Clostridium sporogenesis fused to epidermal growth factor (EGF)-like peptide, reduces, in vitro, the number of cancer cells of four different origins—neuroblastoma, lung, breast, and colon cancer. We reveal that MGL S3 is more toxic for neuroblastoma SH-SY5Y and lung cancer H1299 cells compared to MGL tetani, and causes cell death by the induction of apoptosis. In addition, the observed death of cells treated with MGL S3 is accompanied by the prominent downregulation of ERK activity. By the analysis of transcriptomic data of more than 1500 cancer cell lines and patient samples, we show that the high expression of four genes from the methionine metabolism pathway  is associated with poor prognosis for breast cancer and neuroblastoma patients. Additionally, cells of these origins are characterized by a high correlation between EGFR dependency and DNMT3A/CBS expression. Finally, we demonstrate the ability of MGL S3 to enhance the sensitivity of H1299 cells to EGFR inhibition with gefitinib.Methionine dependence of malignant cells is one of the cancer hallmarks. It is well described that methionine deprivation drives cancer cells death, both in vitro and in vivo. Methionine gamma-lyase (MGL) isolated from different species or obtained by genetic engineering can be used for effective methionine depletion. In this work, we show that MGL S3, a genetically engineered protein comprised of MGL from  Altered metabolism is one of the cancer hallmarks . Cancer The majority of cancer cells of different origin, including breast, colorectal cancer, and leukemic cells, are methionine dependent compared to non-malignant cells that require only homocysteine in the growth media to proliferate normally ,6,7. DieMTR (present in approximately 8% of cancer samples), deletions in methyl-thioadenosine phosphorylase gene MTAP , andc cancer ,15. Integ enzyme ,17.Escherichia coli [In addition to diet, there are several approaches for targeting the methionine metabolism and dependency of cancer cells, including enzymes and small molecules. Enzymatic depletion of methionine can be achieved by MGL or pyridoxal 5′-phosphate. Various wild-type MGLs with different activity, off-target effects, and side toxicity have been obtained by direct purification from bacteria, protozoa, and plants. For anticancer application the recombinant protein, MGL can be obtained by the delivery of the corresponding gene into hia coli ,19,20. NIn addition, recombinant fusion proteins consisting of MGL linked to the different peptides are promising for the selective and effective elimination of cancer cells in vitro and in vivo ,22. AlthClostridium sporogenesis, defining the selection of a genetically-engineered enzyme for EGFR-positive cells. We used four cell lines—breast, lung, colon cancer, and neuroblastoma—as a model for the characterization of cell properties after the methionine reduction, and tested novel drug combinations with prominent anticancer activity.In this study, we have aimed to describe the sensitivity of cancer cells to a genetically engineered MGL fusion protein selective for EGFR-positive cells  by obtaining ERK-KTR reporter cancer cell lines with lentiviral vector delivery. S3 peptide  was addeAccording to the different studies, cancer cell methionine addiction varies dramatically (reviewed in ). For exGiven that the response of cancer cells to bacterial-derived and genetically engineered MGL is not defined by their origin ,8,24, weFirst, cells were exposed to various concentrations (1–5 U/mL) of MGL tetani and MGL S3 and incubated for 72 h, and their viability was measured by XTT assay a. The moBased on the cell viability determined by the XTT assay, the IC50 value of MGL was calculated for each cell line. Among these cell lines, the lowest IC50 of MGL tetani was determined for SH-SY5Y—0.8 U/mL, and the lowest IC50 of MGL S3 for H1299—1.1 U/mL .As the metabolic activity measured by Cell Proliferation tests do not always correspond to the number of viable cells, we have additionally calculated the total cell number of cells by processing photos obtained by fluorescence microscopy in Cell Profiler. Briefly, we have counted cell nuclei marked with DAPI (nuclei stain). We have observed at least a double reduction in cell number in all four studied cell lines in response to 2.5 U/mL of MGL tetani and MGL S3 b.Next, we have calculated IC50 values for MGL S3 and MGL tetani, showing that non-small lung cancer H1299 cells are the most sensitive to MGL S3 (IC50 = 0.6 U/mL), while the breast cancer SK-BR-3 cell line is the most resistant (IC50 = 3.1 U/mL) .Non-small lung cancer H1299 and colon cancer HCT116 are at least two-times more sensitive to MGL S3 compared to MGL tetani, highlighting the increased dependency of these cells on EGFR. The sensitivity of SH-SY5Y neuroblastoma cells to MGL S3 and MGL tetani is likely to be EGFR independent as we observed equal toxicity of these two agents. In contrast, MGL tetani is more cytotoxic to SK-BR-3 than MGL S3. Notably, we have observed massive nuclei damaging in SK-BR-3 cells treated with MGL S3 . On the Interestingly, according to previously published data , SK-BR-3Collectively, these findings indicate that SH-SY5Y, SK-BR-3, H1299, and HCT116 cancer cells are highly susceptible to recombinant MGL, which is in good agreement with previously published studies.Next, we assessed the ability of MGL tetani and MGL S3 to induce apoptosis of cancer cells. We selected two cell lines for further studies: H1299—the most sensitive to MGL S3 and SK-BR-3—the most resistant, according to the cell number calculation assays. To determine whether methionine-depleted cell culture media can lead to the induction of apoptosis in cancer cells, we incubated H1299 and SK-BR-3 cells with 2.5 U/mL of MGL tetani and MGL S3 for 48 h. We found 35–40% of apoptotic SK-BR-3 cells treated with MGL tetani and S3 a. MGL teBCL2 mRNA in SK-BR-3 cells exposed to MGL S3, thus driving our attention to the induced ability of these cells to survive for a relatively long time under methionine deprivation and, possibly, adapt to MGL S3, as we also have detected a three-fold higher IC50 value for MGL S3 compared to MGL tetani in the cell counting assay. Indeed, in multiple studies, an elevated level of BCL2 was linked to cancer cells acquiring resistance to various chemotherapeutic molecules [BCL2 mRNA level dramatically dropped in H1299 cells in response to MGL tetani. MGL S3 caused even more prominent downregulation of BCL2 in these cells. The observed decrease of BCL2 mRNA in response to MGL tetani and MGL S3 is consistent with the results of the apoptosis assay obtained for H1299 cells, as the MGL S3 induces more prominent cell death compared to MGL tetani, along with the stronger induction of BCL2. On the other hand, both MGL proteins caused a similar apoptosis rate (approximately 40%) in SK-BR-3 cells, while BCL2 expression was affected differently. As about, 50% of SK-BR-3 cells survive upon treatment with MGL S3  caused almost the same effect on the downregulation of ERK activity in H1299, HCT116, SH-SY5Y, and SK-BR-3 cells, showing a sufficiency of 1 U/mL to inactivate ERK, and also indicating that methionine deprivation alone cannot completely block cancer cell survival molecular machinery.The level of ERK downregulation by MGL tetani in HCT116 and H1299 cells was comparable to that driven by MGL S3. In contrast, ERK activity was affected only mildly (less than 20%) or remained completely unaffected in SK-BR-3 and SH-SY5Y cells, respectively, upon MGL tetani treatment. All in all, MGL S3 effectively and significantly reduced ERK activity in all studied cell lines.As a next step in the elucidation of the molecular mechanism defining MGL sensitivity and further proceeding to the establishment of promising therapeutic options based on methionine depletion, we have compared the expression of KEGG methionine metabolism genes in two groups of cells—highly and poorly sensitive to MGL-BL929. IC50 values for MGL-BL929 were obtained from  and the The sensitive group (IC50 > 1.26 U/mL) contained 10 cell lines, including HCT116, and the resistant group (IC50 < 0.64 U/mL) contained 10 cell lines, including SK-BR-3. Surprisingly, the comparison of the expression of 16 methionine metabolism genes in two groups revealed no significant difference a underlaN = 104), neuroblastoma , non-small cell lung , and colon  cancer, and their mRNA sequencing data. We revealed that none of the 17 methionine metabolism genes had prognostic relevance for patients with lung and colon cancer. On the contrary, high expression of each of nine genes  was significantly associated with the prognosis of neuroblastoma patients and five  for breast cancer patients , were common markers of poor prognosis for neuroblastoma and breast cancer.Additionally, we have studied four datasets containing information of the overall survival of patients diagnosed with breast , gastric, neuroblastoma, breast, ovarian, and thyroid cancer cells (first cluster) as the most co-dependent on EGFR and methionine cycle genes. The second cluster contained lung cancer and colon/colorectal cancer cells with variable correlation coefficients for the studied parameters. Finally, the third cluster was formed by bone cancer cells that are characterized with the inversed correlation between EGFR dependency and the expression of metabolism genes. CBS and DNMT3A—were placed in one cluster, whilst the other two genes—MTAP and AHCY—were placed in the second cluster. In addition, EGFR dependency correlated with the majority of genes from the first cluster in rhabdoid, gastric, neuroblastoma, breast, ovarian, and thyroid cancer cells. Low, or the absence, of correlation between EGFR dependency and expression is shown for CBS and SRM genes in rhabdoid cancer, for the MTR gene in gastric cancer and for the SRM gene in rhabdoid and neuroblastoma cancer cells. Genes placed in the second cluster have variable correlations with EGFR dependency across the cancer cell lines from both clusters, and thus they should be studied separately for each cancer type. Notably, MGL S3, but not MGL tetani, caused ERK downregulation in neuroblastoma (SH-SY5Y) and breast cancer (SK-BR-3) cells, that represent cancer types that have a strong correlation between EGFR dependency and methionine metabolism genes expression.Interestingly, earlier described genes associated with poor prognosis for neuroblastoma and breast cancer patients b—CBS andThus, an elevated level of methionine metabolism gene expression is present in EGFR-dependent cells, and these cells can possess a higher sensitivity to methionine depletion and EGFR-inhibition or downregulation.EGFR (more than five times) in response to MGL S3 in both cell lines , encoded by corresponding gene AHCY, in the progression of cancer is still not obvious. High expression of AHCY is associated with MYCN-amplified neuroblastoma and contributes to the enhanced proliferation of cancer cells [AHCY, MYCN-amplified neuroblastoma cells have elevated levels of the CBS gene, encoding cystathionine beta synthase. In addition, CBS contribution into the development of tumors and proliferation of malignant cells has been described for chronic myeloid leukemia [DNMT3B, at both the mRNA and protein level has been described for several malignancies, including prostate, lung, breast, and colorectal cancer; glioblastoma; and leukemia [Within the current study, we used cell models to demonstrate the efficacy of MGL S3 for the inhibition of cancer cell growth and showed that it is accompanied by ERK downregulation. By the analysis of several publicly available datasets, we have shown that the sensitivity of cells to MGL does not depend on the levels of expression of 16 methionine metabolism genes. Aimed mainly at the examination of MGL S3 activity in vitro, we also revealed the novel crosstalk between EGFR signaling and methionine metabolism existent in several types of malignant cells. We have shown that, based on the correlation between EGFR dependency  and theer cells ,37. AHCYer cells . Along wleukemia  and brealeukemia , ovarianleukemia , and colleukemia  cancer. leukemia ,45,46,47leukemia . We haveThe establishment of stable reporter cell lines by the lentiviral delivery of the reporter system allowed us to determine cell number and measure ERK activity in the same cells. Previously, we have shown the implication of ERK activity in the response of cancer cells to targeted therapy ,30,48, aAHCY, CBS, DNMT3A, and MTAP, are markers of poor prognosis for neuroblastoma and breast, but not for colon and lung, cancer patients. Finally, we have shown that EGFR inhibition with gefitinib synthesize with MGL S3 in H1299 cells. Thus, the simultaneous targeting of EGFR and methionine metabolism could be a promising anticancer strategy.Moreover, we have described a new unexpected link between EGFR dependency and methionine metabolism gene expression in cancer cell lines. A cluster of cancer cell lines, consisting rhabdoid , gastric, neuroblastoma, breast, ovarian, and thyroid cancer cells, that are co-dependent on EGFR and methionine, was revealed. Moreover, four genes from the methionine metabolism gene set, such, 2 in cell culture medium supplemented with 10% fetal bovine serum , 2 mM L-glutamine , 100 units/mL penicillin, 100 μg/mL streptomycin , and 1 mM sodium pyruvate . RPMI-1640  cell culture medium was used for SH-SY5Y and SK-BR-3 cells and DMEM  for H1299, HCT116, and HEK293T cells. Cell lines were provided by the Heinrich–Pette Institute at the Leibniz Institute for Experimental Virology as a gift and were routinely checked for mycoplasma contamination.Neuroblastoma SH-SY5Y , breast cancer SK-BR-3 (adenocarcinoma with HER2 overexpression), non-small cell lung carcinoma H1299 , colon cancer HCT116 (KRAS mutant), and CTH, human embryonic HEK293T (modified with SV40 large T antigen) cells were maintained at 37 °C and 5% COFor cell viability assessment with Cell Proliferation assay XTT  5000 cells were plated in TC-treated flat-bottom clear 96-well plates (TPP) 24 h before the MGL addition. The final volume of cell culture media containing cells and drugs was 100 µL. After 3 days of incubation, XTT compounds were added to the wells for 4 h and the absorption signal was measured with Multiscan FC .For apoptosis/necrosis measurement with Annexin V and Propidium Iodide, 20,000 cells were plated in TC-treated flat-bottom clear 24-well plates (TPP) 24 h before the MGL addition. After 3 days of incubation, adherent cells were dissociated from the plates with trypsin, washed with phosphate buffer saline, and stained with Annexin V-FITC  in 100 µL of Annexin staining buffer  for 10 min. Prior to measurement on a BD LSRFortessa Flow Cytometer , 300 µL of ice-cold buffer containing propidium iodide was added .6 HEK-293T cells were plated on 100 cm2 Petri dishes 24 h before the transfection. Calcium transfection was used to deliver plasmids into the HEK-293T cells. Cell culture media containing lentiviral particles was collected from HEK-293T cells 24 and 48 h after the transfection with plasmids, coding structural elements of the lentivirus , plasmid coding G protein (envelope) of the vesical stomatitis virus VSV, and a vector plasmid containing a gene of interest. To obtain ERK-KTR cell lines for ERK quantification, pLentiCMV Puro DEST ERKKTRClover plasmid  was used, which encoded the ERK docking domain ELK, nuclei localization site, and nuclei extraction site (with phosphorylation sites) fused to fluorescent protein mClover.An amount of 10Escherichia coli strain BL21(DE3): F-ompT hsdSB gal dcm (DE3) (Novagen) was used for Clostridium tetani MGL and MGL-S3 gene expression. Bacteria were grown in standard conditions and selected with kanamycin (30 μg/mL) after transformation. E. coli BL21(DE3) transformed with pET-mgl-tet and pET-mgl-S3 was used for MGL biosynthesis. Plasmids pET-mgl-S3 were received from original pET-mgl-Sporog, using Gibson Assembly. Primers for cloning were chemically synthesized according to the corresponding amino acid residues VGF52-67 (RCSHGYTGIRCQAVVL) [Clostridium tetani we used, which showed the same effects as MGL from Clostridium sporogenesis to cancer cells and had a close kinetic property [The RCQAVVL)  and fuseproperty .Enzyme synthesis was performed as described in . To puriWe used two ERK-KTR cell lines (H1299 and SH-SY5Y) that were previously established and described in  and simiTotal RNA was isolated with Trizol reagent (Applichem) and concentration and purity were assessed with NanoDrop ND-1000 . An amount of 5000 ng of RNA was used for the synthesis of complementary DNA with a RevertAid First Strand cDNA Synthesis Kit . cDNA was desolved in 500 µL of nuclease-free water  and stored at −20°C.GAPDH).Real time quantitative polymerase chain reaction (qPCR) was performed in three replicates using white hard-shell 96-well PCR plates and CFX96 Touch Real-Time PCR Detection System (Bio-Rad) according to the following protocol: 1 cycle—95 °C, 10 min; 40 cycles—95 °C, 10 s; 57 °C, 15 s; 72 °C, 15 s, followed by melt-curve analysis. For qPCR reaction, 5 µL of cDNA and 200 nM of specific primers were desolved in nuclease-free water and mixed with 3 µL of 5X qPCRmix-HS SYBR to a final volume of 15 µL per well. Specific primers used in this study: BCL2_pr1 TGAACTGGGGGAGGATTGTG; BCL2_pr2 CGTACAGTTCCACAAAGGCA; EGFR_pr1 AGGAGAGGAGAACTGCCAGAA; EGFR_pr2 TCTCGGAATTTGCGGCAGAC; GAPDH_pr1 GAGCCCGCAGCCTCCCGCT; GAPDH_pr2 GCGCCCAATACGACCAAATC. Cycle threshold values for target genes were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase gene (N = 88) [N = 106) [N = 104) [N = 232) [N = 917) [http://r2.amc.nl, accessed on 10 October 2022)”. Dependency data (for EGFR) were obtained from CRISPR screen  [We used four datasets, containing RNA sequencing data of samples obtained from patients diagnosed with neuroblastoma  , non-smaN = 106) , breast N = 104) , and colN = 232)  cancer, N = 917)  downloadmap.org) .Statistical analysis was performed using GraphPad Prism 9 software. The types of tests are defined in the description of the figures along with the number of replicates.AHCY, CBS, DNMT3A, and MTAP) from the methionine metabolism gene set are markers of poor prognosis for neuroblastoma and breast cancer patients. MGL S3 downregulates EGFR mRNA level in breast and lung cancer cell lines, and the EGFR inhibitor gefitinib enhances the sensitivity of H1299 cells to MGL S3. Thus, a novel genetically-engineered protein, MGL S3, could be used for the precise targeting of EGFR-dependent cancer cells, and combined with EGFR inhibitors.MGL S3, a novel methionine gamma lyase, obtained by genetic engineering protein comprised of MGL from Clostridium sporogenesis fused to epidermal growth factor (EGF)-like peptide, reduces cell number, induces apoptosis, and downregulates ERK activity in neuroblastoma, lung, breast, and colon cancer cells in vitro. An elevated level of the methionine metabolism gene expression signature is present in EGFR-dependent cells, such as rhabdoid , gastric, neuroblastoma, breast, ovarian, and thyroid cancer cells. Four genes (


Human noroviruses (NV) are the most prevalent cause of sporadic and pandemic acute gastroenteritis. NV infections cause substantial morbidity and death globally, especially amongst the aged, immunocompromised individuals, and children. There are presently no authorized NV vaccines, small-molecule therapies, or prophylactics for humans. NV 3 C L protease (3CLP) has been identified as a promising therapeutic target for anti-NV drug development. Herein, we employed a structure-based virtual screening method to screen a library of 700 antiviral compounds against the active site residues of 3CLP. We report three compounds, Sorafenib, YM201636, and LDC4297, that were revealed to have a higher binding energy (BE) value with 3CLP than the control (Dipeptidyl inhibitor 7) following a sequential screening, in-depth molecular docking and visualization, physicochemical and pharmacological property analysis, and molecular dynamics (MD) study. Sorafenib, YM201636, and LDC4297 had BEs of -11.67, -10.34, and -9.78 kcal/mol with 3CLP, respectively, while control had a BE of -6.38 kcal/mol. Furthermore, MD simulations of the two best compounds and control were used to further optimize the interactions, and a 100 ns MD simulation revealed that they form stable complexes with 3CLP. The estimated physicochemical, drug-like, and ADMET properties of these hits suggest that they might be employed as 3CLP inhibitors in the management of gastroenteritis. However, wet lab tests are a prerequisite to optimize them as NV 3CLP inhibitors. Human noroviruses (NV), which are members of the Caliciviridae family, are the leading cause of acute gastroenteritis globally, with substantial morbidity and a significant economic burden . NV infe1 glutamine (or glutamate) residue, yielding six nonstructural proteins required for NV replication (The NV genome is made up of a positive-strand RNA with three open reading frames encoding: i) polyprotein, ii) minor capsid protein, and iii) main capsid protein. The polyprotein is processed by a virally encoded 3 C L protease (3CLP), a cysteine protease having cysteine139-histidin30-glutamate54 catalytic triad, an extended binding cleft, and a major substrate selectivity for a Plication . NV 3CLPlication . Peptidylication . In addilication .in silico approach to combat gastroenteritis.Traditional drug development is time-consuming and costly, taking an average of 10–15 years to reach the market and costing an probably 58.8 billion USD in 2015 . These fThe 3D structure of 3CLP, which has the PDB ID 5T6F, was taken from the protein data bank (PDB) . In ordeThe prepared library of compounds in pdbqt format was used for VS. against the active site residues of the 3CLP with the PyRx 0.8 program . The griThe top 20 compounds, including the control (Dipeptidyl inhibitor 7) , were suBy identifying lead molecules, computational approaches help improve the success rate of experimental drug trials. The computational prediction of pharmacokinetic and ADMET properties of small molecules provides the clue to narrowing down the screening and their potential to be drug-like molecules. The efficacy and safety profiles of the selected hits and their pharmacokinetics were predicted using SwissADME  and the https://www.molinspiration.com), was used to evaluate the drug score of selected hits in relation to various human receptors such as ion channels, GPCRs, enzymes, kinases, proteases, and nuclear receptors. On the whole, a greater bioactivity score specifies that the active compound is more likely to be active.The attributes of the bioactivity score predict the overall potential of the three best selected hits to be an effective lead candidate. An online tool ‘Molinspiration chemoinformatics’ , 3CLP interacting residues with its co-crystallized inhibitor  were analyzed, which showed that Gln110, Arg112, Val114, His130, Thr134, Glu54, Met107, Arg108, Ile109, Ile135, Pro136, Gly137, Cys139, Lys152, His157, Ala158, Ala159, Ala160, Thr161, Lys162, and Val168 residues were important in binding with dipeptidyl inhibitor 7 . InteresThe intention of ligand-protein docking is to anticipate the most probable binding modes of the ligand with the catalytic pocket residues of the target protein, with a high BE value (more negative) implying an effective interaction between ‘inhibitor-protein’ complexes . SorafenDespite advances in developing effective antiviral therapies, the currently available antiviral agents have various issues, including high prices, drug resistance, safety, and effectiveness limits . Furtherviz., physicochemical properties, pharmacokinetics, and druglikeness of the Sorafenib, YM201636, and LDC4297.The pharmacokinetics viability and druglikeness characteristics of identified hits computed using the SwissADME and DataWarrior tools indicated that they should be promising lead candidates. in silico ADME assays is critical to achieve druglikeness and decrease risk attrition in the advanced stages. The predicted compounds had acceptable drug-likeness and pharmacokinetic features, and they could not be P-glycoprotein substrates (P-gp). The cytochrome P450 monooxygenase (CYP) enzyme superfamily, which includes cytochrome CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, is crucial in drug metabolism in the liver, and drug biotransformation by O-type oxidation processes, particularly those of 2D6, 2C9, and 3A4, has been anticipated. Sorafenib, YM201636, and LDC4297 were shown to be CYP1A2, CYP2D6, and CYP2C9 inhibitors rather than CYP2C19 inhibitors. These three compounds had substantial lipophilicity and were moderately soluble in water.Evaluation of pharmacokinetic properties for a success therapeutic in the early stages of drug design by Finally, based on BE, sorafenib and YM201636 including the control were chosen for MD simulation studies to assess the stability of complexes. The root mean square deviation (RMSD) is a protein stability metric; the slighter the deviations, the more stable the protein structure . 3CLP-coThe 3CLP-Sorafenib and 3CLP-YM201636 backbones displayed consistent fluctuations in the 3CLP catalytic pocket, most probably due to differing orientations, with the biggest fluctuation regions found between 25–30 and 120–125 residues . The vib2, respectively (The solvent-accessible surface area (SASA) of a protein is the area of its surface that is involved in the interaction with its solvent molecules. Average SASA values for 3CLP-control, 3CLP-Sorafenib, and 3CLP-YM201636 complexes were recorded during the 100 ns simulation. The SASA values for the 3CLP-control, 3CLP-Sorafenib, and 3CLP-YM201636 complexes were 88.02, 84.01, and 85.30 nmectively . SASA anectively .The hydrogen bond is vital to the stability of the ligand-target complex . BetweenThe Gibbs free energy (GFE) landscape of the 3CLP -control, 3CLP-Sorafenib, and 3CLP-YM201636 complexes has been plotted. The blue color represents the location with the least amount of energy. The 3CLP-control and 3CLP-Sorafenib complexes have two distinct global energy minima basins (in blue) , but theGiven the clinical relevance of NV, the study presented here focuses on a high-throughput virtual screen of an antiviral natural compound library against the NV 3CLP using computational technique. The lead compounds sorafenib, LDC4297, and YM201636 met ADMET criteria and interacted with key 3CLP residues. Based on BE, sorafenib and YM201636 were chosen for MD simulation studies, and these compounds displayed stability with the 3CLP. Therefore, these compounds have the potential to be useful in the development of 3CLP inhibitors, and further testing in wet laboratory is warranted.


O-galactoside levels were highest among anthocyanins in both cultivars, reaching >5000 μg·g−1 at the last color transition stage in Huashuo apples, while only >3000 μg·g−1 in Huarui apples. Moreover, the expression of most structural genes, especially DFR, CHI, and 4CL associated with anthocyanin synthesis, were higher in Huashuo apples than in Huarui apples. Combined transcriptomics, metabolomics, and qRT-PCR analysis revealed that six transcription factors from the MYB and bZIP transcription factor families likely play key roles in the dark coloring of Huashuo apples. These results provide deeper insights into apple coloring and suggest a series of candidate genes for breeding anthocyanin-rich cultivars.Red coloration in apples, an important quality trait, is primarily attributed to the accumulation of anthocyanins. Centuries of breeding have produced a wide variety of apples with different levels of anthocyanins in response to genetic and environmental stimuli. The Huashuo apple shows a much darker red color than its sister line, Huarui. Thirteen different anthocyanins were detected in Huashuo and Huarui apples, of which ten were significantly more abundant in Huashuo apples, confirming that the color difference is indeed attributed to high anthocyanins accumulation rather than the types of anthocyanins. In particular, the contents of cyanidin 3- Apple skin color, particularly anthocyanin-based coloration, is a key factor in fruit quality and market acceptance. Anthocyanins play several important roles in plants, including resistance to environmental stresses, protection against diseases, and pollination ,2. AdditO-galactoside, cyanidin 3-O-glucoside, and cyanidin 3-O-arabinoside—among which, cyanidin 3-O-galactoside is the primary component of the apple peel, while the other two are present in various amounts in different apple varieties [O-arabinoside, cyanidin 3-O-rhamnoside, and cyanidin 3-O-rutoside [Flowers with anthocyanin as the primary pigment show a wide range of colors, ranging from orange–red to red, purple, blue, and black. The red peel is mainly composed of three anthocyanins—cyanidin 3-arieties . In addirutoside .PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumarate CoA ligase (4CL). The key branch point from monolignol biosynthesis to anthocyanins biosynthesis is catalyzed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT). CHS, CHI, and F3H are upstream structural genes, while DFR, ANS, and UFGT are downstream structural genes. The latter are usually key genes affecting anthocyanin synthesis, and their expression affects the anthocyanin content in tissues [CHS, F3H, DFR, ANS, and UFGT is positively correlated with anthocyanin accumulation [The anthocyanin synthesis pathway is a branch of phenylpropanoid biosynthesis  that inv tissues ,9. Durinmulation ,11. Howemulation .MdMYB1 [MdMYBA [MdMYB10 [MdMYB110a [MdMYB3 [MdMYB9, MdMYB11 [MdMYB90-like [MdMYB16 [MdMYB111 [MdMYB306-like inhibited the anthocyanins synthesis in apple peels by interacting with MdMYB17 and MdbHLH33 [Transcription factors (TFs) are involved in the color regulation of plant organs. Anthocyanin biosynthesis is regulated by the MYB-bHLH-WD40 (MBW) protein complex ,14. MYB MdMYB1 , MdMYBA  [MdMYBA , MdMYB10[MdMYB10 , MdMYB11dMYB110a , MdMYB3  [MdMYB3 , MdMYB9, MdMYB11 , and MdMB90-like . MYB TFs[MdMYB16  or MdMYBMdMYB111  is overeMdbHLH33 . StudiesMdbHLH33 ,28,29,30MdbHLH33 , WRKY [3MdbHLH33 ,33, and MdbHLH33 ,35.High-throughput methods, such as transcriptomic and metabolomic approaches, have been widely used to explore coloring-related mechanisms in fruits, including apples ,37,38. TTwo apple cultivars, Huashuo (YS) and Huarui (YR), were planted in the orchard located at Zhaotong Apple Industry Research Institute . The trees were five years old, planted at 3.5 × 1.5 m intervals between and along rows, and grafted onto M26 interstock. Nine trees with consistent and healthy growth were selected for sampling from each cultivar, with three trees selected as biological replicates. Samples were collected over the color transition period of 105, 110, 120, and 130 days after full blossom (DAFB); similarly, 15 fruits were collected from the three biological replicate. After separating the peel from the cortex (1 mm thick), some of the mixed peel samples were collected for pigment measurement while the remaining samples were frozen in liquid nitrogen and stored at −80 °C for subsequent metabolome and transcriptome analyses.v/v) for 24 h at 4 °C in the dark. Subsequently, the mixture was filtered, and absorbance of the filtrate was measured at 530 nm using an ultraviolet spectrophotometer . The relative anthocyanin concentration was calculated using the following formula:530nm refers to the absorbance value of the sample at 530 nm, m represents the mass of the sample, and N is the relative anthocyanin concentration of per gram fresh weight (Uint/g FW).Anthocyanin was determined as previously reported, with modifications ,41. Applv/v), vortexed for 5 min, ultrasonicated for 5 min, centrifuged for 5 min, and the supernatants were isolated; this process was repeated twice. The combined supernatants were filtered through a SCAA-104 membrane  and analyzed using a UPLC-ESI-MS/MS system ; MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). The analytical conditions were as follows: UPLC: column, WatersACQUITY BEH C18 ; solvent system, water (0.1% formic acid), methanol (0.1% formic acid); gradient program, 95:5 (v/v) at 0 min, 50:50 (v/v) at 6 min, 5: 95 v/v at 12 min, hold for 2 min, 95:5 (v/v) at 14 min, hold for 2 min; flow rate, 0.35 mL/min; temperature, 40 °C; injection volume, 2 μL. ESI-MS/MS: ion source, ESI, source temperature 550 °C; ion spray voltage (IS) 5500 V; curtain gas (CUR) was set at 35 psi. In Q-Trap 6500+, each ion pair was scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).Anthocyanin metabolites were obtained from Metware Biotechnology Co., Ltd. . Apple peel samples were freeze-dried in a vacuum freeze dryer  for 64 h in the vacuum environment as the temperature gradually rose from −50 °C to 30 °C, and crushed using an MM 400 mixer mill  and zirconia beads for 1.5 min at 30 Hz. The crushed samples (50 mg) were mixed with 0.5 mL of 50% aqueous methanol . The quality and concentration of the RNA samples were determined using an Agilent 2100 Bioanalyser , Qubit 3.0 fluorometer , and NanoDrop 2000 spectrophotometer . High-quality total RNA (1 μg) was used to synthesize first-strand cDNA with the Prime Script RT Reagent Kit with gDNA Eraser .https://phytozome-next.jgi.doe.gov/, accessed on 25 November 2021) using the hisat2 software for assembly. Gene expression levels were estimated using HTseq [The raw RNA-Seq data were cleaned by removing adaptor sequences and low-quality reads using Cutadapt v1.18 and Trimmomatic-0.38 software, respectively ,43. The ng HTseq . Pearsonng HTseq .Weighted gene co-expression network analysis (WGCNA)  was perfSix genes were selected for qRT-PCR analysis to examine the different expression profiles. Methods for RNA extraction and cDNA synthesis are provided in https://cn.string-db.org/cgi/input.pl, accessed on 20 March 2022) with an option value of >0.700 [The target protein sequence was uploaded to the online server STRING to predict the interaction network . The data were analyzed with Excel 2010 ; Sigmaplot v14.0  software was used to create column and line charts. Significant differences in anthocyanin contents or gene expression levels in different samples were determined using Duncan’s multiple range test (p < 0.05) in the ANOVA  program of SPSS 19.0 .To visualize specific relationships, correlation network analysis was performed using the online server OmicStudio tools , CCoAOMT (caffeoyl CoA O-methyltransferase), CCR (caffeoyl CoA reductase), C3H (p-coumarate 3-hydroxylase), HCT (hydroxycinnamoyl-coA shikimate/quinate hydroxycinnamoyl transferase), and CAD , also gradually increased as the color deepened. C4H had two copies with similar expression levels over the entire color transition period; CHI had three copies, one of which had significantly higher levels than the other two during the entire color transition period. FLS had two copies with inconsistent expression levels, although the expression levels of these copies gradually increased throughout the color transition period. Regarding genes associated with lignin synthesis, the expression levels of HCT, CCoAOMT, and CAD in YS were much higher than those in YR.Several genes from the 16th subclass  were assome data . MoreoveFurthermore, some hormone-related genes may also play key roles in anthocyanin accumulation. WGCNA revealed that the absolute value of the correlations between these genes and anthocyanin levels reached >0.8 . Among tSeveral AP2/EREBP, ARF, C2C2, C2H2, HB, bHLH, MYB, and WRKY family TFs correlated with anthocyanin accumulation , six of According to the prediction results of the above key TFs, some TFs can interact with F3H or DFR which are directly involved in anthocyanin biosynthesis. In addition, we selected other genes, 4CL and CHI, which the transcription levels in the whole color transient period in YS were much higher than that in YR, for interaction analysis. Since the correlation between the dynamic accumulation of anthocyanin and the transcription level of F3H was <0.8, we only selected DFR, 4CL, and CHI for protein interaction prediction . DFR MD. The 4CLThe anthocyanin biosynthesis pathway is a well-studied secondary metabolite pathway in plants . The mecO-galactoside, the primary anthocyanin in apple peel, accounts for >85% of the total anthocyanin content. Other anthocyanins, such as cyanidin 3-O-arabinoside and cyanidin 3-O-glucoside have also been identified in apple peel [O-galactoside is unique and only found in red apple peels. In the current study, the anthocyanin content increased as the peel’s red color gradually deepened. Moreover, cyanidin 3-O-galactoside levels were highest among anthocyanins in both YS and YR , and indirectly through the activation of other genes (MdMYB1 and MdbHLH3) that activate anthocyanin biosynthesis [In addition to its key structural genes, anthocyanin biosynthesis is regulated by several TFs, such as members of the MYB, MADS-box, bHLH, WD40, bZIP, and WRKY families ,68. Numeynthesis . The TTGproteins . Among tpromoter . Mao et promoter  reportedbidopsis . AHA10, bidopsis , REVEILLbidopsis , and PRPynthesis . The MYBMdHY5 [MdbZIP44 promoted anthocyanin accumulation by regulating downstream anthocyanin biosynthesis genes in apples [MdMYB10 and downstream anthocyanin biosynthesis genes [Arabidopsis, COP1 is a light-inactivable repressor of photomorphogenic development and negatively regulates HY5 [Another TF family, bZIP, was also reported to play a role in anthocyanin biosynthesis in apples. bZIP family TFs MdHY5  and MdbZn apples . In the is genes . Therefoates HY5 . The COPates HY5 . As is wates HY5 . Thus, aates HY5 , while tates HY5 , so the The role of TFs is essential as they can regulate multiple downstream response genes at the same time. Some of the TFs screened in the current study are known to be associated with the structural gene of anthocyanin synthesis, while others were predicted to interact with other TFs that regulate anthocyanin accumulation. We speculate that the differential expression of the six TFs induce changes in the expression of anthocyanin synthesis-related structural genes and alter endogenous hormones levels, resulting in phenotypic differences between YS and YR ; this coO-galactoside identified as the major anthocyanin. The higher accumulation of anthocyanins in YS, rather than different types of anthocyanins, resulted in a different phenotype compared with YR. The transcriptome data revealed that most anthocyanin biosynthesis-related genes and at least six TFs were significantly correlated with anthocyanin accumulation, which likely contributes to the differential accumulation of anthocyanin in the sister apple lines. Our study findings provide a series of candidate genes with potential applications in the breeding of anthocyanin-rich cultivars.The molecular mechanisms underlying anthocyanin accumulation in YS and YR were analyzed using metabolomics, transcriptomic, qRT-PCR, and interaction analysis. At least 13 anthocyanins were detected in the peels of YS and YR, with cyanidin-3-


Zebularine also suppressed the activation of nuclear factor-κB (NF-κB) and the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, by 69.8%, 74.9%, and 69.6%, respectively, in obstructed kidneys. Furthermore, inhibiting DNA methyltransferase buttressed the nuclear expression of nuclear factor (erythroid-derived 2)-like factor 2, which upregulated downstream effectors such as catalase , superoxide dismutase 1 , and NAD(P)H: quinone oxidoreduate-1  in obstructed kidneys. Collectively, these findings suggest that inhibiting DNA methylation restores the disrupted balance between pro-inflammatory and anti-inflammatory pathways to alleviate renal inflammation and fibrosis. Therefore, these results highlight the possibility of DNA methyltransferases as therapeutic targets for treating renal inflammation and fibrosis.Renal fibrosis, the final pathway of chronic kidney disease, is caused by genetic and epigenetic mechanisms. Although DNA methylation has drawn attention as a developing mechanism of renal fibrosis, its contribution to renal fibrosis has not been clarified. To address this issue, the effect of zebularine, a DNA methyltransferase inhibitor, on renal inflammation and fibrosis in the murine unilateral ureteral obstruction (UUO) model was analyzed. Zebularine significantly attenuated renal tubulointerstitial fibrosis and inflammation. Zebularine decreased trichrome, α-smooth muscle actin, collagen IV, and transforming growth factor-β1 staining by 56.2%. 21.3%, 30.3%, and 29.9%, respectively, at 3 days, and by 54.6%, 41.9%, 45.9%, and 61.7%, respectively, at 7 days after UUO. Zebularine downregulated mRNA expression levels of  End-stage renal disease (ESRD), the final phase of chronic kidney disease (CKD), is a growing public health burden, and thus intensive efforts have been made worldwide to prevent or delay the progression of CKD . Renal fp16 (INK4A), which is hypermethylated in cancer cells [Epigenetic modifications primarily consist of three basic processes: methylation of the DNA, modification of the histone proteins, and the regulation of gene expression by non-coding RNAs . DNA meter cells . Compareer cells .RASAL1 gene was associated with the activation of fibroblasts and fibrogenesis in the kidney [The contribution of DNA methylation to the process of renal fibrosis has received much attention from researchers, but has not yet been fully elucidated. Recent studies have demonstrated the correlation between the methylation status of genomic DNA and the severity of diabetic kidney disease (DKD), suggesting the potential role of DNA methylation in DKD ,15. For e kidney . It shoue kidney ,17. HoweImmunohistochemical staining for DNMT1 showed a marked increase in the tubulointerstitium of kidneys on days 3 and 7 after UUO compared with that in the kidneys of sham-operated mice A,B. HoweTGF-β1 mRNA expression increased markedly in obstructed kidneys, which was also attenuated by zebularine , a relevant marker of myofibroblast activation, and EMT-related genes to investigate the effect of DNMT on EMT during renal fibrosis development. As shown in  kidneys B–G. Morebularine H–J. All interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor (TNF)-α, were enhanced at three and seven days post-UUO , increased markedly in obstructed kidneys, which was prevented in zebularine-treated kidneys A–C. AfteCell death by apoptosis is also involved in renal fibrosis. Thus, we examined apoptotic cells by a terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, as well as the expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 by immunoblot analysis, to investigate the effects of zebularine on apoptosis in UUO kidneys. As shown in The present results demonstrate that the treatment of zebularine, an inhibitor of DNMT, ameliorated renal inflammation and fibrosis in the mouse UUO model, which is an established model of non-metabolic renal fibrosis. Improved kidney fibrosis was accompanied by attenuated EMT and apoptosis in response to UUO. Thus, the present study suggests that DNMT participates in inflammation, EMT, and apoptosis, all of which are involved in the pathogenic process of renal fibrosis.Regarding inflammation, zebularine treatment alleviated the activation of NF-κB and the subsequent expression of inflammatory cytokines in obstructed kidneys . In the Snail1, and in a previous report, Snail1-induced EMT was not reversed by DNMT inhibitors [Snail1, which then cannot induce the expression of EMT-related genes. Therefore, EMT appears to be subjected to epigenetic regulation and to be prevented or reversed by inhibiting DNMT during renal fibrosis. The renoprotective effect of zebularine raises the possibility of aberrant DNA hypermethylation in obstructed kidneys. Since DNMT1 and DNMT3b were highly expressed by UUO operation  production and ROS scavenging is a crucial modulator in the progression of renal fibrosis. In this study, we evaluated the expression of of NF-κB ,33,34. Tof NF-κB . Mechaniof NF-κB . In thisof NF-κB . TherefoThe three major targets of zebularine in the UUO model may be Nrf2, NF-κB, and Nox2. By modulating these molecules, zebularine suppressed oxidative stress, inflammation, and EMT, which led to the prevention of fibrosis in UUO .This study has the following limitations. The first limitation of this study is that the side effect of zebularine, such as hepatic injury, was not measured. The first-pass metabolism of zebularine occurs in the liver. Zebularine is metabolized to uridine by aldehyde oxidase (AO), which is abundant in hepatocyte cytosol . Since tIn conclusion, inhibiting DNMT ameliorated renal fibrosis in obstructed kidneys. Our findings suggest that inhibiting DNA methylation restrains unnecessarily intensified pro-inflammatory pathways, restores disrupted anti-oxidative defense mechanisms, and alleviates EMT and apoptosis, thus ameliorating renal fibrosis. Hence, DNMT may be a valid therapeutic target to protect against renal fibrosis in non-diabetic CKD.All animal experiments were performed following the relevant guidelines and regulations as described in the previous report . BrieflyHistology and immunohistochemistry experiments were performed following the previous reports ,51. Brie2O2 (0.01%) for the color reactions. After counterstaining with haematoxylin, more than 6 fields per kidney tissue, with total 40 fields per experimental group, were randomly selected and photographed manually. Stain-positive cells were detected and quantitated by ImageJ software 1.49 . To assess apoptosis, the TUNEL assay was performed according to the manufacturer’s protocol  and a previous report [We conducted immunohistochemical staining for DNMT1  to investigate the effect of zebularine on DNMT1 expression. To determine the number of myofibroblasts or macrophages, sections were incubated with anti-α-SMA or anti-F/80 antibodies , respectively. After washing in PBS, all sections were incubated for 60 min with peroxidase-conjugated anti-mouse IgG  as a secondary antibody, and then reacted with a mixture of 3,3′-diaminobenzidine (0.05%)-containing Hs report . TUNEL-p2O2 (0.01%) for the color reaction. After counterstaining with haematoxylin, the section was photographed  as a secondary antibody, and then reacted with a mixture of 3,3′-diaminobenzidine (0.05%)-containing Hographed .The total collagen content of the kidney tissue was measured using the acid hydrolysis method following previous reports ,52. BrieThe total proteins in kidney tissues were extracted using the PRO-PREP Protein Extraction Kit , and nuclear proteins in kidney tissues were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit  according to the manufacturer’s instructions. Protein concentrations were determined using a protein assay kit . After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with primary antibodies against the following proteins: collagen IV, catalase (Abcam), TGF-β1, NF-κB, NQO1, nuclear Nrf2, Bcl-2, Bax, DNMT1, DNMT 3b , Nox2 , HO-1 (Thermo Fisher Scientific), SOD1 , β-actin , and Lamin B1 . After a wash, the blots were incubated with a secondary antibody conjugated with horseradish peroxidase. The protein bands were detected with enhanced chemiluminescence reagents and imaged using an Image Quant LAS 4000 . Their intensities were measured by Quantity One 1-D analysis software (Bio-Rad Laboratories), and normalized by β-actin or Lamin B1 in the same sample.The total RNA was isolated from kidney tissues using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s manual. The reverse transcriptase reaction was performed to synthesize cDNA, and qRT-PCR assays were performed using SYBR Premix . Primer sequences for each gene are listed in the All values in this study are represented as the mean ± standard error of the mean (SEM). Differences between groups were determined using one-way analysis of variance with Bonferroni correction.


We measured CSF concentrations of TDP-43 in 194 urban residents, including 92 MMC children aged 10.2 ± 4.7 y exposed to PM2.5 levels above the USEPA annual standard and to high UFPM and 26 low pollution controls (11.5 ± 4.4 y); 43 MMC adults (42.3 ± 15.9 y) and 14 low pollution adult controls (33.1 ± 12.0 y); and 19 amyotrophic lateral sclerosis (ALS) patients (52.4 ± 14.1 y). TDP-43 neuropathology and cisternal CSF data from 20 subjects—15 MMC (41.1 ± 18.9 y) and 5 low pollution controls (46 ± 16.01 y)—were included. CSF TDP-43 exponentially increased with age (p < 0.0001) and it was higher for MMC residents. TDP-43 cisternal CSF levels of 572 ± 208 pg/mL in 6/15 MMC autopsy cases forecasted TDP-43 in the olfactory bulb, medulla and pons, reticular formation and motor nuclei neurons. A 16 y old with TDP-43 cisternal levels of 1030 pg/mL exhibited TDP-43 pathology and all 15 MMC autopsy cases exhibited AD and PD hallmarks. Overlapping TDP-43, AD and PD pathologies start in childhood in urbanites with high exposures to PM2.5 and UFPM. Early, sustained exposures to PM air pollution represent a high risk for developing brains and MMC UFPM emissions sources ought to be clearly identified, regulated, monitored and controlled. Prevention of deadly neurologic diseases associated with air pollution ought to be a public health priority and preventive medicine is key.Environmental exposures to fine particulate matter (PM Anthropogenic UFPM are primarily generated via combustion  and subsequent processes of particle nucleation, coagulation and vapor condensation, while NPs are frequently referred to as engineered or manufactured because they are designed and generated for a particular purpose; e.g., medical [Neurodegenerative disorders with complex environmental and genetic pathogenesis start decades before clinical symptomatology is present ,2,3. Quaase (PD) ,9,10,11.ase (PD) ,13,14,15ase (PD) ,21,22,23 medical . Anthrop medical ,27,28,29p = 0.005 and 0.02, respectively) and the fact that non-P-Tau cases showed significantly faster increases in MMC residents versus controls (p = 0.005) [We have previously shown that cerebrospinal fluid (CSF) concentrations of cytokines and chemokines, cellular prion protein (PrPc), total tau (T-tau), tau phosphorylated at threonine 181 (P-Tau), amyloid Aβ1–42, α-synuclein (t-α-syn and d-α-synuclein), brain-derived neurotrophic factor (BDNF), insulin and leptin can be used to distinguish MMC children from low pollution controls ,31,32,33The overlap of several aberrant CSF proteins in children and young MMC adults, including hyperphosphorylated tau, beta amyloid, alpha synuclein and TDP 43 ,2,4, drononlinearly increasing protein concentrations between ventricular and lumbar CSF are fitting to a Gaussian error function, the differential of the nonlinear concentration distribution function between blood and CSF” [Two research groups were critical in defining the relationship and the interpretation of protein concentrations in cisternal versus lumbar CSF; the work by Reiber ,37 and band CSF”  and thatand CSF” .TDP-43 nuclear depletion and aggregation are hallmarks of ALS and frontotemporal dementia (FTD) ,40,41,42In sum, the purpose of the present study was to assess, in MMC versus lower pollution age-matched subjects, the impact of lifetime exposures to highly polluted environments on CSF TDP-43 burden. Our results identify lumbar CSF TDP-43 increasing exponentially with age in young urbanites with high exposures to PM pollution and TDP-43 cisternal CSF levels of 572 ± 208 pg/mL forecasting TDP-43 pathology in young MMC residents.There is a strong association between TAR DNA-binding protein 43, frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) ,51,52; t2.5) and O3 for the last three decades [2.5 concentrations, averaged over 3 years, for representative MMC monitoring stations in the period between 1990 to April 2020 and their comparison with PM2.5 from the US EPA NAAQS.MMC residents have been chronically exposed to significant concentrations of fine particulate matter  [−3. Given that catalytic converters in cars and unleaded gasoline were not enforced in Mexico until the year 2000, MMC CO and PM2.5 levels before 2000 were among the highest levels of criteria pollutants registered in North America [−3 and, given that the PNC trend decreased to the overall average of 44,000 cm−3 after 2003, exposures of MMC residents reached the average PNC for 40 urban areas across Asia, North America, Europe and Australia precisely after 2003 [UFPM and NPs have a very low mass compared with larger diameter particles; therefore, the PM fraction <100 nm in MMC was evaluated through measurements of particle number concentration (PNC) ,61,62,63issions) . The est America . MMC rester 2003 —commerciter 2003 —residentter 2003 —commerci2, and it lies in an elevated basin 7400 feet above sea level. MMC has nearly 22 million inhabitants, over 50,000 industries and over 5 million vehicles consuming more than 50 million liters of petroleum fuels per day. In this megacity, MMC motor vehicles release abundant amounts of primary PM2.5, elemental carbon, particle-bound polycyclic aromatic hydrocarbons, carbon monoxide, nitrogen oxides and a wide range of air toxins and other toxics, including formaldehyde, acetaldehyde, benzene, toluene and xylenes [0.1 emitters [MMC is an example of uncontrolled urban growth and environmental pollution ,62,63,64 xylenes ,58,59,64 xylenes . In addiemitters .3−) and particles with lipopolysaccharides (PM-LPS), while northern children are exposed to higher concentrations of volatile organic compounds (VOCs) and PM2.5 and its constituents: organic and elemental carbon, including polycyclic aromatic hydrocarbons, secondary inorganic aerosols  and metals  [2.5 [In this study, children and adult cohorts included residents in MMC, while subjects from small control cities represented the typical provincial areas where air pollution emissions are minimal. MMC northern industrialized and southern residential zones were represented in our samples. Southern MMC children are exposed to significant concentrations of ozone, secondary tracers (NOanadium) ,56,62,64anadium) ,64. ThesThis prospective study was approved by the review boards and ethics committees at the Universidad del Valle de Mexico, on the 18 November 2020, and the University of Montana, IRB-206R-09, on the 8 December 2016, and IRB 185-20, on the 23 November 2020.Normal CSF samples were selected from two cohorts admitted for investigation of neurological involvement in the context of a hematological, neoplastic or infectious brain processes: (i) children admitted to the hospital from a clean air city or MMC, with a work-up diagnosis of acute lymphoblastic leukemia (ALL) entering a clinical protocol that included a spinal tap, and (ii) young and older adults admitted to the hospital for a work-up that required a spinal tap with varied diagnoses, including potential neoplastic and infectious CNS involvement. The cohort of young and older adults also included permanent residents in clean environments and lifetime residents in highly polluted MMC. The normal CSF samples studied were destined to be destroyed after the diagnosis of normal CSF in the laboratory. We also examined 19 CSF ALS cases from Dartmouth-Hitchcock Medical Center Cerebral Spinal Fluid Bank for research IRB #29104 and 5 Mexican ALS cases under the Universidad del Valle de Mexico 18 November 2020 IRB.The autopsy cases with cerebello-medullary cisternal tap included 15 MMC subjects and 5 low pollution control cases; all had complete forensic autopsies, including full neuropathological examination. These autopsies were performed between 2004 and 2008 under a Consejo de la Judicatura del Distrito Federal permit (14571/2003).3. All CSF samples in this study were read as normal. We performed the hTDP43 total ELISA  according to the instructions for use. This ELISA uses two monoclonal antibodies directed at amino acids 79–91 and 259–271.Spinal tap was performed on the left lateral decubitus from the intervertebral spaces L3-S1 using a standard 22 spinal needle. Spinal taps were performed between 8 and 10 am. CSF was collected dripping in free air in 1 mL aliquot into Nalge Nunc polypropylene CryoTubes. Lumbar puncture samples were collected during non-traumatic, non-complicated procedures. CSF samples were examined immediately to determine haematological, oncological or infectious involvement and then stored at −80 °C and kept frozen until the current analysis. CSF pleocytosis was defined as CSF white blood cell (WBC) counts of ≥7 cells per mmThis work includes CSF data from a pediatric cohort: 92 children from MMC  and 26 low pollution control children . The selected children had no previous oncologic and/or hematologic CNS or systemic treatments and their CSF samples were read as normal, with CNS involvement being ruled out at the time of their hospitalization. Children’s clinical inclusion criteria were negative known smoking history and environmental tobacco exposure, lifelong residency in MMC or the control city, residency within a radius of 2.5 miles of one of the representative city monitoring stations, full-term birth and unremarkable clinical histories prior to their admission to the hospital. We specifically excluded children with a history of active participation in team sports with high incidences of head trauma, including soccer. Mothers had unremarkable, full-term pregnancies with uncomplicated vaginal deliveries and took no illicit drugs, including alcohol and tobacco. These children had a history of breast feeding for a minimum of 6 months and were introduced to solid foods after the age of 4 months. Participants were from middle class families, living in single-family homes with no indoor pets, used LP gas for cooking and kitchens were separated from the living and sleeping areas. Low and high pollution-exposed children were matched by age, gender and socioeconomic status.We had 19 ALS cases , of which 14 were from NH and 5 from MMC; 43 normal MMC CSF samples ; and 14 samples from residents in low pollution cities (9M/5F average age 33.1 ± 12.0 y).SD = 17.9, age range 16 to 83 y) were examined: 15 MMC male residents, average age 41.13 ± 18.9 y, and 5 controls . The inclusion criteria for this cohort included lifelong residency in MMC or the control cities and a cause of death—i.e., accidents, homicides and suicides—that did not involve the brain directly. Immunohistochemistry was performed using the TDP-43 mab2G10 . This antibody is a recombinant human TDP-43 directed to the amino acids MTEDELREFFSQYGDVM of the TDP43 protein and able to identify prominent physiological nuclear immunostaining and neuronal pathological cytoplasmic immunoreactivity in neurons and glial cells in TDP-43 proteinopathy cases.This work included forensic cases from MMC and low pollution controls. A total of 20 subjects  . The logarithm of TDP-43 increased significantly with age (p < 0.0001) and it was higher for MMC residents (p < 0.0001). Excluding ALS cases, a log-transformed linear regression of TDP-43 concentration relative to age and residency showed that CSF TDP-43 increases significantly with age (p = 0.0032) (Mean CSF TDP-43 concentration was significantly higher in ALS versus MMC and non-MMC controls ( 0.0032) .The average age of the MMC adult cases was 41.1 ± 18.9 years and that of the controls was 46 ± 16 y. A 16-year-old MMC boy was also studied. Remarkably, concentrations of TDP-43 in cisternal CSF with an average of 572 ± 208 pg/mL forecasted cortical and subcortical TDP-43 pathology, while averages of 232 ± 130 pg/mL were associated with negative TDP-43 brain pathology in MMC residents and controls.Representative immunohistochemistry photographs are shown in The pathology was mostly seen in brainstem levels, including the medulla and pons, substantia nigrae and locus coeruleus, and in relation to the mesencephalic reticular formation, ventral tegmental area-parabrachial pigmented nucleus complex and caudal-rostral linear nucleus of the raphe .The cortical and hippocampal pathology was characterized by loss of nuclear TDP-43 expression and/or dash-like IR cytoplasmic neurons. The 24 and 25 y old subjects showed negative IR nuclei in frontal and temporal neurons, cytoplasmic IR and oligodendroglia tangles A,B, whil2.5 above current EPA US standards and highly toxic UFPM have CSF TDP-43 concentrations exponentially increasing with age. TDP-43 is the target protein associated with ALS and fronto-temporal dementia (FTD) [Intracellular accumulation of abnormal misfolded proteins is the critical neuropathological feature of ALS, FTD, Alzheimer’s and Parkinson’s diseases and exposure to particulate matter air pollution has been described as a strong risk factor for Metropolitan Mexico City (MMC) young residents ,2,3,4. Mia (FTD) ,66,67,68ia (FTD) ,2, and nia (FTD) ,69, Hasaia (FTD) ,69.The global connectome architecture of FTD subjects is a relevant issue in early TDP-43 pathology, as it allowed researchers such as Shafiei et al.  to identOur TDP-43 CSF findings and the previously documented quadruple brain pathology ,2,3,4 arThe abnormal CSF proteins found in MMC children and young adults are very concerning because they are involved in several pathophysiological mechanisms of early neural damage with eventual impacts on cognitive, neurological and psychiatric outcomes ,75,76.At the core of our results, we report accumulation of TDP-43 starting at an early age in individuals highly exposed to complex environmental emissions, including UFPM ,2,3,4. TNon-fibrillary TDP-43 accumulates in the rough endoplasmic reticulum (RER), as described by Kon and colleagues , and theNeuropathological information in conjunction with the social brain hypothesis  and the The involvement of TDP-43 in the complex reticular formation in MMC residents must be pursued further, given the complexity of the brainstem reticular nuclei and their connections with supra- and infra-cortical structures involving major integration and relay centers coordinating survival functions and key connections between the cerebral cortex, the cerebellum and the spinal cord ,94,95,96Research regarding TDP-43, including novel nuclear pore articles, muscle TDP-43 involvement and the anatomical targets ,110,111,There is an urgent need for noninvasive, reliable fluid biomarkers to diagnose early TDP-43 pathology to improve the differential diagnosis with overlapping neurodegenerative diseases that undoubtedly exhibit an extreme variability in clinical phenotypes, as described by Virgilio et al., for tau . Since Tp < 0.0001), with NfL values higher for all phenotypes of symptomatic FTD-ALS spectrum (FAS) patients compared to primary psychiatric disorders (PPD) [The work of researchers exploring correlations between fluid biomarkers is highly relevant ,121,122.Since, our MMC cases have AD hallmarks, and given that cognition, gait and balance; auditory-evoked brainstem potentials; and brain MRI changes are striking in the young MMC residents ,124,125—−3 [There were advantages and limitations to this study. A major advantage was the multidisciplinary collaborators and the efforts made to exchange viewpoints regarding the importance of early clinical detection of young individuals at risk, key in understanding the impact of our findings in future multidisciplinary studies. The current particle number concentration (PNC) in MMC is crucial environmental information for the responsible authorities. Residents continue to be exposed to an average of 44,000 particles per cm−3 , with alParticulate matter exposures—specifically, UFPM/NP sustained exposures from utero—are likely key in aberrant neural protein pathology. The CSF TDP-43 results identify logarithmic increases related to age across young megacity urbanites, crucial information in view of the 18% TDP-43 pathology reported in 202 forensic MMC autopsies aged 27.29 ± 11.8 y and the overlap of aberrant hyperphosphorylated tau, beta amyloid, α synuclein and TDP-43. These data are striking in view of the work of Karanth et al.  and CarlWe identified a significant relationship between cisternal CSF TDP-43, an average of 572 ± 208 pg/mL, and TDP-43 brain pathology. This is particularly serious for highly exposed children, as with the 16 y old boy (1013 pg/mL) who had extensive nonmotor and motor TDP43 pathology. As toxicologists working with forensic colleagues, we suggest that forensic cases are very helpful to explore the extent of brain pathology in a population at large, and taking a cisternal sample is simple and quick.Defining early markers of the quadruple aberrant neurodegenerative diseases, including TDP-43 pathology, ought to be the core of our future efforts. MMC residents are showing early clinical symptomatology—including gait and balance alterations, cognitive deficits and MRI volumetric cortical and subcortical abnormalities, all of which may help identify young subjects at higher risk.Exposed children and young adults in highly polluted areas need early neuroprotection and multidisciplinary prevention efforts. Control of combustion and friction UFPM sources and engineered NPs  is becoming increasingly important and urgent to diminish the human and economic costs of a global neurodegenerative epidemic.UFPM/NP exposure should be included in any assessment of the neurodegenerative risk profile of exposed individuals. No matter the portal of entry, chronic delivery of exogenous particles to the brain induces oxidative stress and neuroinflammation.We have described the overlap of multiple neurodegenerative pathologies; the presence of anthropogenic UFPM in fetal brains; and the early development of AD, PD and TDP-43 pathology, along with their progression and their neuropsychiatric consequences: this body of knowledge resulting from multidisciplinary studies cannot be disregarded by those concerned with public health.We urgently need to practice preventive medicine and develop tools to identify children at risk in order to implement neuroprotective strategies. As neurotoxicologists, we ought to define the mechanistic pathways involving complex NPs containing metalloids, metals, organic compounds, plastics, etc., that can cause extensive brain pathology. As physicians, our focus should be protecting the brains of our future citizens and our younger generations, identifying neurotoxic emission sources and being active players in multidisciplinary teams to prevent, ameliorate or halt neurodegenerative diseases.


The majority of breast cancers express the estrogen receptor (ER) and for this group of patients, endocrine therapy is the cornerstone of systemic treatment. However, drug resistance is common and a focus for breast cancer preclinical and clinical research. Over the past 2 decades, the PI3K/Akt/mTOR axis has emerged as an important driver of treatment failure, and inhibitors of mTOR and PI3K are now licensed for the treatment of women with advanced ER-positive breast cancer who have relapsed on first-line hormonal therapy. This review presents the preclinical and clinical data that led to this new treatment paradigm and discusses future directions. A number of endocrine resistance mechanisms have been evaluated in the laboratory and clinic but to date only targeting the cyclin-dependent kinases 4 and 6 (CDK4/6) and PI3K/Akt/mTOR signaling axes has successfully translated to licensed drugs. In this review, we describe the multiple agents that target the PI3K/Akt/mTOR pathway which has and is being evaluated in the clinic for the treatment of breast cancer but with variable success. In particular, the selection of patients and drug toxicity have both proved to be challenged in the clinical development of this treatment class.Endocrine therapy (ET) remains a key systemic treatment for both early and advanced breast cancer. Over the past 40 years, 3 main classes of ET have demonstrated clinical benefit and been licensed by the Food and Drug Administration (FDA) for the treatment of breast cancer; selective estrogen receptor modulators ; selective estrogen receptor degraders ; and aromatase inhibitors  , 2. Howe2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3), naming this enzyme PI3K   . Overallc marker , 56. Murpression , 58. Furpression . PTEN muce to ET , 61.+ breast cancer, the rationale for targeting the PI3K/Akt/mTOR pathway is to overcome resistance to ET by reducing proliferation driven by ligand-dependent activation and inhibiting ligand-independent activation of the ER. Both pan-PI3K inhibitors target all four PI3K class I isoforms and isoform-specific PI3K inhibitors have been developed [PIK3CA-mutant xenograft model [PIK3CA-mutated and murine HER2 amplified PIK3CA-mutated breast cancer models, respectively [In vitro, fulvestrant sensitized ER+ breast cancer cells to PI3K inhibition and subsequently induced apoptosis [In the context of EReveloped . Buparlift model . Similarectively , 65. In poptosis .in vitro activity against both thyroid and breast cancer cell lines with PI3KCA mutations [PTEN mutated xenograft model, MK2206 inhibited tumor growth [PIK3CA-mutated breast cancer [PTEN/PIK3CA-mutant MCF breast cancer cells [The potent, allosteric Akt inhibitor, MK2206, has demonstrated utations –71 and, r growth . Capivast cancer . High Aker cells .+ xenograft model, there was a synergistic interaction between another IGF-1R/IR inhibitor (OSI-906) and fulvestrant [In a screen of breast cancer cell lines, PTEN, Akt, and phosphorylated ribosomal S6 kinase 1 (pS6K1) levels were associated with sensitivity to the mTOR inhibitor, rapamycin . Insulinvestrant .In summary, the ER has complex interactions with the IGF/PI3K/Akt/mTOR signaling cascade and substantial preclinical data have demonstrated the potential for synergistic co-targeting of these pathways.+ breast cancer when used in combination with ET [Several drugs targeting the PI3K/Akt/mTOR pathway have received regulatory approval in the treatment of a number of solid and hematological malignancies. Two of these agents, the mTOR inhibitor, everolimus, and α-specific PI3K inhibitor, alpelisib, are now established and licensed drugs for the treatment of advanced ER with ET –85. See + HER2-negative (HER2−) breast cancer, pancreatic neuroendocrine tumors, and astrocytomas [Since rapamycin (sirolimus) and the first generation of mTOR inhibitors were used in clinical practice, a wide range of agents have been developed, demonstrating more potent specificity . Three gocytomas –124.vs. either everolimus or capecitabine alone. The study was powered to provide estimates of treatment effect, but no formal statistical analysis was preplanned. After a median follow-up of 37.6 months, PFS for the combination arm was estimated at 8.4 months vs. 6.8 months for exemestane monotherapy. Capecitabine outperformed everolimus plus exemestane, although the authors noted that this might be due to imbalances in the baseline characteristics [The combination of mTOR inhibition with ET has been extensively studied in the metastatic breast cancer setting. Following the positive results of the BOLERO-2 trial, everolimus was the first mTOR inhibitor to be approved in the treatment of breast cancer, when used in combination with exemestane . This mueristics . Furthereristics . The comeristics , 87.+ breast cancer, comparing fulvestrant plus everolimus vs. fulvestrant plus placebo. PrE0102, a phase II randomized trial, reported a doubling of the PFS from 5.1 months to 10.3 months in favor of the combination. The objective response and CBRs showed a similar trend, whilst relatively few grade 3 and 4 AEs were observed [Everolimus was further investigated in combination with tamoxifen, in the TAMRAD study, a phase II trial of metastatic, breast cancer resistant to AI therapy . At 6 moobserved .vs. letrozole plus placebo, for postmenopausal patients with advanced ER+ breast cancer [P = 0.003 for interaction). The authors concluded that the lack of PFS benefit could have been attributed to the difference in AI exposure between the populations recruited in HORIZON and BOLERO-2 trials, as well as the suspected alterations developed in endocrine-resistant tumors. In addition, the different drug formulations and dosing schedules between everolimus and temsirolimus, might have contributed to the contrasting findings [In comparison, results from a phase III study of temsirolimus were disappointing. HORIZON compared first-line letrozole plus temsirolimus t cancer . The trifindings .+ breast cancer that had previously progressed on everolimus with either exemestane or fulvestrant were recruited to receive sapanisertib in combination with exemestane or fulvestrant [vs. 2%, respectively. Only a few patients exhibited dose-limiting toxicities, with nausea, diarrhea, fatigue, and hyperglycemia the most common AEs. Vistusertib, another dual mTORC1/2 inhibitor, has also been investigated in combination with fulvestrant in phase II randomized trial that recruited 333 post-menopausal women that had progressed on an AI [Sapanisertib is a potent ATP-competitive inhibitor of mTORC1 and 2 and has been investigated in a non-randomized phase Ib/II study in which a total of 118 patients with metastatic, ERvestrant . CBR wason an AI . The MAN+ breast cancer was investigated in two large, randomized, double-blind, multicentre phase III trials, BELLE-2 and BELLE-3. In the BELLE-2 trial, 1,147 postmenopausal women with advanced, AI-resistant breast cancer were randomized to receive daily doses of either buparlisib or placebo with monthly intramuscular fulvestrant, in 28-day cycles. The study met its primary endpoint, with a modest PFS benefit reported at 6.9 months for buparlisib vs. 5 months for the control arm. However, the authors concluded that in view of the increased toxicities observed in the buparlisib group, including liver toxicity, rash, and hyperglycemia, no further study of this combination should be pursued [vs. 1.8 months). However, the toxicity profile of buparlisib proved unacceptable and two treatment-related deaths were attributed to the combination [Buparlisib is an oral, potent, pan-PI3K inhibitor that has been extensively studied in both solid cancers and haematologic malignancies . Its act pursued . Resultsbination .PIK3CA mutation status and previous exposure to AI, leading to primary or secondary resistance. However, no difference in PFS was achieved in any of the subgroups treated with the combination, and tolerability was challenging [Pictilisib, a pan-PI3K inhibitor with higher affinity for the α and δ isoforms, was studied in combination with fulvestrant in the FERGI trial a randomized, placebo-controlled phase II trial that recruited patients with metastatic breast cancer resistant to ET. Patients were stratified according to llenging . Pilaralllenging .PIK3CA mutations was initially established in several early phase studies, testing the combination of alpelisib with ET in patients with ER+ breast cancer [PIK3CA-mutant, ER+ advanced breast cancer, and prior exposure to ET [PIK3CA mutations showed a significantly improved PFS of 11 months in the fulvestrant/alpelisib group vs. 5.7 months in the fulvestrant/placebo group meeting its primary endpoint. The most common grade 3/4 toxicities involved hyperglycemia, rash, and diarrhea, which attributed to an increased treatment discontinuation rate in patients receiving the combination. Final overall survival results were recently published, showing a difference of 7.9 months in favor of alpelisib, but failing to reach statistical significance. Nonetheless, a stronger treatment effect with borderline significance was observed in patients with lung and/or liver disease for alpelisib [The need for improved efficacy and tolerability has led to a shift in interest toward isoform-specific PI3K-inhibitors. Alpelisib selectively inhibits the PI3Kα isoform and its effectiveness against tumors harboring t cancer , 100. Thre to ET . In thislpelisib .PIK3CA mutations pretreated with a CDK4/6i, was recently published showing 50.4% of patients were without progression or death at 6 months. Although there was no control arm, these data provide some support for the use of alpelisib/fulvestrant as a therapeutic option in patients with PIK3CA-mutated disease following progression on first-line CDK4/6i [+ breast cancer, with an acceptable toxicity profile [vs. letrozole alone showed no improvement in response in the neoadjuvant setting [Although prior treatment with CDK4/6 inhibitor (CDK4/6i) was one of the stratification factors in SOLAR-1, only 20 patients were identified in this subgroup, with the HR for progression or death not reaching statistical significance . However, the BYlieve study, a phase II cohort study of 127 patients with  CDK4/6i . In addi profile . Another profile . Poor to setting , 132.+ breast cancer with encouraging activity [PIK3CA-mutant breast cancer to fulvestrant/taselisib vs. fulvestrant/placebo and met its primary endpoints of a statistically significant improvement in PFS (7.4 months vs. 5.4 months in favor of taselisib). However, given the high rate of serious AEs in the taselisib arm and the modest clinical benefit, taselisib has not become an established therapeutic option [vs. placebo/letrozole has also been explored in the neoadjuvant setting, in the randomized phase II LORELEI trial with a higher ORR observed for the taselisib combination [Lastly, the effectiveness of taselisib, a potent PI3Kα-inhibitor, was assessed in a single-arm, phase II study, in combination with fulvestrant in patients with advanced ERactivity . Furtherc option . The combination .vs. placebo in 140 women with ER+ metastatic breast cancer resistant to an AI. PFS was significantly prolonged in the combination arm (10.3 months vs. 4.8 months in favor of capivasertib) and frequent grade 3/4 AEs in the capivasertib arm were hypertension, diarrhea, rash, and infection [PIK3CA-mutant disease but clinical activity was not encouraging and toxicity significant [In the clinic the most extensively studied Akt inhibitors are capivasertib and ipatasertib, both ATP-selective pan-Akt inhibitors with activity against Akt 1, 2, and 3. The FAKTION randomized phase II study examined the addition of capivasertib to fulvestrant nfection . An ongonfection . Ipatasenfection . The allnificant .Several clinical trials assessing agents that target IGF-1 signaling in combination with ET are ongoing in the setting of both endocrine-sensitive and resistant diseases , 135.+ breast cancer. The triplet regimen was administered to 24 post-menopausal women with the endocrine-resistant disease in phase Ib of the study and was well-tolerated, with disease control observed in 57% of patients, and PRs reported in 19% [+ breast cancer is yet to report [Xentuzumab is a humanized, IGF-1 and IGF-2 neutralizing antibody that has been tested in combination with everolimus and exemestane in a phase Ib/II trial of advanced, ERd in 19% . In a phd in 19% . XENERA-d in 19% . A phaseo report .A different approach involves the employment of monoclonal antibodies directed toward the IGF-1R. Three agents, ganitumab, cixutumumab, and dalotuzumab have been tested in phase II trials in combination with mTOR inhibitors and ET, with no supportive evidence of their treatment benefit to date –141. In In summary, the mTOR inhibitor, everolimus, and PI3Kα inhibitor, alpelisib, are now licensed therapies that have been shown to improve treatment outcomes for metastatic breast cancer when used in combination with hormonal therapy. Other avenues of investigation are ongoing, in particular combining either Akt inhibitors or agents targeting IGF-1 signaling with hormonal therapy.+ breast cancer has met with significant challenges. PI3K/Akt/mTOR activation is not ubiquitous in ER+ breast cancer and can happen at several different nodes in the pathway as described above. PI3K/Akt/mTOR pathway blockade is associated with a variety of significant toxicities, including hyperglycemia, which, as described below, may be a mechanism of resistance. Additionally, multiple other resistance mechanisms exist, both intrinsic to the PI3K/Akt/mTOR pathway and in related pathways. Identification of suitable predictive biomarkers is therefore paramount for providing a personalized approach to treatment and to amplifying the chances of demonstrating efficacy in late-phase clinical trials.The clinical development of therapeutics that target the PI3K/Akt/mTOR pathway in ER+ breast cancer [+/HER2 positive (HER2+) and ER+ basal-like subtypes appeared to fare worse in retrospective analyses [AKT1E17K mutations may predict longer PFS, although this needs prospective investigation [The utility of predictive biomarkers is, in part, dependent on the target of interest within the PI3K/Akt/mTOR pathway. mTOR inhibitors were the first treatment class to be licensed and, as described above, everolimus has shown clinical benefit when given in combination with exemestane for metastatic ERt cancer . Howeveranalyses , 143–145tigation .PIK3CA, the gene encoding the PI3Kα isoform p110α catalytic subunit, as an early potential predictive biomarker for PI3K inhibitors [+ breast cancer has yielded negative trial results [PIK3CA mutations for the pan-class I PI3K inhibitor, buparlisib, in metastatic ER+ breast cancer [PIK3CA as a predictive biomarker for this therapeutic class has been inconsistent, both in approach and results. The pan-PI3K inhibitor trials used varied biomarkers including assessing PIK3CA hotspot mutations within exons 1, 4, 7, 9, and 20, specific point mutations within these exons such as PIK3CAE545K, or composite “PI3K pathway activation” that included either PIK3CA mutation or PTEN loss of function. Additionally, the PIK3CA mutational analysis included both tumor biopsy samples and circulating tumor DNA (ctDNA), further complicating matters.Preclinical work identified mutations within hibitors , 147. Mu results , 149. Tht cancer , 96. Ass+ breast cancer, with improved efficacy and toxicity profiles [PIK3CA mutations, detected either using ctDNA or in tumor tissue, were predictive of improved PFS for alpelisib plus fulvestrant [The development of isoform-specific inhibitors followed the early failures of pan-PI3K inhibitors in ERprofiles , 108. Asvestrant .+ breast cancer were less clear. Although PIK3CA mutations were predictive for a statistically significant improvement in PFS, the HR in the PIK3CA wild-type group was essentially the same as the mutant group, albeit lacking statistical significance [PIK3CA mutations was independent of both site and type of mutation. Despite their predictive value in metastatic disease, PIK3CA mutations were not predictive in neoadjuvant trials of either alpelisib or taselisib [Results for taselisib plus fulvestrant in the SANDPIPER-3 trial in metastatic ERificance . In bothaselisib , 110.PIK3CA, have also been investigated for PI3K inhibitors. Preclinical evidence suggests that PTEN loss should sensitize cells to PI3K/Akt inhibition, although this has not been demonstrated unambiguously in the clinic for either pan-PI3K or PI3Kα inhibitors [Potential predictive biomarkers, other than hibitors , 150. Alhibitors . LuminalPIK3CA, PIK3R1, Akt, or mutation/loss of PTEN could predict treatment response for Akt inhibitors [in vitro and in vivo, PIK3CA and PTEN alterations have not been predictive of treatment response in clinical trials of these agents [+ breast cancer and AKT1E17K mutations [Preclinical evidence suggests mutations in hibitors –154. Howe agents , 155–157utations . There autations , 159.Understanding primary or secondary resistance to targeted therapies can inform the patient selection and novel combinations. Resistance mechanisms may be intrinsic to the pathway or arise through extrinsic, related signaling. For example, mTOR’s auto-regulatory feedback loop attenuates its own response, with inhibition of mTORC1 leading to increased activity of PI3K . For PI3KRAS) mutations associates with PIK3CA mutations in cancer. KRAS mutations appear to confer resistance to both alpelisib and ipatasertib in early phase trials [BRAF)-mutant colorectal cancer, evidence for ER+ breast cancer is lacking [The EGFR/Ras/Raf/MAPK kinase (MEK)/ERK and PI3K/Akt/mTOR signaling cascades co-regulate many downstream effectors in parallel. The presence of Kirsten RAS 2 viral oncogene homolog  inhibitors, and a ketogenic diet were all capable of restoring inhibition of PI3K/Akt/mTOR signaling. Exogenous insulin nullified the benefit of a ketogenic diet in these mice, suggesting insulin should be avoided in managing this toxicity. To date, in the clinic, standard practice for managing hyperglycemia has been to use metformin [Hyperglycaemia is a common on-target toxicity of PI3K inhibitors. PI3K is a necessary downstream effector of the IR and hence inhibition leads to insulin resistance . The resetformin , 169. Hoetformin . A case etformin . Of noteetformin , 172. Se+ breast cancer showed that FMD could induce cancer cell regression, both in vitro and in vivo [Calorie restriction and fasting have long been recognized as limiting tumor growth in animal studies  and it h in vivo . Subsequ in vivo . FMD alo in vivo . HoweverIn summary, multiple mechanisms of resistance and several putative biomarkers have been identified for PI3K and mTOR inhibitors in breast cancer, mostly focusing on specific genetic alterations. However, new data suggests that the feedback hyperglycemia from targeting this pathway may also play a key role in resistance to treatment and hence therapeutic opportunity.+ breast cancer continue to emerge, there may be further opportunities for efficacious novel combinations with mTOR and PI3K inhibitors in appropriately selected populations. To date, published data from clinical trials have used fulvestrant, AI, or tamoxifen as the ET backbone to mTOR, Akt, or PI3K inhibition. Fulvestrant is the only SERD that is licensed for clinical practice but novel, oral SERDs are now in clinical development with improved pharmacokinetics and bioavailability [vs. fulvestrant or AI in patients who progressed on CDK4/6i  [As new drugs for the treatment of ERlability . Four agD trial) . Early pD trial) .+ breast cancer, albeit acquired resistance to CDK4/6i still almost inevitably develops [PIK3CA-mutant disease, suggested augmented anti-tumor activity with the triplet regimen. Confirmed PR and CBR were measured at 36% and 76%, respectively, for the inavolisib combination, while hyperglycemia, stomatitis, gastrointestinal, and hematological toxicities were among the most common AEs. Further insight into the potential of this triplet combination is expected to be provided by a phase III trial that is currently recruiting (NCT04191499) [Co-targeting of the ER and CDK4/6 has become the standard of care for patients with advanced, ERdevelops . Preclindevelops . Prelimidevelops . Similar4191499) .PIK3CA-mutant, ER+ breast cancer in the single-arm PIPA trial. The authors concluded that a response rate of 37.5% was promising for superiority to the palbociclib/fulvestrant doublet and warrants further investigation [Taselisib has also been tested as a triplet therapy along palbociclib and fulvestrant in tigation . The Akttigation . This pltigation . Finallytigation . The fav+ breast cancer also include further combinations with IGF-targeted agents, antagonists of the androgen receptor (AR), FGFR inhibitors, and checkpoint immunotherapy. As outlined above, data from clinical studies of xentuzumab and other anti-IGF-1R antibodies have, however, been disappointing to date. The role of AR expression in breast cancer and the efficacy of its inhibition has been better understood and established in the ER-negative disease [PIK3CA mutations and have suggested prognostic value in breast cancer, irrespective of the hormonal status [+) breast cancer to PI3K pathway inhibition and its potential as a predictive biomarker has provided a rationale for the combined use of anti-androgens with drugs targeting PI3K/Akt/mTOR [+ with PTEN loss (NCT03207529) [In the clinic, prospective future directions for targeting PI3K/Akt/mTOR in ER disease . Preclinl status , 186. FuAkt/mTOR , 188. An3207529) .PIK3CA-mutant solid cancers. Unfortunately, at this point results were disappointing without indication of significant clinical activity [Another approach takes advantage of the interplay between the fibroblast growth factor (FGF)/FGFR and PI3K/Akt/mTOR pathways, as FGFR overexpression leads to activation of PI3K/Akt signaling . Aberranactivity .In summary, targeting the PI3K/Akt/mTOR pathway to subvert resistance to ET in breast cancer is now proven to be of clinical benefit with everolimus and alpelisib already used routinely in clinical practice. A new generation of therapeutics awaits full evaluation in this setting, including drugs targeting Akt and IGF-1 signaling. However, challenges remain, in particular, the need for the development of improved biomarkers for patient selection and clinical evaluation of strategies to abrogate mechanisms resistance, such as feedback hyperglycemia.


The aim of this multicentre international study was to investigate the impact of the COVID-19 pandemic on paediatric appendicitis, specifically the proportion of children with complex appendicitis, alterations in the diagnostic work-up and treatment strategies, and its outcomes.The COVID-19 pandemic had a huge impact on healthcare systems worldwide, forcing policymakers to reorganize hospital resources to prioritize COVID care. Recommendations were made by surgical societies to postpone elective surgery and apply non-operative alternatives if available for surgical diseasesAppendix S1). The study was overseen by an international study steering group (RG/AZ/AP/NH/TW/FM/AG/PA) that developed the study protocol. This study was endorsed by the European Paediatric Surgeons’ Association (EUPSA), which assisted in the recruitment of participating hospitals through the EUPSA Network Office. Principal investigators of participating sites obtained local ethical approval in accordance with local requirements. The study was reported according to the STROBE guidelines4.An international retrospective study was conducted at 40 hospitals from 23 countries (Appendix S2), based on clinical, biochemical, and radiological criteria. Local investigators were asked to define the start of the COVID-19 pandemic at their institution based on the start of the interval during which regular healthcare was affected by the pandemic. The COVID group included patients treated between the start of the COVID period and 31 December 2020. The control group consisted of patients treated during the corresponding interval in 2019. To understand healthcare protocols and management strategies for acute appendicitis before and during the pandemic at each centre, all participating sites were asked to complete a survey that was sent on 23 March 2021 (Appendix S3).Patients (aged less than 18 years) treated for acute appendicitis between January 2019 and December 2020 were screened for eligibility. Those who had non-operative treatment without an imaging-confirmed diagnosis of acute appendicitis were excluded. Diagnosis of acute appendicitis was defined by intraoperative and histopathological confirmation of appendicitis, and, in the event of non-operative treatment .Comparative analyses were undertaken by calculating differences in proportions and 95 per cent confidence intervals. Subgroup analyses based on time interval of presentation, age, and region were performed for appendicitis severity and complications. For all subgroup analyses, Bonferroni correction was applied to adjust for multiple testing. Statistical analyses were carried out using SPSSAppendix S4. Therefore, 8593 patients were included, 4113 in the COVID group and 4480 in the control group. Baseline characteristics were similar in the two groups (Appendix S5).Between January 2019 and December 2020, some 10 655 children were treated for acute appendicitis, of whom 2062 were excluded for the reasons outlined in Appendix S1).The survey showed that, in the majority of participating centres, non-operative treatment and same-day discharge were not standard care during the pandemic, and that there was no change in referral pathways or shift of patients with complex disease to the participating centres (versus 45.0 per cent in the control group (difference 2.7 (95 per cent c.i. 0.6 to 4.8) per cent; P = 0.014) (Table 1). This increased proportion of complex appendicitis was apparent only during the first 3 months of the pandemic (difference 5.6 (1.8 to 9.3) per cent; P = 0.003, adjusted P = 0.007) and was predominantly caused by an absolute decrease in patients with simple appendicitis. The subgroup analysis based on region showed that the proportion of patients treated for complex appendicitis increased by 3.5 (1.2 to 5.8) per cent in Europe . No differences were found for other continents (Table 2).In the COVID group, 47.7 per cent of patients were treated for complex appendicitis P = 0.037). During the pandemic, 7.7 per cent of patients had non-operative treatment compared with 7.3 per cent in the control group (difference 0.4 (–0.7 to 1.5) per cent; P = 0.495). Outcomes of non-operative treatment are recorded in Appendix S6. Among those treated surgically, 74.3 per cent in the COVID group and 71.2 per cent in the control group underwent laparoscopic appendicectomy (difference 3.1 (1.2 to 5.1) per cent; P = 0.002) (Table 1).In the COVID group, 86.0 per cent of children underwent imaging during diagnostic work-up compared with 84.4 per cent in the control group (difference 1.6 (95 per cent c.i. 0.1 to 3.1) per cent; Tables 1 and 2).Both the primary and subgroup analyses showed no differences in the number of patients experiencing any complication between the COVID and control groups, nor in the severity of complications. In both groups, intra-abdominal abscess was the most frequent postoperative complication , whereas others could not detect any difference. In the present study, the increased proportion of complex appendicitis seems to be the result of an absolute decrease in patients with simple appendicitis. A possible explanation could be the resolution of mild cases of simple appendicitis in patients who did not seek medical care and recovered spontaneously or were treated with antibiotics by general practitioners16. After the first months of the pandemic, proportions of simple and complex appendicitis were comparable to those in the control group, which was predominantly the result of an absolute increase in patients with simple appendicitis. This could be explained by the fact that the threshold for seeking medical care for mild appendicitis possibly decreased after the first few months of the pandemic, as lockdown measures were slowly lifted and COVID-19-related fear declined. These findings are in line with those of other population-based studies17–19 that noted an absolute decrease in both adult and paediatric patients presenting with simple appendicitis early in the pandemic.Several small single-centre studiesThis international multicentre study is limited by possible information and selection bias, which is inherent to retrospective selection of patients and data collection. Furthermore, the regional subgroup analysis was limited by a skewed distribution, as the majority of patients were included in Europe. Finally, the survey found no shift of patients with complex disease to participating centres, but this might still have occurred. The major strength of this study is the international collaboration and subsequent large sample size of more than 8500 patients.CONNECT collaborative study group: Martin L. Metzelder, Sophie Langer Ashrarur R Mitul, Sabbir Karim, Nazmul Islam Anna Poupalou, (Université Libre de Bruxelles (ULB), HUDERF Hospital , Brussels, Belgium)Marc Miserez, Edward Willems Erika V. P. Ortolan, Pedro Luiz Toledo de ArrudaLourenção, Mark Bremholm Ellebæk, Susanna Petersen Janne Suominen, Mikko Pakarinen Françoise Schmitt Arnaud Bonnard, Louise Montalva, Garance Martin Antonella Nahom di Veroli, Zaki Assi Alessio Pini-Prato, Ilaria Falconi Daniele Alberti, Giovanni Boroni, Beatrice Montanaro Antonino Morabito, Andrea Zulli, Riccardo Coletta Carmelo Romeo, Enrica Antonelli, Francesca Nascimben Piergiorgio Gamba, Alberto Sgro Alessandro Raffaele, Maria Ruffoli Ivan Aloi, Simone Frediani Marco Gasparella, Paola Midrio Mohit Kakar Shireen A Nah, Yohesuwary Gunarasa Toni Risteski, Vesna Naunova Cvetanoska, Lazo Jovcheski Kjetil Juul Stensrud, Henrik Røkkum, Pål Aksel Næss Aline Vaz-Silva, Joana Patena Forte, Pedro F. Morais, Joana Queirós Pereira Sanja Sindjic Antunovic, Marija Lukac Marion Arnold, Martina Ichino, Andrew Victor Bernstein, Hettie le Roux Leopoldo Martinez, Carlos Delgado-Miguel, Paolo Bragagnini Rodriguez, Paula Salcedo Arroyo, Yurema Gonzalez Ruiz Anna Svenningsson Joep PM Derikx, Roel Bakx Rene MH Wijnen, Claudia MG Keyzer Gerda W Zijp, EA Huurman Wim van Gemert, Olivier Theeuws Ivo de Blaauw, Sanne MBI Botden, Maja Joosten Evert-Jan Boerma, Donald Schweitzer Osman Uzunlu Ingo Jester, Ben Martin, Hetal N Patel Dina Fouad, Christine Lam Clint D. Cappiello, Carla Lopez, Veronica Natale, Emily Lee T.W. received a grant from the Swedish Medical Research Council. The Swedish Medical Research Council had no role in any part of the study.znac239_Supplementary_DataClick here for additional data file.