Tertiary lymphoid structures and B cells determine clinically relevant T cell phenotypes in ovarian cancer.

Intratumoral tertiary lymphoid structures (TLSs) have been associated with improved outcome in various cohorts of patients with cancer, reflecting their contribution to the development of tumor-targeting immunity. Here, we demonstrate that high-grade serous ovarian carcinoma (HGSOC) contains distinct immune aggregates with varying degrees of organization and maturation. Specifically, mature TLSs (mTLS) as forming only in 16% of HGSOCs with relatively elevated tumor mutational burden (TMB) are associated with an increased intratumoral density of CD8+ effector T (TEFF) cells and TIM3+PD1+, hence poorly immune checkpoint inhibitor (ICI)-sensitive, CD8+ T cells. Conversely, CD8+ T cells from immunologically hot tumors like non-small cell lung carcinoma (NSCLC) are enriched in ICI-responsive TCF1+ PD1+ T cells. Spatial B-cell profiling identifies patterns of in situ maturation and differentiation associated with mTLSs. Moreover, B-cell depletion promotes signs of a dysfunctional CD8+ T cell compartment among tumor-infiltrating lymphocytes from freshly isolated HGSOC and NSCLC biopsies. Taken together, our data demonstrate that - at odds with NSCLC - HGSOC is associated with a low density of follicular helper T cells and thus develops a limited number of mTLS that might be insufficient to preserve a ICI-sensitive TCF1+PD1+ CD8+ T cell phenotype. These findings point to key quantitative and qualitative differences between mTLSs in ICI-responsive vs ICI-irresponsive neoplasms that may guide the development of alternative immunotherapies for patients with HGSOC.

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition.

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

A comparative study of beef quality after ageing longissimus muscle using a dry ageing bag, traditional dry ageing or vacuum package ageing.

The objective of this study was to investigate beef quality of longissimus muscle after ageing in dry ageing bags, traditional dry ageing or vacuum for 8 or 19 days. Lower ageing weight loss, odour score and microbial growth were found in meat aged in dry ageing bags than after traditional dry ageing. The sensory panel detected no differences for most of the sensory attributes between samples using the two dry ageing methods, except for the odour of the cutting surface. The dry-aged steaks had more umami and butter fried meat taste compared with vacuum-aged steaks. Ageing time affected most of the sensory traits in this study, which improved as ageing time increased from 8 to 19 days. In a consumer test, meat aged for 21 days in dry ageing bags was preferred than the samples aged in vacuum. This may be due to the higher tenderness and juiciness obtained during storage in dry ageing bags than meat aged in vacuum.

Improvac does not modify the expression and activities of the major drug metabolizing enzymes cytochrome P450 3A and 2C in pigs.

In the present study, we investigated hepatic mRNA expression and activities of CYP3A and 2C in entire, surgically castrated and pigs vaccinated with Improvac. Additionally, we examined the mRNA expression of the two nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), known to regulate CYP3A and 2C mRNA expression, respectively. Activities of CYP3A and 2C were estimated as a rate of 7-benzyloxy-4-trifluoromethylcoumarin and 7-benzyloxyquinoline metabolism (CYP3A) and tolbutamide metabolism (CYP2C). We found no effect of Improvac treatment or surgical castration on either CYP3A or 2C activities. Similarly, the mRNA expressions of CYP3A29, 2C33 and PXR were not changed. CAR mRNA expression differed only between entire and surgically castrated male pigs (p=0.005), being greater in surgically castrated pigs. Our results indicated that neither CYP3A nor 2C are affected by Improvac.

Simultaneous determination of cytochrome P450 1A, 2A and 3A activities in porcine liver microsomes.

The aim of this study was to develop a robust method for the simultaneous determination of the activities of three porcine CYP450 enzymes in hepatic microsomes. A cocktail consisting of three selective CYP450 probe substrates, 7-ethoxyresorufin (CYP1A), coumarin (CYP2A) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC; CYP3A), was incubated with porcine liver microsomes. The presence of 7-ethoxyresorufin appears to significantly influence the kinetics of coumarin hydroxylation and BFC O-debenzylation. These results indicate that the use of 7-ethoxyresorufin in substrate cocktails together with coumarin and BFC should be avoided.