Phosphoproteomic and transcriptomic analyses reveal multiple functions for Aspergillus nidulans MpkA independent of cell wall stress.

The protein kinase MpkA plays a prominent role in the cell wall integrity signaling (CWIS) pathway, acting as the terminal MAPK activating expression of genes which encode cell wall biosynthetic enzymes and other repair functions. Numerous studies focus on MpkA function during cell wall perturbation. Here, we focus on the role MpkA plays outside of cell wall stress, during steady state growth. In an effort to seek other, as yet unknown, connections to this pathway, an mpkA deletion mutant (ΔmpkA) was subjected to phosphoproteomic and transcriptomic analysis. When compared to the control (isogenic parent of ΔmpkA), there is strong evidence suggesting MpkA is involved with maintaining cell wall strength, branching regulation, and the iron starvation pathway, among others. Particle-size analysis during shake flask growth revealed ΔmpkA mycelia were about 4 times smaller than the control strain and more than 90 cell wall related genes show significantly altered expression levels. The deletion mutant had a significantly higher branching rate than the control and phosphoproteomic results show putative branching-regulation proteins, such as CotA, LagA, and Cdc24, have a significantly different level of phosphorylation. When grown in iron limited conditions, ΔmpkA had no difference in growth rate or production of siderophores, whereas the control strain showed decreased growth rate and increased siderophore production. Transcriptomic data revealed over 25 iron related genes with altered transcript levels. Results suggest MpkA is involved with regulation of broad cellular functions in the absence of stress.

Functional modeling identifies paralogous solanesyl-diphosphate synthases that assemble the side chain of plastoquinone-9 in plastids.

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.

Composite modeling of leaf shape along shoots discriminates Vitis species better than individual leaves.

PREMISE: Leaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. Here, we measured the leaf morphology of more than 200 grapevines (Vitis spp.) over four years and modeled changes in leaf shape along the shoot to determine whether a composite leaf shape comprising all the leaves from a single shoot can better capture the variation and predict species identity compared with individual leaves. METHODS: Using homologous universal landmarks found in grapevine leaves, we modeled various morphological features as polynomial functions of leaf nodes. The resulting functions were used to reconstruct modeled leaf shapes across the shoots, generating composite leaves that comprehensively capture the spectrum of leaf morphologies present. RESULTS: We found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict the species identity of previously unassigned grapevines, which were verified with genotyping. DISCUSSION: Observations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape-an assemblage of modeled leaf snapshots across the shoot-is a better representation of the dynamic and essential shapes of leaves, in addition to serving as a better predictor of species identity than individual leaves.

Chlorpyrifos and its metabolite modulates angiogenesis in the chorioallantoic membrane of chick embryo.

Background Chlorpyrifos (CPF) is an organophosphate insecticide, acaricide, and miticide used primarily to control foliage and soilborne insect pests on a variety of food and feed crops. Since trace amounts of these compounds are found in water and food products, they easily enter into the organ system unnoticed. In the same way, the compound or its metabolite gets transmitted from the parent to the embryo mainly through blood vessels. Since blood vessels form the major route of transport, it is pertinent to study the effect of these compounds during angiogenesis. The effect of CPF and 3,5,6-trichloro-2-pyridinol (TCPy) on the angiogenesis of chick embryo was evaluated in the chorioallantoic membrane (CAM) using an ex vivo model. Methods Nine-day-old incubated eggs where inoculated with various doses of CPF and TCPy. After 48 h of incubation, the CAM layers were retrieved and analyzed using angiogenesis software to obtain the density of blood vessels. Histomorphometric studies were performed to measure the thickness of vessel walls. The expression of VEGF, VEGFR2, and N-cadherin genes responsible for angiogenesis were analyzed. Results The exposure to the parent compound CPF and its metabolite TCPy promoted angiogenesis in groups administered with lower concentration of the pesticide and its metabolite, whereas a decline in angiogenesis was observed at higher concentrations. These observations were made by analyzing the density, histomorphometry results, and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) results. The density, thickness, and lumen size of blood vessels in the groups with low concentration of CPF and TCPy were 28.34, 9 µm, and 30 µm, respectively, whereas in the groups with higher CPF and TCPy concentrations, they were 12, 3 µm, and 9 µm, respectively. Conclusions Hence, CPF and its metabolites interfere with angiogenesis in the CAM of chick embryos. Because of their estrogen-mimicking ability, pesticides are the prime etiological suspects of increasing alteration in blood vessel formation. These results may be of help in future studies on the effect of CPF in embryonic growth, wound healing, diabetes, and tumors.

Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation.

In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease.

Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson's-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease.

Development of highly sensitive extractive spectrophotometric determination of nickel(II) in medicinal leaves, soil, industrial effluents and standard alloy samples using pyridoxal-4-phenyl-3-thiosemicarbazone.

Pyridoxal-4-phenyl-3-thiosemicarbazone (PPT) is proposed as a new sensitive reagent for the extractive spectrophotometric determination of nickel(II). PPT reacts with nickel(II) in the pH range 4.0-6.0 to form a reddish brown colored complex, which was well-extracted into n-butanol. The absorbance value of the Ni(II)-PPT complex was measured at different time intervals at 430nm, to ascertain the stability of the complex. The system obeyed Beer's law up to 0.5-5.0microgmL(-1) of nickel(II), with an excellent linearity in terms of the correlation coefficient value of 0.99. The molar absorptivity and Sandell's sensitivity of the extracted species are 1.92 x 10(4)Lmol(-1)cm(-1) and 0.003057microgcm(-2) respectively at 430nm. The detection limit of the method is 0.069microgmL(-1). To assess precision and accuracy of the developed method, determinations were carried out at different concentrations. The relative standard deviation of all measurements does not exceed 2.62%. The developed method has been satisfactorily applied for the determination of nickel(II), when present alone or in the presence of diverse ions, which are usually associated with nickel(II) in medicinal leaves, soil and industrial effluent samples. Various standard and certified reference materials (CM 247 LC, IN 718, BCS 233, 266, 253 and 251) have also been tested for the determination of nickel for the purpose of validation of the present method. The results of the proposed method are compared with those obtained from an atomic absorption spectrometer (AAS).

Chronic ferritin expression within murine dopaminergic midbrain neurons results in a progressive age-related neurodegeneration.

Ferritin elevation has been reported by some laboratories to occur within the substantia nigra (SN), the area of the brain affected in Parkinson's disease (PD), but whether such an increase could be causatively involved in neurodegeneration associated with the disorder is unknown. Here, we report that chronic ferritin elevation in midbrain dopamine-containing neurons results in a progressive age-related neurodegeneration of these cells. This provides strong evidence that chronic ferritin overload could be directly involved in age-related neurodegeneration such as occurs in Parkinson's and other related diseases.

Up-regulation of gamma-glutamyl transpeptidase activity following glutathione depletion has a compensatory rather than an inhibitory effect on mitochondrial complex I activity: implications for Parkinson's disease.

Up-regulation of activity of gamma-glutamyl transpeptidase (GGT) has been reported to occur in the Parkinsonian substantia nigra, the area of the brain affected by the disease. Increased GGT activity has been hypothesized to play a role in subsequent mitochondrial complex I (CI) inhibition by increasing cysteine as substrate for cellular uptake. Intracellular cysteine has been proposed to form toxic adducts with dopamine which can be metabolized to compounds which inhibit CI activity. We have demonstrated that in addition to CI inhibition, GGT activity is up-regulated in dopaminergic cells as a consequence of glutathione depletion. Inhibition of GGT rather than resulting in increased CI inhibition results in exacerbation of this inhibitory effect. This suggests that increased GGT activity is likely an adaptive response to the loss of glutathione to conserve intracellular glutathione content and results in a compensatory effect on CI activity rather than in its inhibition as has been previously widely hypothesized.

Radiographic evaluation of mandible to predict the gender and age.

PURPOSE: This study is been conducted using digital panoramic radiographs for predicting age in various age groups and the accuracy of the parameters were accessed as age advances. MATERIALS AND METHODS: The selected 300 panoramic images were divided into 3 age group of Group A (25-34 years), Group B (35-44 years), and Group C (45 -54 years). Each group comprised of 100 subjects in which 50 were males & 50 females. The age changes were evaluated using five parameters collectively, which were: Gonial angle, Antegonial angle, Mental foramen, Mandibular canal, Mandibular foramen. These parameters were evaluated on panoramic radiographs for age prediction and changes in their position as age advances. RESULTS: Among all the parameters changes in Mandibular canal and mandibular foramen was found to be highly significant (p value ≤0.05) as age advances. CONCLUSION: These parameters can be used to predict the age of the individual as there were significant changes in Mandibular canal and Mandibular foramen as age advances. For Further studies large sample size, and recent modalities in radiography like CBCT or CT scan are required.

Leveraging barrel medic genome sequence for the development and use of genomic resources for genetic analysis and breeding in legumes

BACKGROUND: A total of 62,591 cowpea expressed sequence tags (ESTs) were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Singlenucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. RESULTS: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% in most of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost

Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease.

An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly down-regulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex; activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition.