Why do plants blush when they are hungry?

Foliar anthocyanins, as well as other secondary metabolites, accumulate transiently under nutritional stress. A misconception that only nitrogen or phosphorus deficiency induces leaf purpling/reddening has led to overuse of fertilizers that burden the environment. Here, we emphasize that several other nutritional imbalances induce anthocyanin accumulation, and nutrient-specific differences in this response have been reported for some deficiencies. A range of ecophysiological functions have been attributed to anthocyanins. We discuss the proposed functions and signalling pathways that elicit anthocyanin synthesis in nutrient-stressed leaves. Knowledge from the fields of genetics, molecular biology, ecophysiology and plant nutrition is combined to deduce how and why anthocyanins accumulate under nutritional stress. Future research to fully understand the mechanisms and nuances of foliar anthocyanin accumulation in nutrient-stressed crops could be utilized to allow these leaf pigments to act as bioindicators for demand-oriented application of fertilizers. This would benefit the environment, being timely due to the increasing impact of the climate crisis on crop performance.

Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.

Extracellular matrix stiffening is a quintessential feature of cartilage aging, a leading cause of knee osteoarthritis. Yet, the downstream molecular and cellular consequences of age-related biophysical alterations are poorly understood. Here, we show that epigenetic regulation of α-Klotho represents a novel mechanosensitive mechanism by which the aged extracellular matrix influences chondrocyte physiology. Using mass spectrometry proteomics followed by a series of genetic and pharmacological manipulations, we discovered that increased matrix stiffness drove Klotho promoter methylation, downregulated Klotho gene expression, and accelerated chondrocyte senescence in vitro. In contrast, exposing aged chondrocytes to a soft matrix restored a more youthful phenotype in vitro and enhanced cartilage integrity in vivo. Our findings demonstrate that age-related alterations in extracellular matrix biophysical properties initiate pathogenic mechanotransductive signaling that promotes Klotho promoter methylation and compromises cellular health. These findings are likely to have broad implications even beyond cartilage for the field of aging research.

The green light gap: a window of opportunity for optogenetic control of stomatal movement.

Green plants are equipped with photoreceptors that are capable of sensing radiation in the ultraviolet-to-blue and the red-to-far-red parts of the light spectrum. However, plant cells are not particularly sensitive to green light (GL), and light which lies within this part of the spectrum does not efficiently trigger the opening of stomatal pores. Here, we discuss the current knowledge of stomatal responses to light, which are either provoked via photosynthetically active radiation or by specific blue light (BL) signaling pathways. The limited impact of GL on stomatal movements provides a unique option to use this light quality to control optogenetic tools. Recently, several of these tools have been optimized for use in plant biological research, either to control gene expression, or to provoke ion fluxes. Initial studies with the BL-activated potassium channel BLINK1 showed that this tool can speed up stomatal movements. Moreover, the GL-sensitive anion channel GtACR1 can induce stomatal closure, even at conditions that provoke stomatal opening in wild-type plants. Given that crop plants in controlled-environment agriculture and horticulture are often cultivated with artificial light sources (i.e. a combination of blue and red light from light-emitting diodes), GL signals can be used as a remote-control signal that controls stomatal transpiration and water consumption.

System-wide analyses reveal essential roles of N-terminal protein modification in bacterial membrane integrity.

The removal of the N-terminal formyl group on nascent proteins by peptide deformylase (PDF) is the most prevalent protein modification in bacteria. PDF is a critical target of antibiotic development; however, its role in bacterial physiology remains a long-standing question. This work used the time-resolved analyses of the Escherichia coli translatome and proteome to investigate the consequences of PDF inhibition. Loss of PDF activity rapidly induces cellular stress responses, especially those associated with protein misfolding and membrane defects, followed by a global down-regulation of metabolic pathways. Rapid membrane hyperpolarization and impaired membrane integrity were observed shortly after PDF inhibition, suggesting that the plasma membrane disruption is the most immediate and primary consequence of formyl group retention on nascent proteins. This work resolves the physiological function of a ubiquitous protein modification and uncovers its crucial role in maintaining the structure and function of the bacterial membrane.

A proteome-wide map of 20(S)-hydroxycholesterol interactors in cell membranes.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC-Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy.

A Plasma-Based Protein Marker Panel for Colorectal Cancer Detection Identified by Multiplex Targeted Mass Spectrometry.

INTRODUCTION: Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance. MATERIALS AND METHODS: A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days. RESULTS: Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%. CONCLUSION: These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.

Discovery and Validation of Plasma-Protein Biomarker Panels for the Detection of Colorectal Cancer and Advanced Adenoma in a Danish Collection of Samples from Patients Referred for Diagnostic Colonoscopy.

BACKGROUND: Well-collected and well-documented sample repositories are necessary for disease biomarker development. The availability of significant numbers of samples with the associated patient information enables biomarker validation to proceed with maximum efficacy and minimum bias. The creation and utilization of such a resource is an important step in the development of blood-based biomarker tests for colorectal cancer. METHODS: We have created a subject data and biological sample resource, Endoscopy II, which is based on 4698 individuals referred for diagnostic colonoscopy in Denmark between May 2010 and November 2012. Of the patients referred based on 1 or more clinical symptoms of colorectal neoplasia, 512 were confirmed by pathology to have colorectal cancer and 399 were confirmed to have advanced adenoma. Using subsets of these sample groups in case-control study designs (300 patients for colorectal cancer, 302 patients for advanced adenoma), 2 panels of plasma-based proteins for colorectal cancer and 1 panel for advanced adenoma were identified and validated based on ELISA data obtained for 28 proteins from the samples. RESULTS: One of the validated colorectal cancer panels was comprised of 8 proteins (CATD, CEA, CO3, CO9, SEPR, AACT, MIF, and PSGL) and had a validation ROC curve area under the curve (AUC) of 0.82 (CI 0.75-0.88). There was no significant difference in the performance between early- and late-stage cancer. The advanced adenoma panel was comprised of 4 proteins (CATD, CLUS, GDF15, SAA1) and had a validation ROC curve AUC of 0.65 (CI 0.56-0.74). CONCLUSIONS: These results suggest that the development of blood-based aids to colorectal cancer detection and diagnosis is feasible.

Entering the era of targeted therapy for chronic lymphocytic leukemia: impact on the practicing clinician.

PURPOSE: Chemoimmunotherapy has been the standard of care for chronic lymphocytic leukemia (CLL). However, the introduction of B-cell receptor (BCR) kinase inhibitors such as ibrutinib has the potential to eliminate the role of chemotherapy in the treatment of CLL. How to best incorporate old and new therapies for CLL in this landscape is increasingly complex. METHODS: This article reviews current data available to clinicians and integrates these data to provide a strategy that can be used to approach the treatment of CLL in the era of BCR signaling inhibitors. RESULTS: Current strategies separate patients based on age or functional status as well as genetics [presence or absence of del(17)(p13.1)]. In the era of targeted therapy, this will likely continue based on current available data. Phase III studies support chemoimmunotherapy as the initial standard therapy for patients without del(17)(p13.1). Choice of chemotherapy (fludarabine plus cyclophosphamide, bendamustine, or chlorambucil) and anti-CD20 antibody (rituximab, ofatumumab, or obinutuzumab) varies based on regimen and patient status. For patients with del(17)(p13.1), no standard initial therapy exists, although several options supported by phase II clinical trials (methylprednisolone plus alemtuzumab or ibrutinib) seem better than chemoimmunotherapy. Treatment of relapsed CLL seems to be best supported by ibrutinib-based therapy. Completion of trials with ibrutinib and other new agents in the near future will offer opportunity for chemotherapy-free treatment across all groups of CLL. CONCLUSION: Therapy for CLL has evolved significantly over the past decade with introduction of targeted therapy for CLL. This has the potential to completely transform how CLL is treated in the future.

Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia.

BACKGROUND: In patients with chronic lymphoid leukemia (CLL) or small lymphocytic lymphoma (SLL), a short duration of response to therapy or adverse cytogenetic abnormalities are associated with a poor outcome. We evaluated the efficacy of ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase, in patients at risk for a poor outcome. METHODS: In this multicenter, open-label, phase 3 study, we randomly assigned 391 patients with relapsed or refractory CLL or SLL to receive daily ibrutinib or the anti-CD20 antibody ofatumumab. The primary end point was the duration of progression-free survival, with the duration of overall survival and the overall response rate as secondary end points. RESULTS: At a median follow-up of 9.4 months, ibrutinib significantly improved progression-free survival; the median duration was not reached in the ibrutinib group (with a rate of progression-free survival of 88% at 6 months), as compared with a median of 8.1 months in the ofatumumab group (hazard ratio for progression or death in the ibrutinib group, 0.22; P<0.001). Ibrutinib also significantly improved overall survival (hazard ratio for death, 0.43; P=0.005). At 12 months, the overall survival rate was 90% in the ibrutinib group and 81% in the ofatumumab group. The overall response rate was significantly higher in the ibrutinib group than in the ofatumumab group (42.6% vs. 4.1%, P<0.001). An additional 20% of ibrutinib-treated patients had a partial response with lymphocytosis. Similar effects were observed regardless of whether patients had a chromosome 17p13.1 deletion or resistance to purine analogues. The most frequent nonhematologic adverse events were diarrhea, fatigue, pyrexia, and nausea in the ibrutinib group and fatigue, infusion-related reactions, and cough in the ofatumumab group. CONCLUSIONS: Ibrutinib, as compared with ofatumumab, significantly improved progression-free survival, overall survival, and response rate among patients with previously treated CLL or SLL. (Funded by Pharmacyclics and Janssen; RESONATE ClinicalTrials.gov number, NCT01578707.).

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Lipid and phospholipid profiling of biological samples using MALDI Fourier transform mass spectrometry.

Here we describe a study of the feasibility of lipid and phospholipid (PL) profiling using matrix assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) for two different applications. In this work PL profiles of different mammalian tissues as well as those of whole cell organisms were examined. In particular, comparative analysis of lipid and PL profiles of tissues from mice fed different diets was done and, in another application, MALDI FTMS was used to analyze PL profiles of genetically modified Saccharomyces cerevisiae. Computational sorting of the observed ions was done in order to group the lipid and PL ions from complex MALDI spectra. The PL profiles of liver tissues from mice fed different diets showed a cross correlation coefficient of 0.2580, indicating significant dissimilarity, and revealed more than 30 significantly different peaks at the 99.9% confidence level. Histogram plots derived from the spectra of wild type and genetically modified yeast resulted in a cross correlation coefficient 0.8941 showing greater similarity, but still revealing a number of significantly different peaks. Based on these results, it appears possible to use MALDI FTMS to identify PLs as potential biomarkers for metabolic processes in whole cells and tissues.

Characterizing the phospholipid profiles in mammalian tissues by MALDI FTMS.

Discussed here is an analytical method for profiling lipids and phospholipids directly from mammalian tissues excised from Mus musculus (house mouse). Biochemical analysis was accomplished through the use of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry, where whole tissue sections of mouse brain, heart, and liver were investigated. Lipid and phospholipid ions create complex MALDI mass spectra containing multiple ions with different m/z values corresponding to the same fundamental chemical species. When a computational sorting approach is used to group these ions, the standard deviation for observed relative chemical abundance can be reduced to 6.02%. Relative standard deviations of 10% are commonly accepted for standard chromatographic phospholipid analyses. Average mass measurement accuracy for 232 spectra representing three tissue types from 12 specimens was calculated to be 0.0053 Da. Further it is observed, that the data and the analysis between all the animals have near-identical phospholipid contents in their brain, heart, and liver tissues, respectively. In addition to the need to accurately measure relative abundances of phospholipid species, it is essential to have adequate mass resolution for complete and accurate overall analysis. It is reasonable to make mass composition assignments with spectral resolving power greater than 8000. However, results from the present study reveal 14 instances (C12 carbon isotope) of multiple m/z ions having the same nominal value that require greater resolution in order that overlap will not occur. Spectra measured here have an average resolving power of 12 000. It is established that high mass resolution and mass accuracy coupled with MALDI ionization provide for rapid and accurate phospholipid analysis of mammalian tissue sections.

Real-time monitoring of recombinant bacterial proteins by mass spectrometry.

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.

Ionic liquid matrix-induced metastable decay of peptides and oligonucleotides and stabilization of phospholipids in MALDI FTMS analyses.

Room-temperature ionic liquid matrices (ILMs) have recently been investigated for use in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and proven to be advantageous. Literature accounts of ILM performance for biological samples document increased sensitivity and ionization efficiency. These claims have been investigated here, and are supported for MALDI TOF applications to peptides, oligonucleotides, and phospholipids. Peptides and oligonucleotides however, do not behave in the same way when ILMs are used for MALDI FTMS. As reported here, with 3 tesla MALDI FTMS peptides and oligonucleotides fragment readily. These observations contrast with those found for MALDI time-of-flight mass spectrometry. Fragmentation is apparently slower than the time required to accelerate ions in a MALDI TOF mass spectrometer, but is readily observed by MALDI FTMS. Therefore, fragmentation of these molecules must occur on a relatively slow time scale. As trapping time is extended, increased fragmentation of peptides and oligonucleotides is seen. However, phospholipids do not fragment extensively. Furthermore, use of traditional solid matrices causes significant fragmentation for this category of compound but is suppressed by use of ILMs.

A comprehensive and comparative analysis for MALDI FTMS lipid and phospholipid profiles from biological samples.

Described here is a computationally automated method for translating complex accurate mass spectra into biologically relevant and meaningful data. Rapid profiling of detailed high resolution mass spectra resulting from direct analysis of whole cells and tissues by matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) is discussed. Lipid and phospholipid ions create complex spectra containing multiple m/z values corresponding to the same fundamental chemical species. A computational approach is employed to sort ions, with mass to charge ratios lower than m/z 1000, into groups of similar lipid and phospholipid compositions for comprehensive and rapid analysis. By sorting or binning ions in this manner, variations in the degree of cation exchange can be avoided, thus increasing the comparability of the data. The result is displayed as a histogram that is easily interpretable and comparable with similar analyses and is particularly useful for direct comparison of similar tissues. Spectra of leaves from a healthy Prunus persica (peach) tree are compared with those from leaves infected by the fungus Taphrina deformans. Although the infection can be seen as a difference in leaf structure and by visual inspection of the mass spectra, the method described here details the chemical difference in phospholipid compositions and their relative abundances.

Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS.

Ions attributed to lipids and phospholipids are directly observed by desorption from whole bacteria using intact cell (IC) matrix-assisted laser desorption-ionization (MALDI) Fourier transform mass spectrometry (FTMS). Saccharomyces cerevisiae are grown in rich media broth, concentrated, and applied directly to the MALDI surface without lysis or chemical treatment. FTMS of MALDI ions gives excellent signal to noise ratios with typical resolving powers of 90,000 and mass precision better than 0.002 Da. Use of accurate mass measurements and a simple set of rules allow assignment of major peaks into one of twelve expected lipid classes. Subsequently, fractional mass versus whole number mass plots are employed to enhance visual interpretation of the high-resolution data and to facilitate detection of related ions such as those representing homologous series or different degrees of unsaturation. This approach, coupled with rules based on bacterial biochemistry, is used to classify ions with m/z up to about 1000. Major spectral peaks in the range m/z 200-1000 are assigned as lipids and phospholipids. In this study, it is assumed that biologically-derived ions with m/z values lower than 1000 are lipids. This is not unreasonable in view of the facts that molecular weights of lipids are almost always less than 1000 Da, that the copy numbers for lipids in a cell are higher than those for any single protein or other component, and that lipids are generally collections of distinct homologous partners, unlike proteins or other cell components. This paper presents a new rapid lipid-profiling method based on IC MALDI-FTMS.

Use of double-depleted 13C and 15N culture media for analysis of whole cell bacteria by MALDI time-of-flight and Fourier transform mass spectrometry.

In the present paper, results demonstrating the significant advantages of matrix-assisted laser desorption/ionization (MALDI) analysis of whole cell samples of bacteria grown on double isotopically-depleted (13C and 15N) media are presented. It is shown that several advantages accrue for MALDI with a 9.4 T Fourier transform mass spectrometer (FTMS). Of particular note, for analysis of whole cells, sample preparation is simple and chemical interference is reduced. Moreover, ion coalescence problems are minimized, and data-base identification of proteins facilitated. Furthermore, high resolution mass spectra obtained from such whole cells show significant improvement in apparent mass resolving power and mass measurement accuracy, whether time-of-flight or FTMS MALDI is used. As a consequence, it becomes possible to detect subtle details in the chemistry of the organism, such as the presence of both post-translationally modified and unmodified versions of the same proteins. This approach is also adaptable to direct assay of over-expressed proteins from Escherichia coli cultures and should facilitate studies aimed at the detection of medically important cellular biomarker proteins.

Investigation of MALDI-TOF and FT-MS techniques for analysis of Escherichia coli whole cells.

Recently, it has been demonstrated that bacteria can be characterized using whole cells and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, identification of specific bacterial proteins usually requires analysis of cellular fractions or purified extracts. Here, the first application of Fourier transform mass spectrometry (FTMS) to analysis of bacterial proteins directly from whole cells is reported. It is shown that accurate mass MALDI-FTMS can be used to characterize specific ribosomal proteins directly from Escherichia coli cells. High-accuracy mass measurements and high-resolution isotope profile data confirm posttranslational modifications proposed previously on the basis of low-resolution mass measurements. Seven ribosomal proteins from E. coli whole cells were observed with errors of less than 27 ppm. This was accomplished directly from whole cells without fractionation, concentration, or overt overexpression of characteristic cellular proteins. MALDI-FTMS also provided information regarding E. coli lipids in the low-mass region. Although ions with m/z values below 1000 have been observed by FTMS of whole cells, this represents the first report of detection of ions in the 5000 to 10,000 m/z range by MALDI-FTMS using whole cells.