Transcriptomic analysis and fusion gene identifications of midbody remnants released from colorectal cancer cells reveals they are molecularly distinct from exosomes and microparticles.

Previously, we reported that human primary (SW480) and metastatic (SW620) colorectal (CRC) cells release three classes of membrane-encapsulated extracellular vesicles (EVs); midbody remnants (MBRs), exosomes (Exos), and microparticles (MPs). We reported that MBRs were molecularly distinct at the protein level. To gain further biochemical insights into MBRs, Exos, and MPs and their emerging role in CRC, we performed, and report here, for the first time, a comprehensive transcriptome and long noncoding RNA sequencing analysis and fusion gene identification of these three EV classes using the next-generation RNA sequencing technique. Differential transcript expression analysis revealed that MBRs have a distinct transcriptomic profile compared to Exos and MPs with a high enrichment of mitochondrial transcripts lncRNA/pseudogene transcripts that are predicted to bind to ribonucleoprotein complexes, spliceosome, and RNA/stress granule proteins. A salient finding from this study is a high enrichment of several fusion genes in MBRs compared to Exos, MPs, and cell lysates from their parental cells such as MSH2 (gene encoded DNA mismatch repair protein MSH2). This suggests potential EV-liquid biopsy targets for cancer detection. Importantly, the expression of cancer progression-related transcripts found in EV classes derived from SW480 (EGFR) and SW620 (MET and MACCA1) cell lines reflects their parental cell types. Our study is the report of RNA and fusion gene compositions within MBRs (including Exos and MPs) that could have an impact on EV functionality in cancer progression and detection using EV-based RNA/ fusion gene candidates for cancer biomarkers.

A neural network with a human learning paradigm for breast fibroadenoma segmentation in sonography.

BACKGROUND: Breast fibroadenoma poses a significant health concern, particularly for young women. Computer-aided diagnosis has emerged as an effective and efficient method for the early and accurate detection of various solid tumors. Automatic segmentation of the breast fibroadenoma is important and potentially reduces unnecessary biopsies, but challenging due to the low image quality and presence of various artifacts in sonography. METHODS: Human learning involves modularizing complete information and then integrating it through dense contextual connections in an intuitive and efficient way. Here, a human learning paradigm was introduced to guide the neural network by using two consecutive phases: the feature fragmentation stage and the information aggregation stage. To optimize this paradigm, three fragmentation attention mechanisms and information aggregation mechanisms were adapted according to the characteristics of sonography. The evaluation was conducted using a local dataset comprising 600 breast ultrasound images from 30 patients at Suining Central Hospital in China. Additionally, a public dataset consisting of 246 breast ultrasound images from Dataset_BUSI and DatasetB was used to further validate the robustness of the proposed network. Segmentation performance and inference speed were assessed by Dice similarity coefficient (DSC), Hausdorff distance (HD), and training time and then compared with those of the baseline model (TransUNet) and other state-of-the-art methods. RESULTS: Most models guided by the human learning paradigm demonstrated improved segmentation on the local dataset with the best one (incorporating C3ECA and LogSparse Attention modules) outperforming the baseline model by 0.76% in DSC and 3.14 mm in HD and reducing the training time by 31.25%. Its robustness and efficiency on the public dataset are also confirmed, surpassing TransUNet by 0.42% in DSC and 5.13 mm in HD. CONCLUSIONS: Our proposed human learning paradigm has demonstrated the superiority and efficiency of ultrasound breast fibroadenoma segmentation across both public and local datasets. This intuitive and efficient learning paradigm as the core of neural networks holds immense potential in medical image processing.

Comparative proteomic analysis of three major extracellular vesicle classes secreted from human primary and metastatic colorectal cancer cells: Exosomes, microparticles, and shed midbody remnants.

Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB-Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB-Rs) arise from symmetric cytokinetic abscission. Here, we isolate sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs secreted from human isogenic primary (SW480) and metastatic (SW620) colorectal cancer (CRC) cell lines in milligram quantities for label-free MS/MS-based proteomic profiling. Purified EVs revealed selective composition packaging of exosomal protein markers in SW480/SW620-sEVs/Exos, metabolic enzymes in SW480/SW620-lEVs/MPs, while centralspindlin complex proteins, nucleoproteins, splicing factors, RNA granule proteins, translation-initiation factors, and mitochondrial proteins selectively traffic to SW480/SW620- lEVs/sMB-Rs. Collectively, we identify 39 human cancer-associated genes in EVs; 17 associated with SW480-EVs, 22 with SW620-EVs. We highlight oncogenic receptors/transporters selectively enriched in sEVs/Exos (EGFR/FAS in SW480-sEVs/Exos and MET, TGFBR2, ABCB1 in SW620-sEVs/Exos). Interestingly, MDK, STAT1, and TGM2 are selectively enriched in SW480-lEVs/sMB-Rs, and ADAM15 to SW620-lEVs/sMB-Rs. Our study reveals sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs have distinct protein signatures that open potential diagnostic avenues of distinct types of EVs for clinical utility.

Identification of UBFD1 as a prognostic biomarker and molecular target among estrogen receptor-positive breast cancer.

Estrogen receptor (ER)-positive breast cancer (BRCA) is the most commonly diagnosed molecular subtype of BRCA. It is routinely treated with endocrine therapy; however, some patients relapse after therapy and develop drug resistance, resulting in treatment failure. In the present study, we identified markers of ER-positive BRCA and evaluated their putative function in immune infiltration as well as their clinicopathological significance. The ubiquitin family domain containing 1 (UBFD1) protein was associated with the prognosis of ER-positive BRCA patients. Its expression was higher in ER-positive BRCA tissues compared with adjacent nontumor tissues. Patients with higher UBFD1 expression had a poorer prognosis. UBFD1 is an independent risk factor for ER-positive BRCA patients and its function was primarily associated with hormone activity and inflammation. Taken together, UBFD1 is a potential prognostic biomarker and candidate target of ER-positive BRCA.

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

Astrocytes derived from ASD individuals alter behavior and destabilize neuronal activity through aberrant Ca2+ signaling.

The cellular mechanisms of autism spectrum disorder (ASD) are poorly understood. Cumulative evidence suggests that abnormal synapse function underlies many features of this disease. Astrocytes regulate several key neuronal processes, including the formation of synapses and the modulation of synaptic plasticity. Astrocyte abnormalities have also been identified in the postmortem brain tissue of ASD individuals. However, it remains unclear whether astrocyte pathology plays a mechanistic role in ASD, as opposed to a compensatory response. To address this, we combined stem cell culturing with transplantation techniques to determine disease-specific properties inherent to ASD astrocytes. We demonstrate that ASD astrocytes induce repetitive behavior as well as impair memory and long-term potentiation when transplanted into the healthy mouse brain. These in vivo phenotypes were accompanied by reduced neuronal network activity and spine density caused by ASD astrocytes in hippocampal neurons in vitro. Transplanted ASD astrocytes also exhibit exaggerated Ca2+ fluctuations in chimeric brains. Genetic modulation of evoked Ca2+ responses in ASD astrocytes modulates behavior and neuronal activity deficits. Thus, this study determines that astrocytes derived from ASD iPSCs are sufficient to induce repetitive behavior as well as cognitive deficit, suggesting a previously unrecognized primary role for astrocytes in ASD.

Focused ultrasound ablation surgery for multiple breast fibroadenomas: pathological and follow-up results.

OBJECTIVE: To investigate the histopathological findings and follow-up outcome of focused ultrasound ablation surgery (FUAS) treatment of multiple fibroadenomas (FA). METHODS: A total of 20 patients with 101 multiple FAs were enrolled. After one session FUAS ablation, 21 lesions (≥15.0 mm) were surgically removed within one week for histopathological analysis, including 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, H&E staining, nicotinamide adenine dinucleotide (NADH) -flavretin enzyme staining, Transmission electron microscope (TEM) and scanning electron microscope (SEM). The remaining 80 lesions were followed up at 3, 6 and 12 months after treatment. RESULTS: All ablation procedures were performed successfully. Pathologic findings showed that irreversible damage of FA was confirmed. TTC, H&E and NADH staining and TEM/SEM demonstrated tumor cell death and tumor structural destruction at the gross, cellular, and subcellular levels, respectively. The median shrinkage rate at 12 months post-FUAS was 66.4 (43.6, 89.5) %. CONCLUSION: Histopathological analysis for FAs after FUAS treatment proved that FUAS could effectively induce irreversible coagulative necrosis of FA, and the tumor volume would gradually shrink in follow-up. FUAS was safe and effective to treat multiple FAs with good cosmesis.Key pointsThis study was the first study of detailed histopathological analysis for FAs after FUAS treatment.FUAS can effectively induce irreversible coagulative necrosis of fibroadenoma cells.FUAS ablation of multiple fibroadenomas is safe and effective.

Humanized mouse models for inherited thrombocytopenia studies.

Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal platelet function, which can subsequently lead to impaired hemostasis. In the past decades, humanized mouse models (HMMs), that are mice engrafted with human cells or genes, have been widely used in different research areas including immunology, oncology, and virology. With advances of the development of immunodeficient mice, the engraftment, and reconstitution of functional human platelets in HMM permit studies of occurrence and development of platelet disorders including IT and treatment strategies. This article mainly reviews the development of humanized mice models, the construction methods, research status, and problems of using humanized mice for the in vivo study of human thrombopoiesis.

Humanized mouse models (HMMs) refer to immunodeficient mice that have been used for the investigation of human hematopoiesis and immunity for years. With engrafted human hematopoietic stem cells (HSCs), the differentiation process of HSCs and re-construction of platelets can be monitored in the mice. Until now, several strains of HMMs have been used in the studies of inherited thrombocytopenia (IT), a genetic disorder associated with low platelet count in the blood. In this study, we reviewed the development of these HMMs in IT studies, compared the different sources of HSCs transplanted into HMMs and summarize the strategies of HSC transplantation in HMMs. The Kit^−/− immunodeficient mice showed effectively long-term and stable implantation of human HSC without irradiation and higher implantation levels, and they also support multilinear differentiation of human HSC, such as platelets and red blood cells. The source and count of HSCs and the transplantation strategy may also impact the result. This study provides a basis information for HMMs used in IT and will help other investigators in this field choosing the right research plan.

Breast-conserving surgery is an appropriate procedure for centrally located breast cancer: a population-based retrospective cohort study.

BACKGROUND: The evidence of breast-conserving therapy (BCT) applied in centrally located breast cancer (CLBC) is absent. This study aims to investigate the long-term survival of breast-conserving therapy (BCT) in centrally located breast cancer (CLBC) compared with mastectomy in CLBC and BCT in non-CLBC. METHODS: Two hundred ten thousand four hundred nine women with unilateral T1-2 breast cancer undergoing BCT or mastectomy were identified from the Surveillance, Epidemiology, and End Results database. Kaplan-Meier survival curves were assessed via log-rank test. Propensity score matching (PSM) was used to balance baseline features, and the multivariable Cox model was used to estimate the adjusted hazard ratio [HR] and its 95% confidence interval [CI] for breast cancer-specific survival (BCSS) and overall survival (OS). RESULTS: With a median follow-up of 91 months, the BCSS and OS rates in patients who received BCT were greater than those patients treated with mastectomy in the entire CLBC set. Multivariable Cox analyses showed that CLBC patients who received BCT had better BCSS (HR = 0.67, 95%CI: 0.55-0.80, p < 0.001) and OS (HR = 0.78, 95%CI: 0.68-0.90, p = 0.001) than patients who received a mastectomy, but there were no significant differences of BCSS (HR = 0.65, 95%CI: 0.47-0.90, p = 0.009) and OS (HR = 0.82, 95%CI: 0.65-1.04, p = 0.110) after PSM. In patients treated with BCT, CLBC patients had a similar BCSS (HR = 0.99, 95%CI: 0.87-1.12, p = 0.850) but a worse OS (HR = 1.09, 95%CI: 1.01-1.18, p = 0.040) compared to that of the non-CLBC patient, but there was no significant difference both BCSS (HR = 1.05, 95%CI: 0.88-1.24, p = 0.614) and OS (HR = 1.08, 95%CI: 0.97-1.20, p = 0.168) after PSM. CONCLUSION: Our findings revealed that BCT should be an acceptable and preferable alternative to mastectomy for well-selected patients with CLBC.

Chronic methamphetamine interacts with BDNF Val66Met to remodel psychosis pathways in the mesocorticolimbic proteome.

Methamphetamine (Meth) abuse has reached epidemic proportions in many countries and can induce psychotic episodes mimicking the clinical profile of schizophrenia. Brain-derived neurotrophic factor (BDNF) is implicated in both Meth effects and schizophrenia. We therefore studied the long-term effects of chronic Meth exposure in transgenic mice engineered to harbor the human BDNFVal66Met polymorphism expressed via endogenous mouse promoters. These mice were chronically treated with an escalating Meth regime during late adolescence. At least 4 weeks later, all hBDNFVal66Met Meth-treated mice exhibited sensitization confirming persistent behavioral effects of Meth. We used high-resolution quantitative mass spectrometry-based proteomics to biochemically map the long-term effects of Meth within the brain, resulting in the unbiased detection of 4808 proteins across the mesocorticolimbic circuitry. Meth differentially altered dopamine signaling markers (e.g., Dat, Comt, and Th) between hBDNFVal/Val and hBDNFMet/Met mice, implicating involvement of BDNF in Meth-induced reprogramming of the mesolimbic proteome. Targeted analysis of 336 schizophrenia-risk genes, as well as 82 growth factor cascade markers, similarly revealed that hBDNFVal66Met genotype gated the recruitment of these factors by Meth in a region-specific manner. Cumulatively, these data represent the first comprehensive analysis of the long-term effects of chronic Meth exposure within the mesocorticolimbic circuitry. In addition, these data reveal that long-term Meth-induced brain changes are strongly dependent upon BDNF genetic variation, illustrating how drug-induced psychosis may be modulated at the molecular level by a single genetic locus.

Evaluating the learning curve of high intensity focus ultrasound for breast fibroadenoma by CUSUM analysis: a multi-center study.

OBJECTIVE: To explore the learning curve of high intensity focus ultrasound (HIFU) treatment for breast fibroadenoma. METHODS: A database of 110 patients with 255 breast fibroadenomas who underwent HIFU treatment at two different clinical centers (Center 1 and 2) were retrospectively analyzed. The learning curves of HIFU treatment for breast fibroadenoma were drawn by CUSUM analysis in two centers, respectively. According to the inflection point of the learning curves, the treatment was divided into two groups: initial phase and consolidation phase. HIFU treatment parameters were compared between two groups. The effectiveness and safety results were also evaluated. RESULTS: The inflection points of the learning curves were the 60th treatment in Center 1 and the 65th treatment in Center 2. The screening time, treatment time, sonication time and hyperechoic scale change time were significantly shorter in consolidation phase than those in initial phase of the two centers (p < 0.05). There were no differences in non-perfused volume (NPV) ratio and energy effect factor (EEF) between the two groups in Center 1, while in Center 2, these above-mentioned results in consolidation phase led to a greater improvement than those in initial phase. There was no difference of Visual Analogue Scale (VAS) scores and no adverse event observed in both centers. CONCLUSION: HIFU treatment for breast fibroadenoma was effective and safe. The learning curve of HIFU treatment for breast fibroadenoma can be completed after treating 60-65 tumors without increasing the safety risk.

Potential biomarkers for inherited thrombocytopenia 2 identified by plasma proteomics.

Inherited thrombocytopenia 2 (THC2) is difficult to diagnose due to the lack of specific clinical characteristics and diagnostic methods. To identify potential plasma protein biomarkers for THC2, we collected the plasma samples from a THC2 family (9 THC2 and 15 non-THC2 members), enriched the medium and low abundant proteins using Proteominer and analyzed the protein profiles using the liquid chromatography-mass spectrometry in data independent acquisition mode. Initially, we detected 784 proteins in the plasma samples of this family and identified 27 up-regulated and 36 down-regulated in the THC2 group compared to the non-THC2 group (|log2 ratio| >1 and p-value <0.05). To improve the predictive power, top eight dysregulated proteins (B7Z2B4, LTF, HP, ERN1, IGHV1-8, A0A0X9V9C4, VH6DJ, and D3JV41) were selected by an area under the curve-based random forest process to construct a clinical model. Multivariate analysis with random forest and support vector machine showed that the prediction model provided high discrimination ability for THC2 diagnosis (AUC: 1.000 and 0.967, respectively). The potential plasma protein biomarkers will be tested in more THC2 patients and other thrombocytopenia patients to further validate their specificity and sensitivity.

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Transglutaminase-2, RNA-binding proteins and mitochondrial proteins selectively traffic to MDCK cell-derived microvesicles following H-Ras-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.

Human myeloma cell- and plasma-derived extracellular vesicles contribute to functional regulation of stromal cells.

Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate morphologically-intact and biologically functional sEV capable of mediating the regulation of stromal cells, and a model for the characterization of tumour-stromal cross-talk by sEV in MM.

Dynamic transcriptome analysis identifies genes related to fatty acid biosynthesis in the seeds of Prunus pedunculata Pall.

BACKGROUND: Prunus pedunculata Pall, the deciduous shrub of Amygdalus subgenus in Rosaceae, is a new kind of desert oil-bearing tree. It has a long story of being planted in the West and North of China for sand fixation and desert control. In addition, the seeds of P. pedunculata are rich of oil, especially the monounsaturated fatty acid and polyunsaturated fatty acid. However, little is known about the molecular mechanisms of oil accumulation during the seed development of P. pedunculata. RESULTS: The seeds of P. pedunculata from three independent plants at 10, 18, 24, 31, 39, 45, 59 and 73 days after flowering (DAF) were obtained and the oil compositions were evaluated. It showed that oleic acid was the dominant type of oil content in the mature seeds (from 32.724% at 10DAF to 72.06% at 73DAF). Next, transcriptome sequencing for the developing seeds produced 988.795 million high quality reads and TRINITY assembled 326,271 genes for the first transcriptome for P. pedunculata. After the assembled transcriptome was evaluated by BUSCO with 85.9% completeness, we identified 195,342, 109,850 and 121,897 P. pedunculata genes aligned to NR, GO and KEGG pathway databases, respectively. Then, we predicted 23,229 likely proteins from the assembled transcriptome and identified 1917 signal peptides and 5512 transmembrane related proteins. In the developing seeds we detected 91,362 genes (average FPKM > 5) and correlation analysis indicated three possible development stages - early (10 ~ 24DAF), middle (31 ~ 45DAF) and late (59 ~ 73DAF). We next analyzed the differentially expressed genes (DEGs) in the developing seeds. Interestingly, compared to 10DAF the number of DEGs was increased from 4406 in 18DAF to 27,623 in 73DAF. Based on the gene annotation, we identified 753, 33, 8 and 645 DEGs related to the fatty acid biosynthesis, lipid biosynthesis, oil body and transcription factors. Notably, GPAT, DGD1, LACS2, UBC and RINO were highly expressed at the early development stage, ω6-FAD, SAD, ACP, ACCA and AHG1 were highly expressed at the middle development stage, and LACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin genes were highly expressed at the late development stage. CONCLUSIONS: This is the first time to study the developing seed transcriptome of P. pedunculata and our findings will provide a valuable resource for future studies. More importantly, it will improve our understanding of molecular mechanisms of oil accumulation in P. pedunculata.

Exosomes Derived from Human Primary and Metastatic Colorectal Cancer Cells Contribute to Functional Heterogeneity of Activated Fibroblasts by Reprogramming Their Proteome.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor-derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early- (SW480) and late-stage (SW620) colorectal cancer (CRC) cell-derived Exos both activated normal quiescent fibroblasts (α-SMA- , CAV+ , FAP+ , VIM+ ) into CAF-like fibroblasts (α-SMA+ , CAV- , FAP+ , VIM+ ). Fibroblasts activated by early-stage cancer-exosomes (SW480-Exos) are highly pro-proliferative and pro-angiogenic and display elevated expression of pro-angiogenic (IL8, RAB10, NDRG1) and pro-proliferative (SA1008, FFPS) proteins. In contrast, fibroblasts activated by late-stage cancer-exosomes (SW620-Exos) display a striking ability to invade through extracellular matrix through upregulation of pro-invasive regulators of membrane protrusion (PDLIM1, MYO1B) and matrix-remodeling proteins (MMP11, EMMPRIN, ADAM10). Conserved features of Exos-mediated fibroblast activation include enhanced ECM secretion (COL1A1, Tenascin-C/X), oncogenic transformation, and metabolic reprogramming (downregulation of CAV-1, upregulation of glycogen metabolism (GAA), amino acid biosynthesis (SHMT2, IDH2) and membrane transporters of glucose (GLUT1), lactate (MCT4), and amino acids (SLC1A5/3A5)). This study highlights the role of primary and metastatic CRC tumor-derived Exos in generating phenotypically and functionally distinct subsets of CAFs that may facilitate tumor progression.

Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114.

Exosomes are important bidirectional cell-cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X-114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label-free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database-annotated proteins reveals 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 108 RNA-binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface-localized miRNA Let-7a-5p is RNase A-resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.

Transcriptomic and proteomic analyses reveal new insights into the regulation of immune pathways during adenovirus type 2 infection.

BACKGROUND: Human adenovirus (Ad) infection leads to the changes of host cell gene expression and biosynthetic processes. Transcriptomics in adenovirus type 2 (Ad2)-infected lung fibroblasts (IMR-90) cells has previously been studied using RNA sequencing. However, this study included only two time points (12 and 24 hpi) using constrained 76 bp long sequencing reads. Therefore, a more detailed study of transcription at different phases of infection using an up-graded sequencing technique is recalled. Furthermore, the correlation between transcription and protein expression needs to be addressed. RESULTS: In total, 3556 unique cellular genes were identified as differentially expressed at the transcriptional level with more than 2-fold changes in Ad2-infected cells as compared to non-infected cells by using paired-end sequencing. Based on the kinetics of the gene expression changes at different times after infection, these RNAs fell into 20 clusters. Among them, cellular genes involved in immune response were highly up-regulated in the early phase before becoming down-regulated in the late phase. Comparison of differentially expressed genes at transcriptional and posttranscriptional levels revealed low correlation. Particularly genes involved in cellular immune pathways showed a negative correlation. Here, we highlight the genes which expose inconsistent expression profiles with an emphasis on key factors in cellular immune pathways including NFκB, JAK/STAT, caspases and MAVS. Different from their transcriptional profiles with up- and down-regulation in the early and late phase, respectively, these proteins were up-regulated in the early phase and were sustained in the late phase. A surprising finding was that the target genes of the sustained activators failed to show response. CONCLUSION: There were features common to genes which play important roles in cellular immune pathways. Their expression was stimulated at both RNA and protein levels during the early phase. In the late phase however, their transcription was suppressed while protein levels remained stable. These results indicate that Ad2 and the host cell use different strategies to regulate cellular immune pathways. A control mechanism at the post-translational level must thus exist which is under the control of Ad2.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.