Legume Alternative Oxidase Isoforms Show Differential Sensitivity to Pyruvate Activation

Alternative oxidase (AOX) is an important component of the plant respiratory pathway, enabling a route for electrons that bypasses the energy-conserving, ROS-producing complexes of the mitochondrial electron transport chain. Plants contain numerous isoforms of AOX, classified as either AOX1 or AOX2. AOX1 isoforms have received the most attention due to their importance in stress responses across a wide range of species. However, the propensity for at least one isoform of AOX2 to accumulate to very high levels in photosynthetic tissues of all legumes studied to date, suggests that this isoform has specialized roles, but we know little of its properties. Previous studies with sub-mitochondrial particles of soybean cotyledons and roots indicated that differential expression of GmAOX1, GmAOX2A, and GmAOX2D across tissues might confer different activation kinetics with pyruvate. We have investigated this using recombinantly expressed isoforms of soybean AOX in a previously described bacterial system (Selinski et al., 2016, Physiologia Plantarum 157, 264-279). Pyruvate activation kinetics were similar between the two GmAOX2 isoforms but differed substantially from those of GmAOX1, suggesting that selective expression of AOX1 and 2 could determine the level of AOX activity. However, this alone cannot completely explain the differences seen in sub-mitochondrial particles isolated from different legume tissues and possible reasons for this are discussed.

Current status of the hypothesis of a claustro-insular homolog in sauropsids.

The author worked before on the wide problem of the evolution of the vertebrate pallium. He proposed various Bauplan models based in the definition of a set of pallial sectors with characteristic (topologically invariant) mutual relationships and distinct molecular profiles. Out of one of these models, known as the ‘updated tetrapartite pallium model’, a modified definition of the earlier lateral pallium sector (LPall) emerged, which characterized it in mammals as consisting of an unitary claustro-insular transitional (mesocortical) complex intercalated between neocortex or dorsal pallium (DPall) above and olfactory cortex or ventral pallium (VPall) underneath. A distinctive molecular marker of the early-born deep claustral component of the LPall was found to be the transcription factor Nr4a2, which is not expressed significantly in the overlying insular cortex or in adjoining cortical territories (Puelles 2014). Given that earlier comparative studies had identified molecularly and topologically comparable VPall, LPall and DPall sectors in the avian pallium, an avian Nr4a2 probe was applied aiming to identify the reportedly absent avian claustro-insular complex. An early-born superficial subpopulation of the avian LPall that expresses selectively this marker through development was indeed found. This was proposed to be a claustrum homolog, whereas the remaining Nr4a2-negative avian LPall cells were assumed to represent a possible insular homolog (Puelles et al. 2016a). This last notion was supported by comparable selective expression of the mouse insular marker Cyp26b, also found restricted to the avian LPall (Puelles 2017). Some published data suggested that similar molecular properties and structure apply at the reptilian LPall. This analysis was reviewed in Puelles et al. (2017). The present commentary discusses 3-4 years later some international publications accrued in the interval that touch on the claustro-insular homology hypothesis. Some of them are opposed to the hypothesis whereas others corroborate or support it. This raises a number of secondary issues of general interest.

Designing of RNA Molecule Translating for Activitable Melittin as Selective Targeting of Leishmania Infected Cells

Background: Leishmaniasis is characterized by strong inflammatory responses with high levels of inflammatory cytokines that induce microRNA 21 and matrix metalloproteinases. Melittin has inhibitory effects on proliferation of various cells via induction of apoptosis. Melittin can be integrated in cell membranes and induce apoptosis. Thus, designation of biomolecules for the selective destroy of the infected cells is a treatment option. One approach is the precise engineering of constructs for the selective expression of melittin in the infected cells. Methods: For this aim we designed a construct composing melittin nucleotide sequence and nucleotide sequence coding for polyanionic peptide function inhibitory element to further guarantee the selective function of melittin in inflamed tissues and infected cells, were included in a construct as melittin inhibitor via matrix metalloproteinase degradable linker. Results: Reverse complementary sequences were designed so melittin sequences for the selective targeting of Leishmania could be expressed in infected cells using cell microRNA machinery. Conclusion: Translation machinery in infected cells with increased miR-21 could translate melittin, MMP linker and polyanionic inhibitor through a non-canonical pathway. Then, the MMP linker is degraded and selective killing of Leishmania infected cells would happen.

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

Generation of a HuTCR mouse platform-derived MAGE-A1-directed high-affinity TCR with superior potency versus human-derived TCRs.

e14515 Background: As cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are suppressed via negative thymic selection. Therefore, patient- or donor-derived TCRs are typically of low affinity and result in a reduced antitumor effect. Using our proprietary HuTCR platform, which consists of mouse lines carrying the full human TCR α/β loci in combination with common human HLA alleles, we have isolated high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development. Furthermore, we validated MAGE-A1 as a potential cancer therapy target by using immunohistochemistry to evaluate expression in several major tumor types and healthy tissue. Methods: Using scRNAseq, TCRs were isolated from HuTCR mice. Human-derived MAGE-A1-specific TCR sequences were obtained from publicly available databases. All TCRs were expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional avidity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope peptides and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by co-culture with a panel of tumor cell lines varying in MAGE-A1 and/or MHC-class-I expression. MAGE-A1 expression on protein level was evaluated by immunohistochemistry. Results: Immunization of HuTCR mice with the antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR affinities ranging from 0.3 nM to 3 nM. By comparison, human-derived TCRs exhibited generally lower functional avidity from 3 nM to 60 nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cell lines expressing low MAGE-A1 and/or HLA-A2. Immunohistochemical analysis of MAGE-A1 expression in healthy tissues demonstrated highly selective expression of MAGE-A1 in testis, only. Screening for expression confirmed that a significant proportion of several major cancer types expresses MAGE-A1 as reported by various other groups [reviewed in Curr Opin Cell Biol. 2015 December; 37: 1–8]. Conclusions: The HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. In addition, it was confirmed that MAGE-A1 has a highly selective expression pattern in healthy tissues (testis, only), but shows distinct expression in several major human tumor types.