Cultured beef: from small biopsy to substantial quantity.

Cultured meat is an emerging technology with the potential to solve huge challenges related to the environmental, ethical, and health implications of conventional meat production. Establishing the basic science of cultured meat has been the primary focus of the last decade but it is now feasible that cultured meat products will enter the market within the next 3 to 4 years. This proximity to market introduction demands an evaluation of aspects of the cultured meat production process that have not yet been outlined or discussed in significant detail. For example, one technological approach for the production of cultured meat uses adult muscle stem cells, the limited proliferative capacity of which necessitates repeated collection of tissue samples via biopsies of living donor animals. The selection of donor animals and the details of biopsy processes must be optimized, as this is a key bottleneck in the cultured meat production process. The number of stem cells harvested from a biopsy, together with their proliferative capacity, determines a 'multiplicity factor' achieved by a cultured meat production process, thus dictating the reduction in number of animals required to produce a given quantity of meat. This article considers potential scenarios for these critical upstream steps, focusing on the production of cultured beef as an example. Considerations related to donor selection and details of the biopsy process are discussed in detail. The practicalities of various scenarios for cultured beef production, the health of donor animals, and regulatory issues associated with the safety of cultured meat for consumers are also considered. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Array CGH in human leukemia: from somatics to genetics.

During the past decade, array CGH has been applied to study copy number alterations in the genome in human leukemia in relation to prediction of prognosis or responsiveness to therapy. In the first segment of this review, we will focus on the identification of acquired mutations by array CGH, followed by studies on the pathogenesis of leukemia associated with germline genetic variants, phenotypic presentation and response to treatment. In the last section, we will discuss constitutional genomic aberrations causally related to myeloid leukemogenesis.

Constitutional 2p16.3 deletion including MSH6 and FBXO11 in a boy with developmental delay and diffuse large B-cell lymphoma.

We describe a case of a boy with neurodevelopmental delay and a diffuse large B-cell lymphoma (DLBCL) in whom we discovered a germline de novo 2p16.3 deletion including MSH6 and part of the FBXO11 gene. A causative role for MSH6 in cancer development was excluded based on tumor characteristics. The constitutional FBXO11 deletion explains the neurodevelopmental delay in the patient. The FBXO11 protein is involved in BCL-6 ubiquitination and BCL-6 is required for the germinal center reaction resulting in B cell differentiation. Somatic loss of function alterations of FBXO11 result in BCL-6 overexpression which is a known driver in DLBCL. We therefore consider that a causative relationship between the germline FBXO11 deletion and the development of DLBCL in this boy is conceivable.

The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA.

The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present in t(6;9) AML, encodes an invariable dek-can transcript. Sequence analysis of the dek-can cDNA showed that dek and can are merged without disruption of the original open reading frames and therefore the fusion mRNA encodes a chimeric DEK-CAN protein of 165 kDa. The predicted DEK and CAN proteins have molecular masses of 43 and 220 kDa, respectively. Sequence comparison with the EMBL data base failed to show consistent homology with any known protein sequences.

The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription.

The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 genes is critical for B-cell leukemogenesis.

The MN1-TEL fusion protein, encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a transcription factor with transforming activity.

The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

Relocation of the carboxyterminal part of CAN from the nuclear envelope to the nucleus as a result of leukemia-specific chromosome rearrangements.

Fusion genes encoding the 3' part of the can gene are implicated in two types of leukemia. The dek-can fusion gene is present in t(6;9) acute myeloid leukemia and the set-can fusion gene is present in one case of acute undifferentiated leukemia. In order to obtain leads towards the molecular basis of these diseases, we have studied the cellular localization of the DEK-CAN and SET-CAN fusion proteins and their normal counterparts. DEK-CAN and SET-CAN were localized exclusively in the nucleus, and also DEK and SET were found to be nuclear proteins. However, CAN was mainly located at the nuclear and cytoplasmic face of the nuclear envelope. This observation is in accordance with the presence of an amino acid repeat in the C-terminal part of CAN, common to the family of nucleoporins. The C-terminal part also contains a nuclear location domain as shown by deletion analysis. This domain may be important for the presence of CAN at the nucleoplasmic side of the nuclear envelope. The relocation of the carboxyterminal part of CAN due to DEK-CAN and SET-CAN may reinforce a nuclear function of the CAN protein.

Translocation (12;22) (p13;q11) in myeloproliferative disorders results in fusion of the ETS-like TEL gene on 12p13 to the MN1 gene on 22q11.

In myeloid and lymphoid leukemias recurrent chromosomal aberrations can be detected in chromosome region 12p13. We characterized the genes involved in t(12;22) (p13;q11) in two patients with myeloid leukemia and one with myelodysplastic syndrome (MDS). MN1, a gene on chromosome 22q11 was shown to be fused to TEL, a member of the family of ETS transcription factors on chromosome 12p13. The translocation results in transcription of the reciprocal fusion mRNAs, MN1-TEL and TEL-MN1, of which MN1-TEL is likely to encode an aberrant transcription factor containing the ETS DNA-binding domain of TEL. In addition to fusion of TEL to the PDGF beta receptor in t(5;12) in chronic myelomonocytic leukemia (CMML), our data suggest that the involvement of this protein in myeloid leukemogenesis could be dual; its isolated protein-protein dimerization and DNA-binding domains may be crucial for the oncogenic activation of functionally different fusion proteins.

TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance.

PURPOSE: TEL gene rearrangements due to the 12;21 chromosomal translocation are the most common molecular genetic abnormality in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of cases with a B-precursor immunophenotype. The limited number of clinically useful genetic markers in this leukemia subtype prompted us to assess TEL status as a predictor of treatment outcome. PATIENTS AND METHODS: We determined the status of the TEL gene (rearranged or germline) in 188 cases of B-precursor acute leukemia using Southern blot analysis and related the findings to event-free survival. All comparisons of outcome were stratified by treatment regimen, risk classification, age, and leukocyte count. RESULTS: Forty-eight patients (26%) had a rearranged TEL gene. At 5 years of follow-up, an estimated 91% +/- 5% (SE) of this group were event-free survivors, compared with only 65% +/- 5% of the group with germline TEL (stratified log-rank P = .011). For nonhyperdiploid patients, the odds ratio of an adverse event in the germline TEL group to that for the rearranged TEL group was 4.06 (95% confidence interval, 1.86 to 8.84). The relationship of TEL rearrangement to a favorable prognosis was independent of recognized good-risk features in B-precursor leukemia, including age, initial leukocyte count, and hyperdiploidy. CONCLUSION: Rearrangement of the TEL gene distinguishes a large subset of children with favorable-prognosis B-precursor leukemia who cannot be identified by standard prognostic features. It may be possible to treat these patients less aggressively without loss of therapeutic efficacy.

TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis.

The t(12;21)(p13;q22) is identified by routine cytogenetics in less than 0.05% of pediatric acute lymphoblastic leukemia (ALL) patients. This translocation encodes a TEL/AML-1 chimeric product comprising the helix-loop-helix domain of TEL, a member of the ETS-like family of transcription factors, fused to AML-1, the DNA-binding subunit of the AML-1/CBF beta transcription factor complex. Both TEL and AML-1 are involved in several myeloid leukemia-associated translocations with AML-1/CBF beta being altered in 20-30% of de novo acute myeloid leukemia (AML) cases. We now demonstrate that a TEL/AML1 chimeric transcript encoded by a cryptic t(12;21) is observed in 22% of pediatric ALL, making it the most common genetic lesion in these patients. Moreover, TEL/AML1 expression defined a distinct subgroup of patients characterized by an age between 1 and 10 years, B lineage immunophenotype, non-hyperdiploid DNA content and an excellent prognosis. These data demonstrate that molecular diagnostic approaches are invaluable in identifying clinically distinct subgroups, and that the AML1/CBF beta transcription complex is the most frequent target of chromosomal rearrangements in human leukemia.

Tel, a frequent target of leukemic translocations, induces cellular aggregation and influences expression of extracellular matrix components.

Tel is an Ets transcription factor that is the target of chromosome translocations in lymphoid and myeloid leukemias and in solid tumors. It contains two functional domains, a pointed oligomerization domain and a DNA-binding domain. Retroviral transduction of a wild-type Tel cDNA into a clonal subline of NIH3T3 fibroblasts resulted in a striking morphologic change: at confluency, the cells reorganized into a specific "bridge-like" pattern over the entire surface of the culture dish, and started migrating, thereby leaving circular holes in the monolayer. Thereafter, formation of cellular cords became apparent. This sequence of events was inhibited by coating the culture dishes with fibronectin and collagen IV. Retroviral transduction of Tel into MS1 endothelial cells reproduced the aggregation phenotype, but not the cellular cord formation. Tel-mutagenesis showed that both the pointed domain and the DNA-binding domain of Tel are required for the morphologic change. Other Ets family genes, Fli-1 and Ets-1 that are both endogenously expressed in endothelial cells, could not induce this morphologic change. Exogenous Tel expression is associated with transcriptional upregulation of entactin/nidogen, Smad5, Col3a1, CD44 and fibronectin, and downregulation of Col1a1 and secretory leukocyte protease inhibitor. Interestingly, Tel, Smad5, fibronectin, Col1a1 and Col3a1 all have essential roles during vascular development.

Towards effective and safe immunotherapy after allogeneic stem cell transplantation: identification of hematopoietic-specific minor histocompatibility antigen UTA2-1.

Donor T cells directed at hematopoietic system-specific minor histocompatibility antigens (mHags) are considered important cellular tools to induce therapeutic graft-versus-tumor (GvT) effects with low risk of graft-versus-host disease after allogeneic stem cell transplantation. To enable the clinical evaluation of the concept of mHag-based immunotherapy and subsequent broad implementation, the identification of more hematopoietic mHags with broad applicability is imperative. Here we describe novel mHag UTA2-1 with ideal characteristics for this purpose. We identified this antigen using genome-wide zygosity-genotype correlation analysis of a mHag-specific CD8(+) cytotoxic T lymphocyte (CTL) clone derived from a multiple myeloma patient who achieved a long-lasting complete remission after donor lymphocyte infusion from an human leukocyte antigen (HLA)-matched sibling. UTA2-1 is a polymorphic peptide presented by the common HLA molecule HLA-A*02:01, which is encoded by the bi-allelic hematopoietic-specific gene C12orf35. Tetramer analyses demonstrated an expansion of UTA2-1-directed T cells in patient blood samples after several donor T-cell infusions that mediated clinical GvT responses. More importantly, UTA2-1-specific CTL effectively lysed mHag(+) hematopoietic cells, including patient myeloma cells, without affecting non-hematopoietic cells. Thus, with the capacity to induce relevant immunotherapeutic CTLs, it's HLA-A*02 restriction and equally balanced phenotype frequency, UTA2-1 is a highly valuable mHag to facilitate clinical application of mHag-based immunotherapy.