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T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8 + T-cell populations can be obtained from ~33–50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient. Tumor-reactive T cells are generated by coculturing tumor organoids and autologous peripheral blood lymphocytes and are evaluated for their capacity to carry out effector functions after recognition of tumor cells and whether they kill tumor organoids.

Cancer neoantigens that arise from tumor mutations are drivers of tumor-specific T cell responses, but identification of T cell-recognized neoantigens in individual patients is challenging. Previous methods have restricted antigen discovery to selected HLA alleles, thereby limiting the breadth of neoantigen repertoires that can be uncovered. Here, we develop a genetic neoantigen screening system that allows sensitive identification of CD4 + and CD8 + T cell-recognized neoantigens across patients’ complete HLA genotypes. Patient-specific tumor neoantigens are recognized by T cells.

Hemp ( Cannabis sativa L.) is a multifunctional crop that is capable of prompt environmental adaptation. In this study, a monoecious cultivar (Futura 75) and a dioecious one (Finola) were tested in a mountain area in Valsaviore (Rhaetian Alps, Italy; elevation: 1,100 m a.s.l.) during the growing season 2018. Phytochemical behavior was evaluated by different analytical approaches: HPLC-high-resolution mass spectrometry, SDS-PAGE LC-MS/MS, HS-SPME GC-MS, and GC-FID in order to obtain complete profile of two varieties cultivated in altitude. CSR functional strategy used for ecological evaluation revealed that both genotypes are mainly competitors, although Finola is more stress tolerator (C:S:R = 57:26:17%) than Futura (C:S:R = 69:15:16%). The Finola inflorescences were characterized by higher quantities of β-ocimene and α-terpinolene, while α- and ß-pinene accompanied by extremely high ß-myrcene were found as predominant in Futura. Both varieties were rich in sesquiterpenes (45 recognized) among which trans -caryophyllene and α-humulene were the most abundant. Total tetrahydrocannabinol level was lower than 0.1%, while the most abundant cannabinoid was cannabidiolic acid (CBDA): 2.3% found in Finola vs. 2.7% revealed for Futura. The level of corresponding neutral form, cannabidiol, varied drastically: 0.27% (Finola) vs. 0.056% (Futura). Finola showed the unique cannabinoid profile with unexpectedly high cannabidivarin, 2-fold higher that corresponding acidic analogue, whereas the particularity of Futura 75 was the occurrence of cannabigerolic acid (CBGA) in the quantities that was double than those exposed for Finola. The seeds from both chemovars proved to be rich in polyunsaturated fatty acids, and Finola showed a higher ratio ω6/ω3. No difference was found in the protein content, and the SDS-PAGE profile was similar. The most abundant protein was edestin, followed by heat shock protein 70, ß-conglycinin, and vicilin. In conclusion, comprehensive phytochemical and ecological study of two fiber-type varieties cultivated in Italian Alps displayed specific, legal, and safe cannabinoids profile, followed by particular terpene composition, polyunsaturated fatty acids content, and favorable protein profile. This postulates that geographical provenience of hemp should be considered in selecting a variety that would be suitable for a specific end-use nutraceutical application.

Cannabis sativa L. (hemp) is a versatile plant which can adapt to various environmental conditions. Hempseeds provide high quality lipids, mainly represented by polyunsaturated acids, and highly digestible proteins rich of essential aminoacids. Hempseed composition can vary according to plant genotype, but other factors such as agronomic and climatic conditions can affect the presence of nutraceutic compounds. In this research, seeds from two cultivars of C. sativa (Futura 75 and Finola) grown in a mountain environment of the Italian Alps were analyzed. The main purpose of this study was to investigate changes in the protein profile of seeds obtained from such environments, using two methods (sequential and total proteins) for protein extraction and two analytical approaches SDS-PAGE and 2D-gel electrophoresis, followed by protein identification by mass spectrometry. The fatty acids profile and carotenoids content were also analysed. Mountain environments mainly affected fatty acid and protein profiles of Finola seeds. These changes were not predictable by the sole comparison of certified seeds from Futura 75 and Finola cultivars. The fatty acid profile confirmed a high PUFA content in both cultivars from mountain area, while protein analysis revealed a decrease in the protein content of Finola seeds from the experimental fields.

Drug resistance is a principal limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can retrospectively delineate the genetic basis of drug resistance, but this requires large numbers of post-treatment samples to nominate causal variants. Here we prospectively identify genetic mechanisms of resistance to ten oncology drugs from CRISPR base editing mutagenesis screens in four cancer cell lines using a guide RNA library predicted to install 32,476 variants in 11 cancer genes. We identify four functional classes of protein variants modulating drug sensitivity and use single-cell transcriptomics to reveal how these variants operate through distinct mechanisms, including eliciting a drug-addicted cell state. We identify variants that can be targeted with alternative inhibitors to overcome resistance and functionally validate an epidermal growth factor receptor (EGFR) variant that sensitizes lung cancer cells to EGFR inhibitors. Our variant-to-function map has implications for patient stratification, therapy combinations and drug scheduling in cancer treatment. Base editing screens of 11 cancer genes identify four functional classes of variants that collectively underpin sensitivity and resistance to ten commonly used drugs in cancer cell lines.

Organoid models allow for the study of key pathophysiological processes such as cancer biology in vitro. They offer insights into all aspects covering tumor development, progression and response to the treatment of tissue obtained from individual patients. Tumor organoids are therefore not only a better tumor model than classical monolayer cell cultures but can be used as personalized avatars for translational studies. In this review, we discuss recent developments in using organoid models for cancer research and what kinds of advanced models, testing procedures and readouts can be considered.

Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase ( PvACR2 ) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata , two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in Escherichia coli , purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5–8) in sporophytes and gametophytes of P. vittata . Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5–8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

Cannabis sativa (hemp) seeds are considered a functional food for their favorable contents of essential fatty acids, proteins and antioxidants. Beyond phenolics and carotenoids, the bioactivity of proteins has recently been investigated. However, plant genotype and environmental conditions can affect quantity and quality of macronutrients and phytochemicals in seeds, influencing their nutraceutical properties. In this study, the effects of plant variety and seed origin on the protein profile and antioxidant activity of hemp seeds were evaluated. Seeds from two cultivars, Secuieni Jubileu and Finola, were harvested from a mountain field located in Italy and compared with reference seeds used for sowing. Albumin and globulin extracts were obtained using the Osborne method and their antioxidant power was assayed (DPPH and ABTS methods). A matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry method was developed for protein fingerprinting analysis. Albumins from seeds of the mountain site showed higher radical scavenging activity and compounds of lower molecular weight than reference seeds, suggesting a role of proteins in the observed bioactivity. The MALDI-TOF method discriminated samples according to origin and variety, highlighting changes in the protein profile and identifying signals which could be used as markers of hemp cultivars.

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein.

Abstract Background Plasmodium ovale curtisi and wallikeri are perceived as relapsing malarial parasites. Contrary to Plasmodium vivax, direct evidence for this hypothesis is scarce. The aim of this prospective study was to characterize the reappearance patterns of ovale parasites. Methods P. ovale spp. infected patients were treated with artemether-lumefantrine and followed biweekly for up to 1 year for the detection of reappearing parasitemia. Molecular analysis of reappearing isolates was performed to identify homologous isolates by genotyping and to define cases of relapse following predefined criteria. Results At inclusion, 26 participants were positive for P. ovale curtisi and/or P. ovale wallikeri. The median duration of follow-up was 35 weeks. Reappearance of the same P. ovale species was observed in 46% of participants; 61% of P. ovale curtisi and 19% of P. ovale wallikeri infection-free intervals were estimated to end with reappearance by week 32. Based on the predefined criteria, 23% of participants were identified with 1 or 2 relapses, all induced by P. ovale curtisi. Conclusion These findings are in line with the currently accepted relapse theory inasmuch as the reappearance of P. ovale curtisi strains following initial blood clearance was conclusively demonstrated. Interestingly, no relapse of P. ovale wallikeri was observed. The formation of hypnozoites and consequential relapse of tertian malaria is a widely accepted theory that lacks, however, confirmation for Plasmodium ovale spp. This study clinically and molecularly evaluates reappearing P. ovale spp. parasites to provide new evidence.