Can plant defensins be used to engineer durable commercially useful fungal resistance in crop plants?

Plant defensins are cysteine-rich proteins that play an important role in defense against fungal pathogens. Because of their potent antifungal activity, they have a strong potential to be used for engineering disease resistance in crops. Significant advances have been made in elucidating their structure–activity relationships and modes of antifungal action. Their expression in transgenic plants provides resistance to fungal pathogens in crop plants. In this article, we review recent advances and offer future perspectives on the use of these proteins for engineering durable commercially useful disease resistance in transgenic crop plants.     

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer

Background

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer.

Methodology/Principal Findings

We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels.We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients.

Conclusions/Significance

We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.     

Nonsterile immunity to cryptosporidiosis in infants is associated with mucosal IgA against the sporozoite and protection from malnutrition

We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium. Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p = 0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p = 0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 ± 0.932 versus HAZ -1.731 ± 0.984 p = 0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering.Trial Registration: {"type":"clinical-trial","attrs":{"text":"NCT02764918","term_id":"NCT02764918"}}NCT02764918.     

Recognition of partially concealed leopards by wild bonnet macaques (Macaca radiata). The role of the spotted coat

Wild bonnet macaques (Macaca radiata) have been shown to recognize models of leopards (Panthera pardus), based on their configuration and spotted yellow coat. This study examined whether bonnet macaques could recognize the spotted and dark melanic morph when partially concealed by vegetation. Seven troops were studied at two sites in southern India, the Mudumalai Wildlife Sanctuary and the Kalakad-Mundanthurai Tiger Reserve. The forequarters and hindquarters of the two leopard morphs were presented from behind thick vegetation to individuals at feeding stations 25 m away. Flight reaction times and frequency of flight were obtained from video for only those individuals who oriented towards the models prior to hearing alarm calls. Bonnet macaques exhibited faster reaction times and greater frequency of flight after looking at the spotted morph's forequarter than after looking at either its spotted hindquarter or the dark morph's forequarter. The hindquarter of the dark morph was ignored completely. Artificial neural network modeling examined the perceptual aspects of leopard face recognition and the role of spots as camouflage. When spots were integrated into the pattern recognition process via network training, these spots contributed to leopard face recognition. When networks were not trained with spots, spots did not act as camouflage by disrupting facial features.     

Comparative genomics of the pennate diatom Phaeodactylum tricornutum

Diatoms are one of the most important constituents of phytoplankton communities in aquatic environments, but in spite of this, only recently have large-scale diatom-sequencing projects been undertaken. With the genome of the centric species Thalassiosira pseudonana available since mid-2004, accumulating sequence information for a pennate model species appears a natural subsequent aim. We have generated over 12,000 expressed sequence tags (ESTs) from the pennate diatom Phaeodactylum tricornutum, and upon assembly into a nonredundant set, 5,108 sequences were obtained. Significant similarity (E < 1E-04) to entries in the GenBank nonredundant protein database, the COG profile database, and the Pfam protein domains database were detected, respectively, in 45.0%, 21.5%, and 37.1% of the nonredundant collection of sequences. This information was employed to functionally annotate the P. tricornutum nonredundant set and to create an internet-accessible queryable diatom EST database. The nonredundant collection was then compared to the putative complete proteomes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the centric diatom T. pseudonana. A number of intriguing differences were identified between the pennate and the centric diatoms concerning activities of relevance for general cell metabolism, e.g. genes involved in carbon-concentrating mechanisms, cytosolic acetyl-Coenzyme A production, and fructose-1,6-bisphosphate metabolism. Finally, codon usage and utilization of C and G relative to gene expression (as measured by EST redundance) were studied, and preferences for utilization of C and CpG doublets were noted among the P. tricornutum EST coding sequences.     

The Solution Structure of a Cyclic Analog of Neuropeptide Y with High Y<Subscript>1</Subscript> Receptor Affinity by NMR,CD and MD Simulations

The conformation of a cyclic analog of neuropeptide Y [Tyr¹--Lys--Gly--Arg--cyclo^5/8-(Glu⁵--Tyr--Ile--Lys⁸)--Leu--Ile¹⁰--Thr--Arg--Pro--Arg--Tyr¹⁵--NH₂; cEK-NPY] with high Y₁ receptor affinity was studied using ¹H, ¹³C and ¹⁵N 2D-NMR and CD in three diverse media-viz. DMSO-d₆, water (pH 4.0) and 50% hexafluoroacetone (HFA). The conformation of cEK-NPY was interpreted based on chemical shift (¹H, ¹³C and ¹⁵N), temperature coefficients of the NH chemical shifts, ³J_NHα coupling constants and the pattern of intra and inter-residue NOE’s and the CD spectrum. In both DMSO and water, there is a preponderance of a β-strand structure, while HFA promotes an α-helical structure, which is discontinuous in the mid-region of the peptide, due to the constraints of the lactam ring. The solution structures were generated using Restrained Molecular Dynamics simulations and further refined by Mardigras to R factors between 0.55 and 0.65. The role of its conformations in its biological activity is discussed.     

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.     

Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells

Lipocalin 2, an iron-siderophore-binding protein, converts embryonic kidney mesenchyme to epithelia. We found that lipocalin 2 could also convert 4T1-Ras-transformed mesenchymal tumor cells to an epithelial phenotype, increase E-cadherin expression, and suppress cell invasiveness in vitro and tumor growth and lung metastases in vivo. The Ras-MAPK pathway mediated the epithelial to mesenchymal transition in part by increasing E-cadherin phosphorylation and degradation. Lipocalin 2 antagonized these effects at a point upstream of Raf activation. Lipocalin 2 action was enhanced by iron-siderophore. These data characterize lipocalin 2 as an epithelial inducer in Ras malignancy and a suppressor of metastasis.