A green and economic approach to synthesize magnetic Lagenaria siceraria biochar (γ-Fe2O3-LSB) for methylene blue removal from aqueous solution.

In the printing and textile industries, methylene blue (a cationic azo dye) is commonly used. MB is a well-known carcinogen, and another major issue is its high content in industrial discharge. There are numerous removal methodologies that have been employed to remove it from industrial discharge; however, these current modalities have one or more limitations. In this research, a novel magnetized biochar (γ-Fe2O3-LSB) was synthesized using Lagenaria siceraria peels which were further magnetized via the co-precipitation method. The synthesized γ-Fe2O3-LSB was characterized using FTIR, X-ray diffraction, Raman, SEM-EDX, BET, and vibrating sample magnetometry (VSM) for the analysis of magnetic properties. γ-Fe2O3-LSB showed a reversible type IV isotherm, which is a primary characteristic of mesoporous materials. γ-Fe2O3-LSB had a specific surface area (SBET = 135.30 m2/g) which is greater than that of LSB (SBET = 11.54 m2/g). γ-Fe2O3-LSB exhibits a saturation magnetization value (Ms) of 3.72 emu/g which shows its superparamagnetic nature. The batch adsorption process was performed to analyze the adsorptive removal of MB dye using γ-Fe2O3-LSB. The adsorption efficiency of γ-Fe2O3-LSB for MB was analyzed by varying parameters like the initial concentration of adsorbate (MB), γ-Fe2O3-LSB dose, pH effect, contact time, and temperature. Adsorption isotherm, kinetic, and thermodynamics were also studied after optimizing the protocol. The non-linear Langmuir model fitted the best to explain the adsorption isotherm mechanism and resulting adsorption capacity ( q e =54.55 mg/g). The thermodynamics study showed the spontaneous and endothermic nature, and pseudo-second-order rate kinetics was followed during the adsorption process. Regeneration study showed that γ-Fe2O3-LSB can be used up to four cycles. In laboratory setup, the cost of γ-Fe2O3-LSB synthesis comes out to be 162.75 INR/kg which is low as compared to commercially available adsorbents. The results obtained suggest that magnetic Lagenaria siceraria biochar, which is economical and efficient, can be used as a potential biochar material for industrial applications in the treatment of wastewater.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research.

The salivary gland (SG) is an essential organ that secretes saliva, which supports versatile oral function throughout life, and is maintained by elusive epithelial stem and progenitor cells (SGSPC). Unfortunately, aging, drugs, autoimmune disorders, and cancer treatments can lead to salivary dysfunction and associated health consequences. Despite many ongoing therapeutic efforts to mediate those conditions, investigating human SGSPC is challenging due to lack of standardized tissue collection, limited tissue access, and inadequate purification methods. Herein, we established a diverse and clinically annotated salivary regenerative biobanking at the Mayo Clinic, optimizing viable salivary cell isolation and clonal assays in both 2D and 3D-matrigel growth environments. Our analysis identified ductal epithelial cells in vitro enriched with SGSPC expressing the CD24/EpCAM/CD49f+ and PSMA- phenotype. We identified PSMA expression as a reliable SGSPC differentiation marker. Moreover, we identified progenitor cell types with shared phenotypes exhibiting three distinct clonal patterns of salivary differentiation in a 2D environment. Leveraging innovative label-free unbiased LC-MS/MS-based single-cell proteomics, we identified 819 proteins across 71 single cell proteome datasets from purified progenitor-enriched parotid gland (PG) and sub-mandibular gland (SMG) cultures. We identified distinctive co-expression of proteins, such as KRT1/5/13/14/15/17/23/76 and 79, exclusively observed in rare, scattered salivary ductal basal cells, indicating the potential de novo source of SGSPC. We also identified an entire class of peroxiredoxin peroxidases, enriched in PG than SMG, and attendant H2O2-dependent cell proliferation in vitro suggesting a potential role for PRDX-dependent floodgate oxidative signaling in salivary homeostasis. The distinctive clinical resources and research insights presented here offer a foundation for exploring personalized regenerative medicine.

The electrostatic landscape of MHC-peptide binding revealed using inception networks.

Predictive modeling of macromolecular recognition and protein-protein complementarity represents one of the cornerstones of biophysical sciences. However, such models are often hindered by the combinatorial complexity of interactions at the molecular interfaces. Exemplary of this problem is peptide presentation by the highly polymorphic major histocompatibility complex class I (MHC-I) molecule, a principal component of immune recognition. We developed human leukocyte antigen (HLA)-Inception, a deep biophysical convolutional neural network, which integrates molecular electrostatics to capture non-bonded interactions for predicting peptide binding motifs across 5,821 MHC-I alleles. These predictions of generated motifs correlate strongly with experimental peptide binding and presentation data. Beyond molecular interactions, the study demonstrates the application of predicted motifs in analyzing MHC-I allele associations with HIV disease progression and patient response to immune checkpoint inhibitors. A record of this paper's transparent peer review process is included in the supplemental information.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

Identification of dysregulation of sphingolipids in retinoblastoma using liquid chromatography-mass spectrometry.

Retinoblastoma (RB) is a rare ocular cancer seen in children that counts for approximately 3% of all childhood cancers. It is found that mutation in RB1, a tumour Suppressor Gene on chromosome 13 as the cause of malignancy. Retinoblastoma protein is the target for ceramide to cause apoptosis. We studied lipidomics of two RB cell lines, one aggressive cell line (NCC-RbC-51) derived from a metastatic site and one non aggressive cell line (WERI-Rb1) in comparison with a control cell line (MIO-M1). Lipid profiles of all the cell lines were studied using high resolution mass spectrometer coupled to high performance liquid chromatography. Data acquired from all the three cell lines in positive mode were analyzed to identify differentially expressed metabolites. Several phospholipids and lysophospholipids were found to be dysregulated. We observed upregulation of hexosyl ceramides, and down regulation of dihydroceramides and higher order sphingoglycolipids hinting at a hindered sphingolipid biosynthesis. The results obtained from liquid chromatography-mass spectrometry are validated by using qPCR and it was observed that genes involved in ceramide biosynthesis pathway are getting down regulated.

Profiling Kinase Activities for Precision Oncology in Diffuse Gastric Cancer.

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally. This is due to the fact that majority of the cases of GC are diagnosed at an advanced stage when the treatment options are limited and prognosis is poor. The diffuse subtype of gastric cancer (DGC) under Lauren's classification is more aggressive and usually occurs in younger patients than the intestinal subtype. The concept of personalized medicine is leading to the identification of multiple biomarkers in a large variety of cancers using different combinations of omics technologies. Proteomic changes including post-translational modifications are crucial in oncogenesis. We analyzed the phosphoproteome of DGC by using paired fresh frozen tumor and adjacent normal tissue from five patients diagnosed with DGC. We found proteins involved in the epithelial-to-mesenchymal transition (EMT), c-MYC pathway, and semaphorin pathways to be differentially phosphorylated in DGC tissues. We identified three kinases, namely, bromodomain adjacent to the zinc finger domain 1B (BAZ1B), WNK lysine-deficient protein kinase 1 (WNK1), and myosin light-chain kinase (MLCK) to be hyperphosphorylated, and one kinase, AP2-associated protein kinase 1 (AAK1), to be hypophosphorylated. LMNA hyperphosphorylation at serine 392 (S392) was demonstrated in DGC using immunohistochemistry. Importantly, we have detected heparin-binding growth factor (HDGF), heat shock protein 90 (HSP90), and FTH1 as potential therapeutic targets in DGC, as drugs targeting these proteins are currently under investigation in clinical trials. Although these new findings need to be replicated in larger study samples, they advance our understanding of signaling alterations in DGC, which could lead to potentially novel actionable targets in GC.

sBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides.

BACKGROUND: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. METHODS: Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. CONCLUSIONS: Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

Endoxifen downregulates AKT phosphorylation through protein kinase C beta 1 inhibition in ERα+ breast cancer.

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direct ENDX administration (5 µM), but not low concentrations observed during tamoxifen treatment (<0.1 µM), profoundly altered the phosphoproteome of the aromatase expressing MCF7AC1 cells with limited impact on the total proteome. Computational analysis revealed protein kinase C beta (PKCß) and protein kinase B alpha or AKT1 as potential kinases responsible for mediating ENDX effects on protein phosphorylation. ENDX more potently inhibited PKCß1 kinase activity compared to other PKC isoforms, and ENDX binding to PKCß1 was confirmed using Surface Plasma Resonance. Under conditions that activated PKC/AKT signaling, ENDX induced PKCß1 degradation, attenuated PKCß1-activated AKTSer473 phosphorylation, diminished AKT substrate phosphorylation, and induced apoptosis. ENDX's effects on AKT were phenocopied by siRNA-mediated PKCß1 knockdown or treatment with the pan-AKT inhibitor, MK-2206, while overexpression of constitutively active AKT diminished ENDX-induced apoptosis. These findings, which identify PKCß1 as an ENDX target, indicate that PKCß1/ENDX interactions suppress AKT signaling and induce apoptosis in breast cancer.

MicroRNA Expression Profile in Early-Stage Breast Cancers.

BACKGROUND: Breast cancer is one of the leading causes of cancer deaths in women. Early diagnosis offers the best hope for a cure. Ductal carcinoma in situ is considered a precursor of invasive ductal carcinoma of the breast. In this study, we carried out microRNA sequencing from 7 ductal carcinoma in situ (DCIS), 6 infiltrating ductal carcinomas (IDC Stage IIA) with paired normal, and 5 unpaired normal breast tissue samples. We identified 76 miRNAs that were differentially expressed in DCIS and IDC. METHODS: Additionally, we provide preliminary evidence of miR-365b-3p and miR-7-1-3p being overexpressed, and miR-6507-5p, miR-487b-3p, and miR-654-3p being downregulated in DCIS relative to normal breast tissue. We also identified a miRNA miR-766-3p that was overexpressed in early-stage IDCs. The overexpression of miR-301a-3p in DCIS and IDC was confirmed in 32 independent breast cancer tissue samples. RESULTS: Higher expression of miR-301a-3p is associated with poor overall survival in The Can-cer Genome Atlas Breast Cancer (TCGA-BRCA) dataset, indicating that it may be associated with DCIS at high risk of progressing to IDC and warrants deeper investigation. CONCLUSION: We also analyzed competing endogenous networks associated with differentially expressed miRNAs and identified LRRC75A-AS1 and MAGI2-AS3 as lncRNAs that potentially play an important role in early-stage breast cancers.

Automated Sample Preparation Workflow for Tandem Mass Tag-Based Proteomics.

Although tandem mass tag (TMT)-based isobaric labeling has become a powerful approach for multiplexed protein quantitation, automating the workflow for this technique has not been easy to achieve for widespread adoption. This is because preparation of TMT-labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction, and alkylation to tryptic digestion, desalting, labeling, and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic, especially with large-scale clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for an automated proteomic workflow employing TMT labeling with the manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using an automated platform and a conventional manual protocol. Single-shot liquid chromatography with tandem mass spectrometry analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiency of tryptic digestion, alkylation, and TMT labeling were similar in both manual and automated processes. In addition, comparison of quantitation accuracy and precision showed similar performance in an automated workflow compared to manual sample preparation by an expert. Overall, we demonstrated that the automated platform performs at a level similar to a manual process performed by an expert for TMT-based proteomics. We anticipate that this automated workflow will increasingly replace manual pipelines and has the potential to be applied to large-scale TMT-based studies, providing robust results and high sample throughput.

Aberrations in ion channels interacting with lipid metabolism and epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most prevalent malignant gastrointestinal tumor. Ion channels contribute to tumor growth and progression through interactions with their neighboring molecules including lipids. The dysregulation of membrane ion channels and lipid metabolism may contribute to the epithelial-mesenchymal transition (EMT), leading to metastatic progression. Herein, transcriptome profiles of patients with ESCC were analyzed by performing differential gene expression and weighted gene co-expression network analysis to identify the altered ion channels, lipid metabolism- and EMT-related genes in ESCC. A total of 1,081 differentially expressed genes, including 113 ion channels, 487 lipid metabolism-related, and 537 EMT-related genes, were identified in patients with ESCC. Thereafter, EMT scores were correlated with altered co-expressed genes. The altered co-expressed genes indicated a correlation with EMT signatures. Interactions among 22 ion channels with 3 hub lipid metabolism- and 13 hub EMT-related proteins were determined using protein-protein interaction networks. A pathway map was generated to depict deregulated signaling pathways including insulin resistance and the estrogen receptor-Ca2+ signaling pathway in ESCC. The relationship between potential ion channels and 5-year survival rates in ESCC was determined using Kaplan-Meier plots and Cox proportional hazard regression analysis. Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) was found to be associated with poor prognosis of patients with ESCC. Additionally, drugs interacting with potential ion channels, including GJA1 and ITPR3, were identified. Understanding alterations in ion channels with lipid metabolism and EMT in ESCC pathophysiology would most likely provide potential targets for the better treatment of patients with ESCC.

Urinary Proteomics for Discovery of Gastric Cancer Biomarkers to Enable Precision Clinical Oncology.

For precision in clinical oncology practice, detection of tumor-derived peptides and proteins in urine offers an attractive and noninvasive alternative for diagnostic or screening purposes. In this study, we report comparative quantitative proteomic profiling of urine samples from patients with gastric cancer and healthy controls using tandem mass tags-based multiplexed mass spectrometry approach. We identified 1504 proteins, of which 246 were differentially expressed in gastric cancer cases. Notably, ephrin A1 (EFNA1), pepsinogen A3 (PGA3), sortilin 1 (SORT1), and vitronectin (VTN) were among the upregulated proteins, which are known to play crucial roles in the progression of gastric cancer. We also found other overexpressed proteins, including shisa family member 5 (SHISA5), mucin like 1 (MUCL1), and leukocyte cell derived chemotaxin 2 (LECT2), which had not previously been linked to gastric cancer. Using a novel approach for targeted proteomics, SureQuant, we validated changes in abundance of a subset of proteins discovered in this study. We confirmed the overexpression of vitronectin and sortilin 1 in an independent set of urine samples. Altogether, this study provides molecular candidates for biomarker development in gastric cancer, and the findings also support the promise of urinary proteomics for noninvasive diagnostics and personalized/precision medicine in the oncology clinic.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics.

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

Whole Exome Sequencing Reveals Novel Variants in Unexplained Erythrocytosis.

Erythrocytosis is characterized by an increase in red cells in peripheral blood. Polycythemia vera, the commonest primary erythrocytosis, results from pathogenic variants in JAK2 in ∼98% of cases. Although some variants have been reported in JAK2-negative polycythemia, the causal genetic variants remain unidentified in ∼80% of cases. To discover genetic variants in unexplained erythrocytosis, we performed whole exome sequencing in 27 patients with JAK2-negative polycythemia after excluding the presence of any mutations in genes previously associated with erythrocytosis (EPOR, VHL, PHD2, EPAS1, HBA, and HBB). We found that the majority of patients (25/27) had variants in genes involved in epigenetic processes, including TET2 and ASXL1 or in genes related to hematopoietic signaling such as MPL and GFIB. Based on computational analysis, we believe that variants identified in 11 patients in this study could be pathogenic although functional studies will be required for confirmation. To our knowledge, this is the largest study reporting novel variants in individuals with unexplained erythrocytosis. Our results suggest that genes involved in epigenetic processes and hematopoietic signaling pathways are likely associated with unexplained erythrocytosis in individuals lacking JAK2 mutations. With very few previous studies targeting JAK2-negative polycythemia patients to identify underlying variants, this study opens a new avenue in evaluating and managing JAK2-negative polycythemia.

Characterization of Lipid Alterations by Oncogenic PIK3CA Mutations Using Untargeted Lipidomics in Breast Cancer.

Lipids play crucial biological roles in health and disease, including in cancers. The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pivotal promoter of cell growth and proliferation in various types of cancer. The somatic mutations in PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, are frequently present in cancer cells, including breast cancer. Although the most prominent mutants, represented by single amino acid substitutions in the helical domain in exon 9 (E545K) and the kinase domain in exon 20 (H1047R) are known to cause a gain of PI3K function, activate AKT signaling and induce oncogenic transformation, the effect of these mutations on cellular lipid profiles has not been studied. We carried out untargeted lipidomics using liquid chromatography-tandem mass spectrometry to detect the lipid alterations in mammary gland epithelial MCF10A cells with isogenic knockin of these mutations. A total of 536 species of lipids were analyzed. We found that the levels of monosialogangliosides, signaling molecules known to enhance cell motility through PI3K/AKT pathway, were significantly higher in both mutants. In addition, triglycerides and ceramides, lipid molecules known to be involved in promoting lipid droplet production, cancer cell migration and invasion, were increased, whereas lysophosphatidylcholines and phosphatidylcholines that are known to inhibit cancer cell motility were decreased in both mutants. Our results provide novel insights into a potential link between altered lipid profile and carcinogenesis caused by the PIK3CA hotspot mutations. In addition, we suggest untargeted lipidomics offers prospects for precision/personalized medicine by unpacking new molecular substrates of cancer biology.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Epigenetic reprogramming of cell cycle genes by ACK1 promotes breast cancer resistance to CDK4/6 inhibitor.

Hormone receptor-positive, HER2-negative advanced breast cancers exhibit high sensitivity to CDK4/6 inhibitors such as palbociclib. However, most patients inevitably develop resistance, thus identification of new actionable therapeutic targets to overcome the recurrent disease is an urgent need. Immunohistochemical studies of tissue microarray revealed increased activation of non-receptor tyrosine kinase, ACK1 (also known as TNK2) in most of the breast cancer subtypes, independent of their hormone receptor status. Chromatin immunoprecipitation studies demonstrated that the nuclear target of activated ACK1, pY88-H4 epigenetic marks, were deposited at cell cycle genes, CCNB1, CCNB2 and CDC20, which in turn initiated their efficient transcription. Pharmacological inhibition of ACK1 using its inhibitor, (R)-9b dampened CCNB1, CCNB2 and CDC20 expression, caused G2/M arrest, culminating in regression of palbociclib-resistant breast tumor growth. Further, (R)-9b suppressed expression of CXCR4 receptor, which resulted in significant impairment of metastasis of breast cancer cells to lung. Overall, our pre-clinical data identifies activated ACK1 as an oncogene that epigenetically controls the cell cycle genes governing the G2/M transition in breast cancer cells. ACK1 inhibitor, (R)-9b could be a novel therapeutic option for the breast cancer patients that have developed resistance to CDK4/6 inhibitors.