Paternal effect does not affect in vitro embryo morphokinetics but modulates molecular profile

Abstract Background Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. Methods Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. Results Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. Conclusions This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

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Efficacy of dihydroartemisinin/piperaquine in patients with non-complicated Plasmodium falciparum malaria in Yaoundé, Cameroon

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab281 ◽

2021 ◽

Author(s):

Mélissa Mairet-Khedim ◽

Sandrine Nsango ◽

Christelle Ngou ◽

Sandie Menard ◽

Camille Roesch ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Southeast Asia ◽

Falciparum Malaria ◽

Gene Polymorphisms ◽

Plasmodium Falciparum Malaria ◽

Molecular Profile ◽

Uncomplicated Plasmodium Falciparum Malaria ◽

In Vitro Survival ◽

Phenotypic Susceptibility

Abstract Background Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. Objectives This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. Patients and methods Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. Results The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine. Conclusions Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia.

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Functional and Molecular Characterization of PD1+ Tumor-Infiltrating Lymphocytes From Lung Cancer Patients

10.21203/rs.3.rs-152467/v1 ◽

2021 ◽

Author(s):

Jesse Lipp ◽

Limei Wang ◽

Nathalie Harrer ◽

Stefan Mueller ◽

Sabina Berezowska ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

T Cell ◽

Wnt Signaling ◽

Cell Activation ◽

Effector Function ◽

Transcriptional Analysis ◽

Molecular Profile ◽

Dependent Manner

Abstract Background The majority of infiltrating T-cells (TILs) in lung cancer are contained in the memory compartment and overexpress PD1 and have been associated with dysfunction. Antibody-mediated cancer immunotherapy targets inhibitory surface molecules, such as PD1, PD-L1, and CTLA-4, aiming to re-invigorate dysfunctional T cells.Methods Using fluorescence-activated cell sorting (FACS), we purified CD45RO+ memory CD8+ and CD4+ TILs and their patient-matched non-tumor counterparts from treatment-naïve NSCLC patient biopsies to better evaluate the effect of PD1 expression on the functional and molecular profile of tumor-resident T cells. Moreover, we compared the functional, molecular, and clonal composition of TIL preparations after TCR-dependent in vitro expansion with their freshly isolated counterparts in matched patients.Results We show that PD1+CD8+ TILs have elevated expression of the transcriptional regulator ID3 and that the overall cytotoxic potential of CD8 T cells can be improved by knocking down ID3, defining it as a potential regulator of T cell effector function. PD1+CD4+ memory TILs remain functionally intact and despite overexpressing key transcriptional activators known to negatively regulate CD8 function such as TOX and TOX2, display transcriptional patterns consistent with both follicular helper and regulator function and robustly facilitate B cell activation and expansion in response to TCR-dependent stimulation. Furthermore, we show that expanding ex vivo-prepared TILs in vitro in a TCR-dependent manner broadly preserves their functionality with respect to tumor cell killing, expansion and activation of B cells, and TCR repertoire. Although purified PD1+CD8+ TILs generally maintain an exhausted phenotype upon expansion in vitro, transcriptional analysis reveals a downregulation of markers of T cell dysfunction, including the co-inhibitory molecules PD1 and CTLA-4 and the transcription factors ID3, TOX and TOX2, while genes involved in cell cycle and DNA repair are upregulated. We find reduced expression of WNT signaling components to be a hallmark of PD1+CD8+ exhausted T cells in vivo and in vitro and demonstrate that restoring WNT signaling, by pharmacological blockade of GSK3β, can improve effector function. Conclusions These data unveil novel targets for tumor immunotherapy and have promising implications for development of a personalized adoptive TIL-based cell therapy for lung cancer.

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CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

Neuro-Oncology ◽

10.1093/neuonc/noz175.185 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi47-vi47

Author(s):

Marilin Koch ◽

Stefan Czemmel ◽

Felix Lennartz ◽

Sarah Beyeler ◽

Justyna Przystal ◽

...

Keyword(s):

Molecular Mechanisms ◽

Nuclear Translocation ◽

Clonogenic Survival ◽

Dominant Negative ◽

Molecular Profile ◽

Transcriptional Networks ◽

Molecular Alterations ◽

Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

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129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab129 ◽

2013 ◽

Vol 25 (1) ◽

pp. 212

Author(s):

G. Machado ◽

A. Ferreira ◽

I. Pivato ◽

A. Fidelis ◽

J. F. Srpicigo ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

T Test ◽

Molecular Profile ◽

Control Group ◽

Fluid Medium ◽

Uterine Flushing

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality. Financial support: Embrapa, CNPq, CAPES.

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Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15999200918110638 ◽

2020 ◽

Vol 15 ◽

Author(s):

Shalmali Pendse ◽

Vaijayanti Kale ◽

Anuradha Vaidya

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Ex Vivo ◽

Cell Types ◽

Molecular Profile ◽

Cell Contact ◽

Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

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A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain

Endocrinology ◽

10.1210/en.2015-1390 ◽

2015 ◽

Vol 156 (11) ◽

pp. 4105-4114 ◽

Cited By ~ 28

Author(s):

Erika Atucha ◽

Ioannis Zalachoras ◽

José K. van den Heuvel ◽

Lisa T. C. M. van Weert ◽

Diana Melchers ◽

...

Keyword(s):

Memory Consolidation ◽

Glucocorticoid Receptors ◽

Target Genes ◽

Inhibitory Avoidance ◽

Male Rat ◽

Molecular Profile ◽

Avoidance Task ◽

Antagonistic Effects ◽

Acute And Chronic Stress

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a “selective modulator” type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

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Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin

Blood ◽

10.1182/blood.v118.21.1251.1251 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1251-1251

Author(s):

Debra Hoppensteadt ◽

Walter Jeske ◽

Angel Gray ◽

Jeanine M. Walenga ◽

Rakesh Wahi ◽

...

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Molecular Profile ◽

Thrombin Time ◽

Time Period ◽

Pharmacodynamic Profile ◽

The Us ◽

Fibrinolysis Inhibitor ◽

Tissue Factor Pathway

Abstract Abstract 1251 Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence. Disclosures: No relevant conflicts of interest to declare.

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Different molecular profiles are associated with breast cancer cell homing compared with colonisation of bone: evidence using a novel bone-seeking cell line

Endocrine Related Cancer ◽

10.1530/erc-13-0158 ◽

2014 ◽

Vol 21 (2) ◽

pp. 327-341 ◽

Cited By ~ 47

Author(s):

Faith Nutter ◽

Ingunn Holen ◽

Hannah K Brown ◽

Simon S Cross ◽

C Alyson Evans ◽

...

Keyword(s):

Breast Cancer ◽

Bone Metastasis ◽

Expression Profiles ◽

Molecular Profile ◽

Calcium Signal ◽

Cell Homing ◽

Gene And Protein Expression ◽

Breast Cancer Bone Metastasis

Advanced breast cancer is associated with the development of incurable bone metastasis. The two key processes involved, tumour cell homing to and subsequent colonisation of bone, remain to be clearly defined. Genetic studies have indicated that different genes facilitate homing and colonisation of secondary sites. To identify specific changes in gene and protein expression associated with bone-homing or colonisation, we have developed a novel bone-seeking clone of MDA-MB-231 breast cancer cells that exclusively forms tumours in long bones following i.v. injection in nude mice. Bone-homing cells were indistinguishable from parental cells in terms of growth ratein vitroand when grown subcutaneouslyin vivo. Only bone-homing ability differed between the lines; once established in bone, tumours from both lines displayed similar rates of progression and caused the same extent of lytic bone disease. By comparing the molecular profile of a panel of metastasis-associated genes, we have identified differential expression profiles associated with bone-homing or colonisation. Bone-homing cells had decreased expression of the cell adhesion molecule fibronectin and the migration and calcium signal binding protein S100A4, in addition to increased expression of interleukin 1B. Bone colonisation was associated with increased fibronectin and upregulation of molecules influencing signal transduction pathways and breakdown of extracellular matrix, including hRAS and matrix metalloproteinase 9. Our data support the hypothesis that during early stages of breast cancer bone metastasis, a specific set of genes are altered to facilitate bone-homing, and that disruption of these may be required for effective therapeutic targeting of this process.

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Molecular Analysis of Fetal and Adult Primary Human Liver Sinusoidal Endothelial Cells: A Comparison to Other Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21207776 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7776

Author(s):

Muhammad Ahmer Jamil ◽

Heike Singer ◽

Rawya Al-Rifai ◽

Nicole Nüsgen ◽

Melanie Rath ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Molecular Profile ◽

Vascular Endothelial ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Genome Wide ◽

Differential Gene ◽

Methylation Profiling

In humans, Factor VIII (F8) deficiency leads to hemophilia A and F8 is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the specificity and characteristics of these cells in comparison to other endothelial cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human primary LSECs together with other fetal primary endothelial cells from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differentially regulated pathways and biofunctions in fetal LSECs. We used targeted bisulfite resequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in-house iPS generated vascular endothelial cells to primary LSECs; a higher similarity was found to fetal than to adult LSECs. In this study, we provide a detailed molecular profile of LSECs and a guide to testing the effectiveness of production of in vitro differentiated LSECs.

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