Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Proofs of different kind are presented of the existence of highly active bacteria producing IAA from tryptophan on plant surfaces and in plant homogenates. Both homogenates and washing solutions of nonsterile pea plant parts are active in producing IAA from tryptophan. Activity is much enhanced by the addition of glucose or by preincubating the preparations; it is abolished by sterile filtration, by some bactericidic and bacteriostatic substances, by chloramphenicol, streptomycin, and albucid (penicillin being only partly effective). Preparations of sterile plants do not produce IAA from tryptophan within the detection limit of the Salkowski test. The bacteria are even present on seed surfaces, in the air, and in aceton or ammonium sulfate precipitations of homogenates. Main products of the bacterial tryptophan conversion, as demonstrated by paper chromatography, are indolepyruvic acid, indoleacetic acid, indolecarboxaldehyde, and indolecarboxylic acid. In presence of glucose indolepyruvic acid is by far dominating. Many hitherto known results about tryptophan conversion to IAA by higher plants are likely to be falsified by epiphytic bacteria.     

High‐throughput identification of conditional MHCI ligands and scaled‐up production of conditional MHCI complexes

Despite the need to monitor the impact of Cancer Immunotherapy (CI)/Immuno‐Oncology (IO) therapeutics on neoantigen‐specific T‐cell responses, very few clinical programs incorporate this aspect of immune monitoring due to the challenges in high‐throughput (HTP) generation of Major Histocompatibility Complex Class I (MHCI) tetramers across a wide range of HLA alleles. This limitation was recently addressed through the development of MHCI complexes with peptides containing a nonnatural UV cleavable amino acid (conditional MHCI ligands) that enabled HTP peptide exchange upon UV exposure. Despite this advancement, the number of alleles with known conditional MHCI ligands is limited. We developed a novel workflow to enable identification and validation of conditional MHCI ligands across a range of HLA alleles. First, known peptide binders were screened via an enzyme‐linked immunosorbent assay (ELISA) assay. Conditional MHCI ligands were designed using the highest‐performing peptides and evaluated in the same ELISA assay. The top performers were then selected for scale‐up production. Next‐generation analytical techniques (LC/MS, SEC‐MALS, and 2D LC/MS) were used to characterize the complex after refolding with the conditional MHCI ligands. Finally, we used 2D LC/MS to evaluate peptide exchange with these scaled‐up conditional MHCI complexes after UV exposure with validated peptide binders. Successful peptide exchange was observed for all conditional MHCI ligands upon UV exposure, validating our screening approach. This approach has the potential to be broadly applied and enable HTP generation of MHCI monomers and tetramers across a wider range of HLA alleles, which could be critical to enabling the use of MHCI tetramers to monitor neoantigen‐specific T‐cells in the clinic.     

The influence of epiphytic bacteriae on auxin metabolism

Summary Plants are settled by epiphytic bacteriae able to convert tryptophan to IAA. This bacterial activity is abolished by chloramphenicol and streptomycin but not by penicillin. Tryptophan conversion to IAA by plant parts or enzyme preparations is far more intensive in non-sterile conditions than in sterile ones. This is true for all investigated objects: Helianthus annuus, Phaseolus vulgaris, Pisum sativum, Triticum vulgare, Zea mays, Enteromorpha compressa, Fucus vesiculosus, Furcellaria fastigiata. From pea plants, 58 strains of IAA producing bacteriae were isolated and partly identified.While non-sterile plants (Pisum, Zea) contain considerable amounts of IAA (extraction, thin layer chromatography, biotest), hardly any traceable auxin can be extracted of sterile plants. But sterile plants re-infected with mixtures or single strains of suitable bacteriae again contain considerable amounts of extractable IAA.

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Zusammenfassung Pflanzen sind von epiphytischen Bakterien besiedelt, die Tryptophan zu IES umsetzen können. Diese bakterielle Aktivität wird unterbunden durch Chloramphenicol und Streptomycin, aber nicht durch Penicillin. Der Tryptophanumsatz zu IES durch Pflanzenteile oder Enzympräparate ist unter unsterilen Bedingungen weit intensiver als unter sterilen. Das trifft für alle untersuchten Objekte zu: Helianthus annuus, Phaseolus vulgaris, Pisum sativum, Triticum vulgare, Zea mays, Enteromorpha compressa, Fucus vesiculosus, Furcellaria fastigiata. Von Erbsenpflanzen konnten 58 Stämme IES-produzierender Bakterien isoliert und zum Teil identifiziert werden.Während unsterile Pflanzen (Pisum, Zea) beträchtliche IES-Mengen enthalten (Extraktion, Dünnschichtchromatographie, Biotest), kann aus sterilen Pflanzen kaum nachweisbares Auxin extrahiert werden. Aber sterile, mit Mischungen oder einzelnen Stämmen geeigneter Bakterien re-infizierte Pflanzen enthalten wieder beträchtliche Menge extrahierbarer IES.

     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Homogenates of epicotyls or roots of nonsterile pea plants incubated with tryptophan produce IAA within 1 to 4 hours, which was detected by means of the Avena curvature test and thin layer chromatography. Three results prove this short-term IAA production to be mainly caused by epiphytic bacteria: 1) Homogenates of sterile plant parts catalyze a conversion of tryptophan to IAA, a hundredfold lower. 2) Chloramphenicol or streptomycin very actively reduce the IAA gain obtained with nonsterile homogenates. 3) Washing solutions of nonsterile plant parts which do not contain plant enzymes but only epiphytic bacteria, produce IAA from tryptophan, too. IAA synthesis from tryptophan in vitro by enzymes of the pea plant occurs with lower intensity than hitherto known; possibly it is physiologically unimportant. It is discussed to what extent the hitherto existing research work about the IAA biogenesis in higher plants might be incriminated by disregarding tbe rôle of epiphytic bacteria.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metaholism

During an ether extraction (20 h, 4°C), nonsterilc pea epicotyls deliver at least 5 times more auxin than sterile ones. A great part of the additional auxin originates from a bacterial auxin production during the extraction. Presence of chloramphenicol or streptomycin during the extraction lowers the auxin amount extractable from nonsterile epicotyls. In extraction conditions (i.e. covered with ether, 20 h, 4°C), epiphytic bacteria contained in homogenates of nonsterile plant parts produce IAA from added tryptophan. Furthermore, first results are presented underlining the fact that epiphytic bacteria already produce auxin at the surface of nonsterile plant parts (before an extraction).     

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF_19-27) and NY-ESO-1_157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.