRibosomal protein L5 (RPL5), a member of the ribosomal protein (RP) family, belongs to the 60s ribosomal subunit and acts as a sensor of ribosomal stress . BecauseIn this study, we aimed to explore the potential role of RPL5 in breast cancer and its underlying mechanisms. We hypothesized that RPL5 regulated the progression of breast cancer via regulating ERS and autophagy. Mechanistically, the regulatory function of RPL5 was exerted by modulating E2F transcription factor 1 (E2F1). The study will pvovide a novel insight for breast cancer therapy.Clinical specimens (n = 30) were collected from patients with breast cancer at The Third Affiliated Teaching Hospital of XinJiang Medical University . Patients received anticancer therapy were excluded in this study. After surgery, the tissues were immediately stored at −80°C. The study protocol was approved by the Ethics Committee of The Third Affiliated Teaching Hospital of XinJiang Medical University  prior to the study . All the participants provided informed consent. Correlation between RPL5 and clinical characteristics is shown in 2O2 for 15 min. The blocking was performed using normal goat serum. Then, the slides were incubated with anti-RPL5 or anti-E2F1 at 4°C overnight and incubated with goat anti-rabbit IgG at 37°C for 30 min. After washing with PBS, the sliders were stained with DAB solution and counterstained with hematoxylin.IHC assay was performed according to the previously described . Paraffi2. Autophagy inhibitor 3-MA  was used as the positive control.The breast cancer cell line MCF-7 and normal breast epithelial cell line MCF-10A were obtained from the American Type Culture Collection (ATCC). The cells were incubated in DuIbecco’s modified eagIe’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C with 5% CORPL5 overexpressing vector, E2F1 overexpressing vector, E2F1-specific siRNAs, empty vector, and siRNA were purchased from GenePharm, Shanghai. MCF-7 cells were seeded into 6-well plates and transfection was conducted with Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol for 48 h.3 cells/well and cultured at 37°C for 0, 12, 24, and 48 h, respectively. The cells were then treated with 10 µL CCK-8 reagent and cultured at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader.Transfected cells were seeded into 96-well plates at the concentration of 1х 10Click-iT EdU Imaging Kit (Invitrogen) was used to analyze tumor cell growth according to manufacturer’s instrument . The cel−ΔΔCt method, with GAPDH as the housekeeping control.Total RNA was extracted from tissues and cells using TRizol reagent (Invitrogen). Reverse transcription was performed using the PrimeCcript RT Master Mix (Takara). qPCR was then performed using FastStart™ PCR Master (Roche). The relative expression of RPL5 and E2F1 (fold-change) was calculated using the 2Protein was extracted from the MCF-7 cells, and the total protein concentration was estimated using a BCA kit. Next, the protein was separated using 12% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. The next day, the membranes were incubated with secondary antibodies. The bands were captured using ECL Western Blotting Substrate (Solarbio).The expression of RPL5 and E2F1 was analyzed using StarBase database. The survival rate related to RPL5 was acquired from Kaplan online database. The DNA motif of E2F1 and potential binding sites between E2F1 and GRP78 were predicted using the JASPAR online tool.Renilla luciferase activity was used as the internal control.The GRP78 promoter sequence was cloned into the pGL3 vector. Mutant sequences of the GRP78 promoter were also cloned into the pGL3 vector. Cells were co-transfected with WT or MUT GRP78 and siRNA or si-E2F1 by using Lipofectamine 3000 for 48 h. The luciferase activity was measured using a luciferase kit (Promega). CHIP analysis was performed according to a previous study . The celAll experiments were repeated at least 3 times. Data were analyzed using GraphPad Prism 6.0 and are presented as the mean ± SD. Differences were evaluated using Student’s t-test and ANOVA. Correlation coefficients between RPL5 and E2F1 were analyzed using the Pearson method. Statistical significance was set at p < 0.05.In this study, we investigated the potential role of RPL5 in breast cancer and underlying mechanisms. Western blotting was used to analyze ERS-related factors and autophagy-related factors. We found that RPL5 regulated ERS and autophagy of breast cancer cells via regulating E2F1. The study will pvovide a novel insight for the treatment of breast cancer.According to the data of StarBase database, RPL5 was downregulated in breast invasive cancer ). In thiAfter transfection, RPL5 expression was significantly upregulated in the ove-RPL5 group, compared with the control and vector groups . FurtherAfter transfection with RPL5 overexpression plasmids, the protein expression levels of ERS markers, including GRP78, p-PERK, p-EIF2α, ATF4, and CHOP, were all remarkably increased ). In addThe data from Starbase illustrated that E2F1 was highly expressed in breast invasive cancer ). The reE2F1 was significantly downregulated by si-E2F1-1 and si-E2F1-2 . si-E2F1In TM-treated cells, E2F1 knockdown enhanced the protein levels of GRP78, p-PERK, p-EIF2α, ATF4, and CHOP ). MoreovAfter transfection, E2F1 expression was significantly reduced by RPL5 overexpression, which was rescued by E2F1 overexpression . FurtherIn the present study, we explored the role of RPL5 in breast cancer. We found that RPL5 inhibited tumor cell growth and autophagy. Moreover, RPL5 promoted ERS and suppressed autophagy by regulating E2F1.Mutations in RPs have been found to play a role in cancer development . The rolUnder homeostatic conditions, PERK binds GRP78 in an inactive state. Upregulation of GRP78 is involved in cancer progression ,23. Misf31–33]. High E2F1 expression is found in breast cancer tissues and is associated with a poor prognosis [E2F1, which belongs to the E2F family, is a transcription factor that transcriptionally induces adenoviral E2. It is the most important protein for cells to enter the S phase and is involved in cell apoptosis, DNA damage response, metabolism, and chemotherapeutic resistance [rognosis . E2F1 prrognosis ,36. Morerognosis . Additiorognosis ,39. Inhirognosis . HoweverRPL5 was downregulated and E2F1 was upregulated in breast cancer. Overexpression of RPL5 inhibited tumor cell growth and knockdown of E2F1 transcriptionally activated GRP78. Moreover, RPL5 modulated ERS and autophagy by regulating E2F1 expression. This study suggests that RPL5 might be a potential target for breast cancer therapy.


Commercial UHT-pasteurized 2% milk and chocolate milk were independently inoculated with 4 strains of P. odorifer at 1 to 2 log cfu/mL and stored at 4°C and 7°C for 30 d. Changes in P. odorifer cell densities were determined by standard serial dilution with spread plating on tryptic soy agar with yeast extract and incubation at 25°C for 48 h. Spore counts were determined following thermal treatment at 80°C for 12 min. Thermal resistance of a cocktail of P. odorifer in milk was determined after treatments at 63°C for 30 min and 72°C for 15 s. Strains of P. odorifer grew rapidly at 7°C and reached a maximum cell density of ~8 log cfu/g in both 2% and chocolate milk within 12 d. All strains grew more slowly at 4°C and had not reached maximum cell density by 21 d. With extreme temperature abuse , P. odorifer will sporulate in milk; however, thermally resistant subpopulations, including spores, did not develop in milk at 4°C until after stationary phase was achieved (>24 d). Vegetative cells of P. odorifer were verified to be sensitive to pasteurization (>7 log reduction); therefore, P. odorifer would not be expected to contribute to reduced shelf life of fluid milk products containing rework, even with extended storage before rework.Rework is a common practice used in the dairy industry as a strategy to help minimize waste from processing steps or errors that might otherwise render the product unsaleable. Dairy processors may rework their high-temperature, short-time (HTST) fluid milk products up to code date (21 d) at a typical dilution rate of ≤20% rework into ≥80% fresh raw milk. Bacterial spores present in raw milk that can survive pasteurization and grow at refrigeration temperatures are often responsible for milk spoilage. However, the potential impact of growth and thermal resistance of organisms in reworked product has not been investigated. Our objective was to characterize growth, sporulation, and thermal resistance of  Bacillus and Paenibacillus spores are commonly found in raw milk and are collected during milking from the farm environment (Paenibacillus spp. are the dominant genus found at elevated levels (≥6 log cfu/mL) in pasteurized milk at the end of shelf life  at 6°C and to reach final cell densities of >4 log cfu/mL within 14 d (P. odorifer in SMB has been modeled at 4°C (0.4 log cfu/mL per day); however, minimum growth temperature for P. odorifer is typically reported as 5°C (The majority (59%) of odorifer . Spores elf life . Paenibahin 14 d  and >6 lhin 14 d . The grod as 5°C .P. odorifer contamination, growth, and sporulation could pose an elevated risk in reworked product. First is the contamination level of P. odorifer spores in raw milk. Psychrotrophic spore density in fluid milk averages 1 spore/6 mL of raw milk  and chocolate milk stored under conditions used by the dairy industry before reworking into fresh product. Growth rates, maximum cell density, and thermal resistance are critical parameters to determine the potential impact of P. odorifer on fluid milk products containing rework.Our overall objective was to determine the potential effect of P. odorifer representing diverse rpoB allele types (AT) were used in this study: FSL A6-0363 (AT40), FSL R10-2726 (AT35), FSL E2-0150 (AT2), and JWC-2503 (AT1922). The first 2 strains were originally isolated from pasteurized milk and the latter 2 from pasteurized chocolate milk. Isolates were revived from frozen storage (−80°C) by transferring to tryptic soy broth with 0.3% yeast extract  with incubation at 25°C for 24 h. Cultures were streaked for isolation on tryptic soy agar plates supplemented with 0.3% yeast extract  and incubated at 25°C for 48 h. A single isolated colony of each strain was independently transferred into TSBYE and incubated at 25°C for 24 h. The resulting culture was serially diluted in 0.1% peptone water (Neogen) to a final cell density of 6 log cfu/mL, which served as the inoculum.Strains of P. odorifer strain to a final cell density of 1 to 2 log cfu/mL. Inoculated milk samples were aliquoted (200 mL) into sterile 237-mL plastic jars (Uline) in triplicate and incubated at 4°C and 7°C for 31 d. The cell density of P. odorifer was determined by standard serial dilution (0.1% peptone water) and spread plating on TSAYE with incubation at 25°C for 48 h. Growth rates were determined using JMP Pro v16 software (SAS Institute Inc.) by applying a line of fit to the exponential growth phase for each strain.Ultra-high temperature pasteurized reduced fat (2%) fluid milk  and lowfat (1%) chocolate milk (Nestlé USA Inc.) products were purchased at retail. Milk  was aseptically transferred to Whirl-Pak bags (Nasco) and stomached for 30 s. Each bag was inoculated with a single P. odorifer strain was transferred to 25 mL of reduced-fat milk and incubated at 25°C for 24 h. Cultured milk samples were combined and shaken for 30 s to create a P. odorifer cocktail and stored at 4°C for 24 h before use. Aliquots (900 μL) of reduced fat milk were transferred into microcentrifuge tubes and inoculated with 100 μL of P. odorifer cocktail to a cell density of 8 log cfu/mL. A dry heating block (Benchmark Dry Bath) was used to treat milk samples at 63°C for 0 to 30 min, 70°C for 0 to 10 min, 72°C for 15 s (HTST), and 80°C for 12 min (spore count). Thermal treatments were performed in triplicate. A thermocouple  was held in an uninoculated milk sample to monitor temperature to establish come-up time. Heat-treated samples were immediately transferred to ice and survivors were enumerated using plating methods described above.For thermal inactivation studies, each P. odorifer strains over the course of the 21-d shelf life of fluid milk products at 4°C and 7°C is shown in P. odorifer in whole milk (7°C = 1.01 cfu/mL per day) and SMB (6°C = 0.6 cfu/mL per day) have been reported  than in chocolate milk (7.74–7.83 log cfu/mL). This difference in growth was most obvious for JWC-2503 . This stproducts . The obs sucrose . There ireported . IncreasP. odorifer in milk after 21 d of storage in order to quantify the potential microbial contribution when mixed with raw milk for reprocessing. Previous research on fluid milk spoilage has focused on microbial growth at 6 to 7°C to mimic temperatures during distribution, at retail, and throughout storage in home refrigerators, whereas the storage temperature for milk held for rework is tightly controlled at 4°C. After 21 d of storage, P. odorifer ranged from 6.8 to 7.5 log cfu/mL at 4°C and 7.6 to 8.2 log cfu/mL at 7°C , the count of the cocktail was reduced to 5.4 log cfu/mL (2.5 log reduction) indicating high sensitivity of P. odorifer to thermal inactivation. After a 5-min holding time at 63°C, 3.5 log cfu/mL of P. odorifer remained viable. No further reduction of P. odorifer was achieved with additional treatment at 63°C for up to 30 min. Time and temperature combinations of 63°C for 5 to 30 min, 70°C for 0 to 10 min, and 72°C for 15 s did not differ in their lethality toward the P. odorifer cocktail, with survivors ranging between 3.0 and 3.1 log cfu/mL. These results indicated presence of a thermally resistant subpopulation of P. odorifer. The inoculation preparation procedure  for the thermal inactivation study likely supported initiation of P. odorifer sporulation. Additional inoculated milk samples were heat-treated at 80°C for 12 min, demonstrating a spore population of 2.0 log cfu/mL in the milk. Thermal resistance of Paenibacillus spores has been recorded in the literature; however, the emphasis has been on spores that display excessive thermal resistance. For example, Paenibacillus spores survived heat treatments up to 120°C for 30 s   of r rework . Initialfor 30 s . Further°C, 2 s) . OverallP. odorifer strains in fluid milk at 4°C was performed to monitor development of thermally resistant subpopulations with extended storage (P. odorifer isolates displayed a similar rapid growth rate (0.40–0.52 log cfu/mL per day) in fluid milk during the first 10 d of storage. However, between 10 and 15 d, no growth was observed for any of the isolates, and cell density stalled at 5.3 to 6.2 log cfu/mL. Paenibacillus odorifer transitioned into a second slower growth phase (0.09–0.14 log cfu/ml/d) from 15 d to 25 or 30 d. This growth pattern was not obvious in the previous growth studies at 4°C reported in P. odorifer growth at 7°C in milk  and was presumed to be completely composed of vegetative cells (Paenibacillus odorifer FSL R10-2726 had a resistant subpopulation of 1.95 log cfu/mL (vegetative count: 8.00 log cfu/mL) in one sample replicate, whereas JWC-2503 and FSL A6-0363 had low levels of resistant cells (0.3 and 2.4 log cfu/mL) in 2 replicates. With continued storage (31 d), thermally resistant subpopulations of P. odorifer were detected in milk samples inoculated with FSL R10-2726, JWC-2503, and FSL A6-0363, ranging between 0.30 and 3.12 log cfu/mL. Paenibacillus odorifer FSL E2-1050 did not develop a thermally resistant subpopulation. Data suggest that strains developed resistant subpopulations as they approached their maximum cell density (stationary phase), whereas FSL E2-1050 was still actively replicating  was based on the thermal resistance of a variety of mesophilic and thermophilic sporeformers  and therefore are unlikely reduce the shelf life of milk products containing rework in alignment with the Pasteurized Milk Ordinance (Paenibacillus odorifer is unlikely to contribute to the reduced shelf life of fluid and flavored milk products containing rework.The overall objective of our research was to evaluate the potential impact of rdinance . Paeniba


Streptomyces. The 16S rRNA gene sequences have shown that the isolate 1S1 clustered with Streptomyces longisporoflavus. The strain Streptomyces sp. 1S1 was cultivated and extracted with ethyl acetate. The GC–MS study of the extract indicated the presence of certain fatty acyl compounds e.g., tetradecanoic acid, 9-octadecenoic acid, hexadecanoic acid, and 9,12,15-octadecatrienoic acid. Using chromatographic techniques, three compounds were isolated and by spectroscopic methods e.g., IR, MS and NMR structurally elucidated. The three compounds were identified as a triacylglyceride, 9-octadecenoic acid, and hexadecanoic acid. The study reinforces the evidence of the potential of Streptomyces sp and the ability to produce several antimicrobial compounds.Red Sea represents one of the most remarkable marine ecosystems. However, it is also one of the world's least explored areas of marine biodiversity. The aims of this investigation were therefore, to isolate marine microorganisms from the seashore sediments and water in shallow region from west Yemen coast, to assess their antimicrobial potential, to identify the highly active isolate, and to purify and identify the bioactive compounds from it. In this regard, twenty-five bacterial strains have been isolated from twenty samples and tested for their antimicrobial ability against some pathogenic bacteria and yeast by using the agar disk diffusion and agar well diffusion assay. Out of the total 25 marine actinomycetes isolates only 13 exhibited interesting antimicrobial activity. The morphological, biochemical, and phylogenetic characteristics of the potential isolate 1S1 were compatible with their classification in the genus  They are taxonomically diverse, biologically active, and colonize all aquatic living habitats, from the deep waters to the shallowest estuaries .Bacteria, actinomycetes, and fungi among marine microorganisms in general, are considered important sources of many naturally occurring biomolecules and therapeutic products . Due to ). Moreover, it is reported that marine sediment sources are valuable for the isolating novel actinomycetes capable of producing beneficial novel products  were collected from ten different sites along the west coast of Yemen in 2013–2014 and 2016–2017. Marine samples were taken from the Red Sea coast between 30 cm and 1000 cm deep. The temperature, pH and salinity of each sample were measured with thermometer, pH and salinity meter. The samples were stored at 4 °C using sterile polythene containers (Data are not shown).2.3) at 4 °C and finally preserved at −20 °C in 20% sterile glycerol in distilled water (v/v)  .2.4Culture characteristics of pure actinomycetes colonies such as elevation, surface, aerial and substrate mycelium color and pigments production were recorded on SCA media according to Bergey’s Manual of Determinative Bacteriology . To stud2.52.5.1Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 9538, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 2453 and locally Streptococcus pneumoniae and as fungus Candida albicans ATCC 2091. The antimicrobial activity of the marine isolates was tested using two methods namely, agar disc diffusion method (ADM) (cup method) and agar spot inoculation. Marine actinomycetes isolates were streaked on (SCA) and incubated at 28 °C for 7 days, target microorganisms were seeded on nutrient agar (NA) for bacteria and Sabouraud dextrose agar (SDA) for fungi.Tests microorganisms as bacteria were 2.5.2Discs of (SCA) with growth of marine isolates were cut by cork borer (6 mm) and transferred to the surface of seeded target of microorganism's plates under aseptic conditions. These plates were kept for 1–2 h in a refrigerator to facilitate diffusion and then incubated at 37 °C for bacteria and at 28 °C for fungi. Antimicrobial activity was recorded in term of inhibition zones against target microorganism’s growth around the agar disc of marine isolates .2.6The actinomycete isolate (1S1) which showed the greatest antimicrobial activity, was tested for its extracellular antibiotic production capabilities under submerged fermentation conditions. The selected active actinomycete was inoculated into starch casein broth and incubated at 28 °C using a shaking incubator (200–250 rpm) for seven days. After incubation, the broth was filtered through Whatmannn No.1 filter paper, then the biomass of actinomycetes was removed by centrifugation at 3000 xg for 10 min. The supernatant was collected, concentrated, and extracted with equal volume of the ethyl acetate. The ethyl acetate/broth mixture was shaken for several hours in proper flasks. Using a separating funnel, the ethyl acetate layer was then removed from the water-based broth. The extraction process was repeated three times until colorlessness of the ethyl acetate extract. The collected ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and evaporated using rotary evaporator . To preserve the extract, it was kept in sealed glass vials at 4 °C in a refrigerator . The ext2.7Streptomyces longisporoflavus  that showed the greatest antimicrobial activity against the selected human pathogens. For genetic characterization of the active isolates, the following steps were performed: Genomic DNA was extracted from the pure selected isolate . DNA preroflavus .Fig. 2St2.8The ethyl acetate extract was further subjected to other chromatographic separation techniques e.g., thin layer chromatography (TLC) and open column chromatography (CC) in order to isolate the main constituents. Ethyl acetate extract was chromatographed on a silica gel column  using chloroform, ethyl acetate gradient elution (9.5:0.5) until (0:100). The collected fractions (20 ml each) were pooled together depending on their TLC behavior to give six main fractions .Scheme 12 and 3 (35 mg) were rechromatographed on a reversed-phase column   using methanol:water (5:95) as a mobile phase to afford a major substance (compound 1) (5 mg). On the other hand, under the same chromatographic conditions (CC2), fraction 6 (13 mg), gave compound 2 (3 mg). While fraction 1 (15 mg) and 4 (20 mg) were separated on two reversed-phase columns   using methanol:water (15:85) as a mobile phase to give two major substances (compound 3) (3 mg) and compound 4 (3.5 mg) (Fractions (3.5 mg) . Accordi2.9m/z.The GC–MS examinations have been directed on gas chromatograph (Hewlett-Packard 5890 series II) connected to mass spectrometer . Fused silica packed in capillary Elite-5MS column  has been used as stationary phase. The mobile phase utilized was helium at a stream rate of 1 ml/min. Temperature of the injector was set at 200 °C. The Analysis began with heating up the oven at 60 °C and then steadily increasing it to 300 °C. The final step of the process was to keep the oven at 300 °C for a 20-minute period. An election impact ionization system with an ionization energy of 70 eV has been utilized for GC–MS detection. A scan rate of 0.6 s was utilized to cover the mass range of 35 to 600 2.101H, 13C and 2D-NMR spectra were recorded on a Bruker AMX-500 spectrometer. The chemical shift values are expressed in (ppm) units using tetramethylsilane (TMS) as internal standard and the coupling constants (J) are expressed in hertz (Hz). Spectroscopic grade of CDCl3 was used as a solvent.The 33.1Streptomyces including the microbial strain 1S1 , and the remaining were unknown (Data are not shown).From twenty samples collected (10 samples from sediment and 10 samples from seawater) from 10 different sites along Tehama western coast of Yemen, 65 marine actinomycetes isolates were isolated (42 isolated from sediment & 23 isolates from seawater). Only 25 isolates were subjected for investigation, identification and antimicrobial screening in this study. Identification of the 25 strains by morphological, microscopical, and cultural characteristics revealed that 14 isolates (56%) were species of the genus rain 1S1 , 7 of is3.2The results are presented in 3.3Streptomyces genus based on mycelium formation and spiral chain development  and open column chromatography (CC) in order to isolate the main constituents. Ethyl acetate extract was chromatographed on a silica gel column and the collected fractions were pooled together depending on their TLC behavior to give six main fractions .3.6Compound 1 has been acquired in the form of a white amorphous powder that dissolved readily in chloroform. The IR spectrum demonstrated the presence of strong bands at 1750 and 1165 cm−1 indicating an ester function  and strong bands at 2910 and 2845 cm−1 indicating the presence of methylene groups. The EIMS spectrum of compound 1 displayed an [M + ] ion at m/z 722 consistent with the molecular formula C45H86O6. Several NMR-data including 1H, 13C, DEPT-135, HSQC, 1H–1H COSY and HMBC were achieved for the structure elucidation of this compound. The 13C NMR spectrum showed the presence of 45 carbon signals including signals at δ 173.49, 173,40 and 172.98 assigned to three ester carbonyl carbon atoms, a signal at δ 69.02 for methine carbon, two signals at δ 62.22 and 62.19 for methylene carbons indicating a triglyceride (ester of glycerol with three tetradecanoic acids). The 13C NMR-spectrum showed also 18 peaks between 29.10 and 29.95 ppm suggesting the presence of many CH2 C-atoms. That was assured by the DEPT 135˚ and HSQC spectra. In addition, a corresponding peak at 1.24–1.32 ppm in the 1H NMR showed that compound 1 represents probably a fatty acid ester. All the other signals at δ 22.70 to 36.88 were caused by CH2-groups C-atoms in a long chain. In addition, three methyl carbons at δC 14.26, 14.43 and 14.40 were indicated for three terminal methyl groups of three tetradecanoic acids.1H NMR spectrum of the compound 1 showed characteristic signals at δ 0.87, 0.88 and 0.89 as three triplets of three protons as an evidence of terminal CH3-groups, at δ1.29 as a broad singlet for a long chain of CH2-protons, at δ 2.88, 2.97 and 3.11 for CH2-groups α to carbonyl group, two signals at δ 4.12 and 4.15 with two double protons for CH2-groups and a signal at δ 4.28 for a methine group. The HSQC spectrum showed that the protons at δ 4.12 and 4.15 are attached on the methylene carbons at δ 62.22 and 62.19 and the proton at δ 4.28 was attached on methine carbon at δ 69.02. Relying on the NMR data and matching with literature data  was obtained as brownish yellow oily substance. A number of important functional groups were visible in the IR spectrum. The IR spectrum revealed a weak band at 3502 cm−1 indicating the presence of OH-group, strong bands at 2920 and 2845 cm−1, suggesting the presence of methylene groups as well as a strong band at 1712 cm−1 showing the existence of a CO-group. The EIMS spectrum of compound 2 showed an [M + ] ion at m/z 282 proportionate with the molecular formula C18H34O2. The 1H NMR spectrum of compound 2 demonstrated one triplet signal of three protons at δ 0.87 indicating the presence of a terminal CH3-group. A broad singlet of twenty protons was displayed at δ1.29 for a long chain of CH2-protons. One multiplet of four protons at δ 2.01 was accounted for two CH2-groups α to C = C group (neighbors of the olefinic protons). The 1H NMR spectrum showed also one multiplet of two protons and one triplet of two protons at δ 1.63 and 2.22 assigned for CH2-groups β and α to CO-group respectively. Moreover, a triplet signal of two protons at δ 5.33 was attributed to olefinic protons. The 13C NMR spectrum showed the presence of 18 carbon signals including two signals in the unsaturated carbon region, at δ 130.02 and δ 130.06 for methine carbons. The signal at δ 175.82 appeared to be a carbonyl carbon atom of a carboxylic acid moiety. Additionally, one methyl carbon at δ 14.26 was indicated for the terminal methyl group. All the other signals at δ 22.82 to 36.67 were assigned to CH2-carbons in a long chain. Relying on the NMR data and matching with literature data  was obtained as an oily white residue. The IR spectrum showed a weak band at 3500 cm−1 confirming the existence of a OH-group, strong bands at 2928 and 2841 cm−1 indicating the presence of methylene groups, and a strong band at 1715 cm−1 showing the presence of a CO-group. The EIMS spectrum of compound 4 displayed an [M + ] ion at m/z 256 consistent with the molecular formula C16H32O2. The 1H NMR spectrum of compound 4 demonstrated one triplet signal of three protons at δ 0.89 indicating the presence of a terminal CH3-group. A broad singlet of twenty-four protons was displayed at δ1.29 for a long chain of CH2-protons. The 1H NMR spectrum displayed also one multiplet of two protons and one triplet of two protons at δ 1.61 and 2.31 respectively, assigned for CH2-groups β and α to CO-group. The 13C NMR spectrum showed the presence of 16 carbon signals including a signal at δ 174.92 assigned to the a carbonyl C-atom of a carboxylic acid moiety. All the other signals at δ 22.70 to 36.88 were assigned to CH2-carbons in a long chain. In addition, one methyl carbon at δ 14.13 was indicated for the terminal methyl group. Relying on the NMR data and matching with literature data (16H32O2)  .4Streptomyces. Actinomycetes in general are assumed to be the most economic and biotechnologically important prokaryotes producing many secondary and biologically relevant metabolites. According to Jakubiec-Krzesniak (2018) and co-authors, actinobacteria are among the most prolific natural makers of bioactive compounds, and they continue to be a significant source of novel secondary metabolites for medication development  28H26N4O3) Thirteen out of the twenty-five samples showed remarkable antimicrobial activity. Comparison of 16S rRNA gene sequences showed that most antimicrobial active strain 1S1 is a member of the genus athogens . Few stuStreptomyces sp. 1S1 performed using various chromatographic techniques, e.g., open column chromatography (CC), thin layer chromatography (TLC) and gas chromatography (GC), lead to the isolation of three compounds, which were identified as myristic acid triglyceride (trimyristin), 9-octadecenoic acid (oleic acid), and hexadecanoic acid   . The streviously .Mycobacterium, Streptomyces, Rhodococcus, and Nocardia species (Triacylglycerols (TAGs) are widely seen in yeast, fungi, plants, and animals (but not in bacteria) as fatty acid storages. Nevertheless, TAG accumulation appears to be popular among actinomycetes, including  species . Bacteri species . Other f species . FurtherCitrus aurantiifolia showed activity against Mycobacterium tuberculosis H37Rv. The active extract was fractionated by column chromatography, yielding 6 major compounds including palmitic acid which was one of the most active ones.Oleic acid is one of many unsaturated fatty acids, while palmitic acid is one of several saturated fatty acids; both can be found in many plants and marine organisms including marine algae and bacteria. Many fatty acids are known to be antimicrobial . Oleic aStreptomyces sp. 1S1 ethyl acetate extract.Thus, it is anticipated that the fatty acids identified in this work, particularly 9-octadecenoic acid (oleic acid) and hexadecanoic acid , may also be responsible for the antimicrobial effect of the 5Streptomyces. In the current work, twenty-five bacterial species were isolated from twenty samples from the seashore sediments and water of west Yemen coast and evaluated for their antimicrobial action. Our findings showed that the most antimicrobial active strain was Streptomyces sp.. Analysis of the crude extract by GC–MS indicated the presence of certain acyl-fatty compounds. Three compounds were isolated and identified as myristic acid triglyceride, 9-octadecenoic acid, and hexadecanoic acid. In this study, the functionality of the Red Sea bacteria as a source of bioactive, biotechnologically, and pharmaceutically significant compounds was underlined.New bioactive natural products can be discovered by studying the marine actinomycetes of the Red Sea. Such natural products can particularly be found in the genus This research work was funded by Researchers Supporting Project number (RSP-2021/119), King Saud University, Riyadh, Saudi ArabiaThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper


Two differenttemperatures were applied using a slow rate of 20 mL·min–1 (100%) of CO2 passing through each samplefor only 1 h. Using the thermogravimetry (TG)-temperature-programeddesorption (TPD) technique, the captured amounts of CO2 at 5 °C were determined to be in the range of (39.6–103.9)and (28.9–82.1) mg2CO·g–1 for samples of Mg(OH)2 mixed with 20–50% F andcalcined at 400 and 500 °C, respectively, whereas, at 30 °C,the capacity of CO2 captured is slightly decreased to bein the range of (32.2–89.4) and (20.9–55.5) mg2CO·g–1, respectively. The thermaldecomposition of all prepared mixtures herein was examined by TG analysis.The obtained samples calcined at 400 and 500 °C were characterizedby X-ray diffraction and surface area and porosity measurements. Thetotal number of surface basic sites and their distribution over allsamples was demonstrated using TG- and differential scanning calorimetry-TPDtechniques using pyrrole as a probe molecule. Values of (ΔH) enthalpy changes corresponding to the desorption stepsof CO2 were calculated for the most active adsorbent inthis study, that is, Mg(OH)2 + 20% F, at 400 and 500 °C.This study’s findings will inspire the simple preparation andeconomical design of nanocomposite CO2 sorbents for climatechange mitigation under ambient conditions.The utilization of Mg–O–F prepared fromMg(OH) According to the data released by the Scripps Instituteof Oceanography at the University of California in May 2022, the dailyaverage concentration of atmospheric CO2 reached a recordhigh of 421.37 parts per million (ppm).1 Numerous climate scientists and researchers are interested in experimentingwith various CO2-capturing materials. Currently, the CO2 adsorption technique is used on some highly active materials,such as basic metal oxides, to combat global warming. In the pureform or promoted with other materials, MgO is regarded as a cornerstonecomponent in all prepared materials for CO2 capture purposes,among these basic metal oxides.9 This characteristic relates to the abundance and strength of basicsites over the MgO surface.12 Other types of adsorbents, includingthe reduced graphene oxide–MnO2 nanocomposite,13 nickel–lithium silicate for CO2 capture and subsequent production of CH4,14 Li4SiO4-based sorbentsfor high-temperature CO2 capture,15 NiO-functionalized ultra-stable Y zeolite,16 Mg–Al mixed metal oxides for high-temperature CO2 capture,17 alumina-supported layereddouble hydroxides,18 CuAl2O4 nanoplates,19 Ni–CaO dualfunction materials,20 and CaO–Fe2O3–SiO2 composite, have beenextensively examined.21 Few articles havefocused on the study of capturing CO2 in a dynamic modeat different temperatures by flowing CO2 gas at differentrates through the adsorbents.23 Many review articleswith a forward-looking perspective discussed various CO2 capture techniques27 utilizing different adsorbent materials.The world is on the vergeof an environmental catastrophe due toglobal warming as a result of the continuous increase in atmosphericCO2 mixturesfor CO2 capture are cited in the literature. In this regard,this study can be viewed as proactive research into using these mixtures.Furthermore, pure MgF2 is considered to be a suitable andeffective sintering additive for the preparation of certain typesof ceramics,29 in manufacturing transparentIR windows,30 in solar thermal technology,31 and as a catalyst in the form of MgFx2–(OH)x during aldol condensationof furfural and acetone.32Rarely, publishedarticles on using MgO–MgF2 capture at 5and 30 °C, despite a limited CO2 flow time (1 h) throughoutthe samples. The applied method is based on two environmentally friendlymaterials, that is, Mg(OH)2 and NH4F·HF,which were used to prepare the required mixtures without releasingharmful greenhouse gases.On theoccasion of the 27th UN Climate Change Conference of theParties (COP27), which will be held in Sharm el-Sheikh, Egypt, inNovember 2022, two research teams from South Valley University (Egypt)and Queen’s University Belfast (UK) collaborated in a vitaleffort to combat global warming. Herein, our samples were preparedusing a direct and simple method and exhibited a relatively high surfacearea and a superior reactivity toward CO3 from MgO without a promoter has poorkinetics and is limited toa few surface carbonate layers, resulting in only a small fraction(<2%) of the theoretical CO2 sorption capacity of MgO.The slowdown kinetics is caused by the high lattice enthalpy of MgOand the formation of a layer of magnesium carbonate, which acts asa barrier for CO2 molecules. Alkali metal salts are themost effective promoters for MgO-based sorbents.2 This work focuses on the preparation of Mg–O–Fnanocomposite catalysts with a high surface area, a large populationof surface basic sites, and a high CO2 capture efficiencyusing environmentally friendly materials without emitting harmfulgreenhouse gases. Compared to recently published articles utilizingother adsorbents, these samples appear to be more effective CO2 adsorbents under ambient conditions.Early on, it was determined that thegeneration of MgCO22.12 with 20–50%F (by weight) as ammonium hydrogen difluoride (AHDF) NH4F·HF were prepared as follows: calculated amounts of ammoniumhydrogen difluoride were dissolved in deionized water in Teflon beakers and mixed wellwith the corresponding weights of Mg(OH)2 .These mixtures were then evaporated until dry in a water bath. Theresulting mixtures were dried at 100 °C overnight. All thesemixtures were also calcined at two temperatures of 400 and 500 °Cin static air for 3 h.Mixed samples of Mg(OH)2.2–1 was used in the He atmosphere (40 mL·min–1) during the study of the thermal stability of all prepared mixtures,as well as the pure compounds of Mg(OH)2 and NH4F·HF. Thermogravimetry (TG) and DSC experiments of the temperature-programeddesorption (TPD) of CO2 over all samples under study, atdifferent temperatures of CO2 adsorption, were accomplishedusing a heating rate of 10 °C min–1 in a dynamicN2 flow (40 mL·min–1). The instrumentis equipped with a data acquisition and handling system (TA-50WSI),and highly sintered α-Al2O3 was appliedas reference material in DSC experiments.A 50H thermogravimetricanalyzer and differential scanning calorimetry (DSC) instruments (Shimadzu—Japan)were used in this work. A slow heating rate of 3 °C mink = 1.5406 Å) from a generator operatingat 40 kV and 40 mA in the 2θ range between 10 and 80°.The instrument is supported with interfaces of DIFFRACplus SEARCH and DIFFRACplus EVA to facilitate an automatic searchand match of the crystalline phases for identification purposes withthe ICDD database.X-ray diffraction(XRD) patterns of the calcined samples at 400 and 500 °C weremeasured by powder XRD using a Brucker AXS-D8 Advance diffractometer(Germany), equipped with a copper anode generating Ni-filtered CuKaradiation (2 adsorption/desorption isotherms at the liquid nitrogentemperature (−196 °C) using a Micromeritics ASAP 2020system, equipped with an online data acquisition and handling systemoperating BET and Barrett–Joyner–Halenda analyticalsoftware. All samples were degassed at 200 °C and 10–5 Torr for 2 h before measurements (1 Torr = 133.3 Pa).The Brunauer–Emmett–Teller(BET) surface area andporosity of all samples calcined at 400 and 500 °C were measuredby N–1) overeach sample was measured by the TPD of pyrrole  as a probe molecule using TG and DSC techniques.The experimental details can be explained as follows:34 60 mg of each sample is preheated at 390 °C for 1 h in theair before the probe molecule is exposed. 20 ± 2 mg covered sampleswith pyrrole were subjected to thermogravimetric analysis (TGA) andanother sample was subjected to DSC analyses at a heating rate of(10 °C·min–1) in a dry N2 flow(40 mL·min–1), using a 50H Shimadzu thermalanalyzer (Japan). The thermal analyzer is equipped with a data acquisitionand handling system (TA-50WSI). α-Al2O3 was used as the reference material in DSC measurements. The massloss due to the desorption of pyrrole during TG experiments from thebasic sites was determined to measure the total surface basicity assites per gram. Nbasic):35npyrrole is the number of moles of desorbed pyrrole, NA is Avogadro’s number (sites/mol), and wTG is the weight of the TG sample (g).Thetotal number of basic sites . Thesample is cooled down to room temperature (RT), and the reactor isthen transferred to a thermostatic water bath set to the desired temperature,that is, at 5, 10, or 30 °C, as shown in 2 gas stream (99.9%) isallowed to pass over the catalyst bed for 1 h, with a flow rate =20 mL·min–1, permeating between catalyst particlesinside the reactor to exit from the opposite side of the reactor.The residual CO2 flow is trapped by passing through anaqueous 5% solution of KOH. The quantity of CO2 moleculescaptured by each sample was measured immediately, employing the TPDtechnique, using the above-mentioned TG and DSC units as follows:20–25 mg of each sample was subjected to TG and DSC analyseson heating to 425 °C, in the case of samples calcined at 400°C, or to 525 °C in the case of the other samples treatedat 500 °C (at a 10 °C·min–1 heatingrate) in dry N2 (flow rate = 40 mL·min–1). In TG curves, the mass loss due to CO2 desorption wasmeasured as the capturing efficiency of each sample at the specifiedtemperature. Values of the molar enthalpy change (ΔHdes in J mol–1) corresponding to thedesorption of CO2 in each step, at different temperatures,of the most active samples were calculated from the DSC curves.36Before the CO33.1–1) toexamine the thermal decomposition of both Mg(OH)2 and ammoniumhydrogen difluoride (AHDF) NH4F·HF as pure precursors,as well as all their mixtures in the He atmosphere, as presented in 2 began tolose weight upon heating immediately and continuously up to 200 °C.This step is followed by another weight loss at approximately 270°C, which is ascribed to the dehydroxylation of Mg(OH)2.38 At 400 °C, the calculated mass losswas 34.5%, whereas at 500 °C, it was 37.7%. Due to the liberationof physically adsorbed water molecules associated with Mg(OH)2, these values exceeded the theoretical value40 for the conversion of anhydrous Mg(OH)2 to MgO (ca. 30.86%),as presented in 4F·HFundergoes melting at 125 °C, followed by a subsequent fast stepdue to its decomposition around 150 °C,41 as shown in 2 in these mixtures to produce magnesium fluorideand magnesium fluoride hydroxide42 as follows:x) of AHDF added to Mg(OH)2 increased from 20to 50%, respectively. This may be attributable to the increased amountsof the liberated HF that participate in 4F·HF as a fluorinating agentin these mixtures facilitates the formation of both MgF2 and magnesium fluoride hydroxide Mg(OH)F at a low-temperature rangein all prepared samples.43 It is a benefit ofthis method that MgF2 can be prepared more easily. Thisphenomenon has a negative impact on these samples’ surfacearea, as will be discussed later. The third zone (250–390 °C)could be related to both dehydroxylation and dehydration processes,42 followed by a slow weight loss step at a temperatureabove 390 °C (Zone-IV) due to the removal of any residual H2O molecules still bonded on the surface of the solid samples,as follows:2-40%F and Mg(OH)2-50% F, suggests the formation of the Mg–O–Fnanocomposite as a mixture of MgF2 and MgO. XRD analysiswill provide further explanation to confirm this hypothesis.A slowheating rate was chosen and (220), respectively. These diffraction peaks were sharp and predominantin the case of Mg(OH)2-20% F, then their intensity decreasedgradually with increasing (x) % F added to Mg(OH)2, see 2 (ICCD file: 6-0290) inthese mixtures49 was recorded along with the crystallographicplanes (110), (101), (111), (201), (211), and (220), which correspondto the Bragg reflections at 2θ values of 27.265, 35.174, 40.405,43.613, 53.468, and 56.149°, respectively. The sharpness andintensity of these diffractions increased steadily from Mg(OH)2-20% F up to Mg(OH)2-50% F due to the formationof further MgF2 in these nanocomposite mixed samples. Thesame diffraction peaks were recorded in all patterns of samples calcinedat 500 °C, as shown in 2 was clearly observed in these samples at 500 °C, particularlyin those containing more than 20% F, see 2-20% F sample, whether calcined at 400or 500 °C, contains more MgO than MgF2. This phenomenonwill significantly impact the sample’s surface area and CO2 capturing efficiency, as will be discussed later. The meancrystallite size of MgO in all samples was calculated using the (200)reflection to be within the range of 7.9–9.4 nm and between8.6 and 12.0 nm for samples calcined at 400 and 500 °C, respectively.Furthermore, the crystallite size of MgF2 in all sampleswas estimated using the main reflection (111) and was in the rangeof 3.2–19.1 and 21.6–26.7 nm for samples calcined at400 and 500 °C, respectively. The calculated values of the crystallitesize using the Scherrer equation of all samples are cited in 2 crystallite phases inall samples.XRD analysis of theprepared nanocomposite mixtures at 400 and 500 °C was performedin the range of 10–80° (2θ) to confirm the nanocompositestructure of these samples. 3.32 adsorption/desorptionisotherms that were recorded at −196 °C and the pore volumeprofiles of all samples. It is concluded from 51 with type H3 hysteresis loops.52 The calculated values of the surface area (SBET) and the corresponding values of the externalsurface area of samples calcined at 400 °C, as shown in x)% F as NH4F·HF is probably due to the melting ofthis fluorinating agent41 at an early stagein the calcination process of these mixtures. This phenomenon hascaused the surface area of all samples calcined for 3 h at 400 and500 °C to decrease. The adsorption hysteresis of all sampleswas located in the P/P° regionof 0.40–0.45, except for one sample, that is, Mg(OH)2 mixed with 50% F and calcined at 500 °C, see 51 Thepore volume profiles of these samples strongly supported these findings;see F), that is, > 20% F, has resultedinthe generation of new wide pores with diameters of 13, 16, and 18.9nm in the case of samples calcined at 400 °C. Two samples, namely,Mg(OH)2-40% F and Mg(OH)2-50% F, calcined at500 °C, recorded wider mesopores with diameters equal to 33 and33.8 nm, respectively, see x) % F inthese nanocomposite mixtures. This is due to the release of more NH3 during the decomposition of molten NH4F·HF,as shown in The textural properties of the samples calcined at 400 and 500 °Cwere investigated using the BET method. 3.453 and enhancing its capacity to capture CO2.54 The catalytic surface with the highest concentrationsof basic sites is the most effective in capturing CO2.Using TG- and DSC-TPD techniques, pyrrole was used as a probe moleculeto determine the number and strength of surface basic sites over allsamples in this work. Numerous articles have studied the TPD techniqueof pyrrole57 to categorize the different types of basic sitesover solid catalysts.The surface basicity ofany adsorbent is critical for achieving a high catalytic activityin the dehydrogenation reaction of alcohols2 and metal oxides,59 as well asemploying pyrrole as an NMR probe molecule to determine the basicsites’ strength over solid catalysts.60 Very recently, Chen and his co-workers33 proposed some possible intermediates of pyrrole due to its reactionswith the O/Cu (100) surface, based on the TPD curves of pyrrole.Others investigated the use of pyrroleas an IR spectroscopic molecular probe in a surface basicity determinationof MgFx) % F from 20 to 50% added to Mg(OH)2 in thesemixtures was accompanied by the formation of more MgF2 inthese samples, as shown in XRD patterns, see 2 particlespossess a strong Lewis acidity during their formation in magnesiumhydroxyl fluoride.61 Consequently, a steady reductionof the total basic sites can be observed through these samples, whethercalcined at 400 or 500 °C, as shown in the TG curves 2-50% F that was calcined at 400 and500 °C at higher temperatures, see 2 at low temperatures.TG and DSC-TPD profiles of pyrrole recorded for all samples, calcinedat 400 and 500 °C, are shown in G curves 5a,b and 3.52 capture experimentsyield logical and good results. A preliminary test was carried outat three different temperatures, that is, 5, 10, and 30 °C. TG-and DSC-TPD profiles 2-20% F at 400 °C,with the highest surface area using a slow flow rate (20 mL·min–1) of CO2 in a vertical U-tube reactor isconducted. Two different temperatures, 5 and 30 °C, were selectedfor all CO2-capturing tests due to the overlap of resultsrecorded at 10 °C with those at 5 °C, especially at temperatures<350 °C. At 5 °C, the sample captured 103.9 mg2CO·g–1, which is equivalent to2.36 mmol2CO·g–1, whileat 10 °C, it captured 111.9 mg2CO·g–1 and 2.54 mmol2CO·g–1, respectively. Furthermore, at 30 °C, the resultswere clearly different, as shown in 2 at low and relatively hightemperatures, that is, 30 °C, as will be discussed later. 2 captured by Mg(OH)2-x % F samples calcinedat 400 and 500 °C at 5 and 30 °C, respectively. 2 captured by each sample as % mass loss (due to thedesorption of CO2) in milligrams of CO2 pergram and millimoles of CO2 per gram, respectively, in thetemperature ranges of RT–420 °C and RT–520 °C,in the case of samples calcined at 500 °C.In orderto adjust the temperature at which COprofiles 6 represe2 from weak andstrong basic sites, all TG-TPD curves exhibited two continuous massloss steps. This behavior is evident whether CO2 was capturedat 5 or 30 °C. The total % mass loss due to desorption of CO2, in the range of RT–420 °C, dramatically decreasedas the (x) % F added to Mg(OH)2 steadilyincreased. This phenomenon is correlated with the decrease in thesurface area of these samples due to the formation of MgF2 in these nanocomposite mixtures, as shown in 2 desorbed within the same temperature range revealsan additional effect of calcination temperature on the CO2-capturing efficiency of the samples. Heating samples up to 420 °C,the total amount of desorbed CO2 (as mg2CO·g–1 and mmol2CO·g–1) for samples calcined at 400 °Cwere always higher than those calculated for samples at 500 °Cin the same temperature range; see 62 and eliminates surface defects of MgO. This clearly reduces bothweak and strong basic sites over all samples. From the data of CO2 capture at 5 and 30 °C shown in 2 captured by any sample is inversely proportionalto the temperature at which the capturing process is carried out.This is ascribed to the exothermic nature of the adsorption of CO2 over the MgO surface, as confirmed recently.23Due to the continuous desorption ofCO2 is associated with these weak sitesin the form of bicarbonate species.54 Furthermore,strong basic sites are recorded at a higher temperature range,63 that is, 290–450 °C, where CO2 interactswith the strong basic sites to form monodentate and bridged carbonatecarbonates.62 This is clearly exhibited inthe case of Mg(OH)2-20% F calcined at 400 °C and tosomewhat in the case of the other samples, see DSC-TPD profiles, as presented in 2 at 5 and 30 °C wasMg(OH)2-20% F, whether calcined at 400 or 500 °C.Its reactivity could be associated with the presence of surface interfacesbetween both MgF2 with low concentrations distributed inthe bulk of MgO throughout its high surface area.From the above-discussed results,one can conclude that the mostactive sample in capturing COH, Jmol–1) corresponding to each desorptionstep, based on TG- and DSC-TPD profiles in 2-20% F are listed in H calculated for step-1, in the case of Mg(OH)2-20% F at 400 °C, are always higher than the corresponding valuescalculated for step-2 during CO2 capture at 5 and 30 °C.This agrees with the desorbed amounts of CO2 in each step,see H for both steps, as presented in 2 after (1 h) capturing the pure gas, are in close agreementwith those recently published.12The calculatedvalues of the molar enthalpy change 2-20%F calcined at 400 and 500 °C.Finally, 2 capture adsorbentsappearsto be superior to that of many adsorbents previously evaluated forthe same application 2 with different wt % of NH4F·HF, particularly those containing 20–30% F, whethercalcined at 400 or 500 °C, demonstrated a high capacity in capturinga large number of CO2 molecules in a short period of time(1 h), see The performance of our COtion see 5. This a12 and NH4F·HF, a direct andsimple method has been developed for the preparation of nanocompositemixtures of Mg(OH)2-(x) wt % F.Using environmentally friendly materials,such as Mg(OH)22 with a high surface area and a mesoporousstructure. Using the DSC-TPD technique with pyrrole, all samples,whether calcined at 400 or 500 °C, exhibited two surface basicsites as weak and strong basic sites, as measured by pyrrole as aprobe molecule.The obtained samples weremixtures ofMgO and MgF32 capture at 5 and 30 °C totheir high surface area, the high population of surface basic siteswith weak and strong basic sites, and the presence of a low concentrationof MgF2 composing active interfaces in MgO as the maincomponent in these samples, as seen from XRD patterns. On the otherhand, the efficiency was inversely proportional to the temperatureat which the samples were calcined.We correlatedthe high efficiency ofthese samples toward CO42-20% F samplewas the most active in our investigation because it had a larger surfacearea and a greater total number of basic sites than the other samples(30–50% F). Consequently, the Mg(OH)2-20% F sampleis the most effective in capturing CO2.The Mg(OH)2 capture in the flue gas of diluted CO2 concentrationand containing steam (H2O) and gas impurities , which are commonly present in coal plantsand other industrial applications, to investigate the effects of theseimpurities on the sorption performance. In addition, the future workwill include increasing the operating pressure from 1 to 20 or 30atm and the temperature to various values to examine the effect ofincreasing pressure and temperature on the sorption performance.Future work will utilize the prepared Mg–O–Fsamplesfrom this study and design MgO-based looping new systems for CO


In vitro maturation of somatic cells and spermatogonia in organ culture systems has been reported. However, this has been a challenge for organoids derived from dissociated testicular cells. Here, we report generation and maintenance of rat testicular organoids in microwell culture for 28 days. We find that rat organoids can be maintained in vitro only at lower than ambient O2 tension of 15% and organoids cultured at 34°C have higher somatic cell maturation and spermatogonial differentiation potential compared to cultures in 37°C. Upon exposure to known toxicants, phthalic acid mono-2-ethylhexyl ester and cadmium chloride, the organoids displayed loss of tight-junction protein Claudin 11 and altered transcription levels of somatic cell markers that are consistent with previous reports in animal models. Therefore, the microwell-derived rat testicular organoids described here can serve as a novel platform for the study of testicular cell maturation and reproductive toxicity in vitro.An  Male factor infertility is responsible for 40-50% of all cases of infertility worldwide  and arouIn vitro culture systems to model this niche have primarily been limited to conventional two-dimensional co-culture systems , aged P4-P5, were euthanized and testes were removed. All animal procedures were performed as approved by the Animal Care Committee, University of Calgary. Using a pair of forceps, the tunica albuginea was removed to release the tubules, which were then washed in Hank’s Balanced Salt Solution  containing 1% penicillin-streptomycin . The tubules were digested using collagenase type IV  in HBSS (2 mg/mL) for 25 min at 37°C. The tubules were then sedimented by centrifugation at 90x g for 1.5 min and washed with 5 mL HBSS thrice. Finally, the tissue was digested with 0.25% trypsin–EDTA  and DNase I  in HBSS (10 mg/mL) for 5 min to obtain the starting cell population . All exp5 rat testicular cells suspended in 500 μL OFM. Finally, the plates were centrifuged at 500x g for 5 minutes to sediment the cells in the microwells. 500 μL of media were removed from each well and fresh media supplemented with Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix  was used to replenish each well. Microwell plates were divided into three groups and placed in 3 incubators setup for three conditions: (i) ambient O2 tension , (ii) 15% O2 tension at 37°C  and (iii) 15% O2 tension at 34°C . Culture in OFM was carried out for 3-5 days with 50% media changes every second day.AggreWell 400 plates  were treated with Anti-adherence Rinsing Solution  according to the manufacturer’s instructions. Each well was filled with 500 μL of organoid formation medium (OFM)  supplemented with insulin 10 μg/mL, transferrin 5.5 μg/mL, selenium 6.7 ng/mL ; 20 ng/mL epidermal growth factor ; 1% Penicillin-Streptomycin) and the plates were then centrifuged at 2000x g for 2 minutes to release any trapped air bubbles . ODM was composed of Minimum Essential Medium α  supplemented with 10% KnockOut Serum Replacement , hepatocyte growth factor (5 ng/mL) , activin A (100 ng/mL) , follicle stimulating hormone (1 ng/mL) , luteinizing hormone (1 ng/mL) , testosterone (1 μM) , recombinant human BMP-4 (20 ng/mL) , recombinant human BMP-7 (20 ng/mL) , 3,3’,5-triodo-L-thyronine sodium (2 ng/mL) , l-ascorbic acid-2-glucoside (1 mM)  and 1% Penicillin-Streptomycin , 23. Cul) , anti-TNP1 (Transition Protein 1)  . The samples were then permeabilized using a gradient series of methanol  and bloc)  SuppleSupple  , anti-VA) , anti-α-) , anti-3β)  (Supple) , anti-La) , anti-Fi) , anti-Cl) , anti-UCotein 1) , anti-PRotein 1) , anti-ACotein 1)  and antiotein 1)  antibodi+ve and SYCP3+ve cells in each organoid. From each of the three independent experiments (n = 3) performed, a total of 30 organoids were analyzed for VASA+ve and 10 organoids were analyzed for SYCP3+ve cell counts.Immunohistochemistry for VASA was performed on day 7 and 28 organoids, while SYCP3 staining was performed on day 28 organoids. Using confocal microscopy, organoids were selected blindly based on only DAPI and scanned across the z-axis to quantify the number of VASAGapdh. Statistical analysis was performed on the mean of ΔΔCt.RNA was isolated from 1200 organoids using RNeasy Micro Kit  and then reverse transcription was performed using SuperScript™ IV VILO™ Master Mix . RT-qPCR with the primers listed in 2. Controls were treated with equivalent volumes of DMSO. After 48 hours of treatment, at day 21, the organoids were harvested and analyzed with immunofluorescence and RT-qPCR.Day 19 organoids were treated with 1 μM MEHP and 0.25 μM CdCl2 . The control groups for MEHP and CdCl2 were treated with equivalent volumes of DMSO for 1.5 μM of MEHP and 1.25 μM of CdCl2, respectively. Controls of 1.5 μM and 1.25 μM DMSO were treated with equivalent volumes of phosphate buffered saline . At day 7 (48 hours after treatment), the organoids were harvested and approximately 60 organoids suspended in 50 μL ODM were seeded in each well of a 96-well plate as duplicates to perform the MTT assay  according to manufacturer’s instructions. Absorbance at OD 590 nm was measured using SpectraMax i3x plate reader (Molecular Devices). MTT assay was used to measure cellular metabolic activity as an indicator of cell viability.Day 5 organoids were treated with 0.5, 1 and 1.5 μM of MEHP  and 0.01, 0.05, 0.25 and 1.25 μM of CdClt-tests were performed for single comparisons between two groups. For more than two groups, one-way ANOVA with Tukey’s multiple comparison tests were performed. A value of p < 0.05 was set as the limit of statistical significance.All the results described here are from at least three independent experiments performed with three separately prepared rat starting cell population (n = 3). Data were analyzed using the GraphPad Prism 8 software. Unpaired two-tailed +ve Sertoli cells  . Both culture conditions supported the initial generation of testicular organoids (72 hours), with organotypic morphology similar to our previously published porcine and murine model  testicular starting cell population which contained 92.2 ± 1.28% GATA4li cells , 1.7 ± 0rmatids) , 2.6 ± 0ig cells  and 19.9id cells  (n = 3) ar cells , initialne model . Howeverne model , the ratShbg (sex hormone binding globulin) (Sertoli and Leydig cells), which was increased 5.7-fold  in 34°C cultures, no significant differences in transcription levels were observed between the two groups for the somatic cell markers Fshr (follicle stimulating hormone receptor) (Sertoli cells), Star (steroidogenic acute regulatory protein), Cyp17a1 (cytochrome P450 family 17 subfamily a member 1) (Leydig cells) and Hsd17b3 (hydroxysteroid 17-beta dehydrogenase 3) (Sertoli and Leydig cells)     –47. At dt testes . The num < 0.05) , 49. TheAmh (anti-mullerian hormone) and Sertoli and Leydig cell markers Fshr, Shbg, Star, Hsd17b3 and Kitlg were analyzed for day 0, 7, 14, 21, 28 organoids. The transcription levels of Amh were undetectable within a week  while expression of Cyp17a1 showed no significant changes over the duration of the culture . Fshr, Shbg and Kitlg were upregulated by 2.7-, 17.9- and 3.3-fold at day 21     (Supple2) to be tested on organoids. Then, relative cell viability assessments were performed on day 7 (treatment began on day 5) organoids treated with the aforementioned dosages with a 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, revealing that 1 μM MEHP and 0.25 μM CdCl2 were the highest doses without adverse effects on viability      were used to select the dosages of 0.5, 1 and 1.5 μM for phthalic acid mono-2-ethylhexyl ester (MEHP); and 0.01, 0.05, 0.25 and 1.25 μM for the heavy metal cadmium chloride   . Unlike in vitro ; and a Canadian Institutes of Health Research (CIHR) fellowship (MFE-176542) and an ACHRI postdoctoral fellowship to NLML.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.