Rotenone-insensitive NAD(P)H dehydrogenases in plants: Immunodetection and distribution of native proteins in mitochondria.

Antisera produced against peptides deduced from potato nda1 and ndb1, homologues of yeast genes for mitochondrial rotenone-insensitive NADH dehydrogenases, recognise respective proteins upon expression in Escherichiacoli. In western blots of potato (Solanum tuberosum L.) mitochondrial proteins, the NDB and NDA antibodies specifically detect polypeptides of 61 and 48 kDa, respectively. The proteins are found in mitochondria of flowers, leaves and tubers. Different signal intensities are seen relative to other respiratory chain components when organs are compared, indicating variations in relative abundance of dehydrogenases within the plant. The antibodies detect single polypeptides, of similar size as in potato, in mitochondria from several plant species. No specific cross-reaction was found in chloroplasts, but a weak NDA signal of 50 kDa was found in microsomes, possibly associated with peroxisomes. Two- dimensional native/SDS-PAGE analyses indicate that both NDA and NDB proteins reside as higher molecular mass forms, possibly oligomeric. The NDB immunoreactive protein is released by sonication of mitochondria, but resistant to extraction by digitonin and partially to Triton X-100. In comparison, the NDA protein remains bound to the inner membrane at sonication or digitonin treatment, but can be solubilised with Triton. Investigation of a beetroot (Beta vulgaris L.) induction system for external NADH dehydrogenase indicates that the NDB antibody does not recognise the induced external NADH dehydrogenase in this species, but possibly an external NADPH dehydrogenase

NAD(P) turnover in plant mitochondria

An analytical procedure based on alkaline extraction and HPLC analysis was adapted for quantification of pyridine nucleotides in plant mitochondria. The amounts of NAD and NADP extracted from seven different species varied from 1.0 to 3.7 and 0 to 0.5 nmol (mg protein) –1 , respectively. Although NADP was found in four species, its reduced form was in all cases below the detection limit of 0.1 nmol (mg protein) –1 . The NAD pool was mainly oxidized in the absence of substrates. However, oxidation of substrates followed by anaerobiosis caused 50–92% NAD pool reduction, indicating that the majority of the NAD+ was metabolically active. The NAD reduction level in potato tuber mitochondria oxidizing malate varied with assay conditions. The highest level of reduction (>80%) was reached at anaerobiosis, at pH 6.5 and 7.2, conditions favouring malic enzyme (ME), whereas the lowest reduction level (0%) was observed at pH 7.5, conditions favouring malate dehydrogenase (MDH). Mitochondria incubated at 0°C without respiratory substrate showed a loss of endogenous NAD + which correlated with a decline in the rate of oxidation of NAD+ -linked substrates. The lost NAD+ was mainly recovered as breakdown products in both the surrounding medium and the mitochondria. When submitochondrial fractions were incubated with NAD + or NADP + , the highest rate of NAD(P)+metabolism was detected in the outer membrane fraction. The metabolites detected, adenosine monophosphate (AMP), nicotinamide mononucleotide (NMN) and adenosine, imply that several enzymes involved in pyridine nucleotide degradation, including an NAD pyrophosphatase, are localized to the outer membrane

Machine-learning-based evaluation of intratumoral heterogeneity and tumor-stroma interface for clinical guidance

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Laurinavicius, A., Rasmusson, A., Plancoulaine, B., Shribak, M., & Levenson, R. Machine-learning-based evaluation of intratumoral heterogeneity and tumor-stroma interface for clinical guidance. American Journal of Pathology, 191(10), (2021): 1724–1731, https://doi.org/10.1016/j.ajpath.2021.04.008.Assessment of intratumoral heterogeneity and tumor-host interaction within the tumor microenvironment is becoming increasingly important for innovative cancer therapy decisions because of the unique information it can generate about the state of the disease. However, its assessment and quantification are limited by ambiguous definitions of the tumor-host interface and by human cognitive capacity in current pathology practice. Advances in machine learning and artificial intelligence have opened the field of digital pathology to novel tissue image analytics and feature extraction for generation of high-capacity computational disease management models. A particular benefit is expected from machine-learning applications that can perform extraction and quantification of subvisual features of both intratumoral heterogeneity and tumor microenvironment aspects. These methods generate information about cancer cell subpopulation heterogeneity, potential tumor-host interactions, and tissue microarchitecture, derived from morphologically resolved content using both explicit and implicit features. Several studies have achieved promising diagnostic, prognostic, and predictive artificial intelligence models that often outperform current clinical and pathology criteria. However, further effort is needed for clinical adoption of such methods through development of standardizable high-capacity workflows and proper validation studies.Supported by the European Social Fund grant 09.3.3-LMT-K-712

Antimycin A treatment decreases respiratory internal rotenone-insensitive NADH oxidation capacity in potato leaves

BACKGROUND: The plant respiratory chain contains several energy-dissipating enzymes, these being type II NAD(P)H dehydrogenases and the alternative oxidase, not present in mammals. The physiological functions of type II NAD(P)H dehydrogenases are largely unclear and little is known about their responses to stress. In this investigation, potato plants (Solanum tuberosum L., cv. Desiree) were sprayed with antimycin A, an inhibitor of the cytochrome pathway. Enzyme capacities of NAD(P)H dehydrogenases (EC 1.6.5.3) and the alternative oxidase were then analysed in isolated leaf mitochondria. RESULTS: We report a specific decrease in internal rotenone-insensitive NADH dehydrogenase capacity in mitochondria from antimycin A-treated leaves. External NADPH dehydrogenase and alternative oxidase capacities remained unaffected by the treatment. Western blotting revealed no change in protein abundance for two characterised NAD(P)H dehydrogenase homologues, NDA1 and NDB1, nor for two subunits of complex I. The alternative oxidase was at most only slightly increased. Transcript levels of nda1, as well as an expressed sequence tag derived from a previously uninvestigated closely related potato homologue, remained unchanged by the treatment. As compared to the daily rhythm-regulated nda1, the novel homologue displayed steady transcript levels over the time investigated. CONCLUSIONS: The internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves. However, the decrease is not due to changes in expression of known nda genes. One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted

Metabolomic evaluation of pulsed electric field-induced stress on potato tissue

Metabolite profiling was used to characterize stress responses of potato tissue subjected to reversible electroporation, providing insights on how potato tissue responds to a physical stimulus such as pulsed electric fields (PEF), which is an artificial stress. Wounded potato tissue was subjected to field strengths ranging from 200 to 400 V/cm, with a single rectangular pulse of 1 ms. Electroporation was demonstrated by propidium iodide staining of the cell nucleae. Metabolic profiling of data obtained through GC/TOF-MS and UPLC/TOF-MS complemented with orthogonal projections to latent structures clustering analysis showed that 24 h after the application of PEF, potato metabolism shows PEF-specific responses characterized by the changes in the hexose pool that may involve starch and ascorbic acid degradation.The Royal Physiographic Society in Lund, SwedenPortuguese Foundation of Science (FCT), PortugalDepartment of Cell and Organism Biology, Lund Universit

Pulsed electric field-induced cell permeabilisation of potato tissue lead to sustained metabolic changes

Metabolite profiling was used to characterize stress responses of potato tissue subjected to reversible electroporation, providing insights on how potato tissue responds to a physical stimulus such as pulsed electric fields (PEF), which is an artificial stress. Wounded potato tissue was subjected to field strengths ranging from 200 to 400 V/cm, with a single rectangular pulse of 1 ms. Electroporation was demonstrated by propidium iodide staining of the cells nucleae. Metabolic profiling of data obtained through GC/TOF-MS complemented with orthogonal projections to latent structures (OPLS) clustering analysis showed that 24 h after the application of PEF, potato metabolism shows PEF-specific responses characterized by the changes in the hexose pool that may involve starch and ascorbic acid degradation

Efficient, Unbiased Quality Assurance Of Automated Tissue Analysis Applicable To Daily Pathology Practice

Introduction/ Background Quantification of tissue biomarkers is increasingly demanded for diagnosis and is commonly performed by expert pathologists using microscopy of stained tissue at high magnification. This manual scoring is a reasonably fast, supervised procedure, but it suffers from inter- and intra-observer differences due to a) differences in selection of regions of interest, b) differences in quantity estimation, c) intra-tissue variability of biomarker expression. Computers and whole slide microscopy scanners have made it feasible to perform high-capacity analysis of high resolution images of tissue. Image analysis (IA) enables better reproducibility, but conversely, the unsupervised analysis introduces challenges regarding accuracy. Furthermore, borderline cases will always have to be rigorously inspected by pathologists. Many IA evaluation methods exist, but for pathology, a supervised comparison of experimental segmentation to an appropriately obtained standard criterion is the optimal strategy. The production of standard criterion necessitates evaluation of whole slide images to eliminate any possible region sampling bias while inter- and intra- observer bias can only be minimized by replacing any manual estimates by objective measurements. A logical step is thus to change the task of the pathologist from quantity estimation to verifying the output an automated procedure reports. Still, verification of entire tissue slides is in daily pathology practice too time-consuming. To minimize the workload pathology is turning to stereological methods which aim to efficiently quantify matter unbiasedly and have been proved useful for supervised validation of automated analysis for Ki67 scoring of breast cancer. However, the workload still needs to be reduced to a level comparable to the manual scoring procedure. Aims We aim to enable high accuracy, objective evaluation of automated image analysis with a workload and workflow feasible for daily pathology practice. This regards both production of reference data for image analysis tool calibration and continuous quality control inspection of borderline cases. Methods This study investigates proportionate sampling, a very efficient stereological sampling scheme utilizing weighted sampling of regions of automated image analysis for manual evaluation of automated IA. The sampling of regions to be inspected by a pathologist draws upon the IA to assign probability weights to all regions. This results in a highly efficient, unbiased sampling and quality assurance estimate for the automated image analysis. Results Presented here is proof-of-concept of an efficient, unbiased image analysis evaluation methodology. The task of the pathologist is changed from quantity estimation to instead annotate discrepancies between the output from the IA and the tissue in a few sampled regions. From the annotations an unbiased quality assurance estimate of the IA can be estimated including levels of accuracy obtainable and expected workloads. This confirms that the stereological proportionate sampling enables manual verification of automated whole slide image analysis for unbiased reference dataset creation and quality control inspection in borderline cases. Furthermore, the methodology is easily integrated into both image analysis platforms for production of reference data sets and laboratory information systems for daily pathology practices.

Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

<p>Abstract</p> <p>Background</p> <p>Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus <it>Trichoderma viride</it>. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (<it>Nicotiana tabacum </it>L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.</p> <p>Results</p> <p>Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids (PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes.</p> <p>Conclusion</p> <p>We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by <it>Trichoderma spp</it>. to attack other microorganisms, and thus adding to the beneficial effect that <it>Trichoderma </it>has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to <it>in vivo </it>conditions.</p