This study developed a new model using Drosophila melanogaster larvae to study chemotherapy-induced peripheral neuropathy (CIPN). The goals were to examine the effects of Taxol and Cisplatin chemotherapy drugs on mitochondrial dynamics and function, and determine if pretreatment with another drug could prevent the effects of chemotherapy. Behavioral assays showed that as drug concentration increased, motor and sensory function decreased and mortality rose. Imaging of larval neurons found that the chemotherapy drugs reduced mitochondrial movement, especially at higher Cisplatin doses. The results provide preliminary evidence that this model could help uncover mechanisms of CIPN and identify potential treatments.
Development of Nimbolide from Seeds of Azadirachta Indica Extract Assisted by...IOSRJPBS
The recent studies on Nimbolide, a robust cervical anticancer agent was identified for the separation of Nimbolide from Azadirachta indica berry collected from Ranga Reddy- Dist, the seeds were initially isolated, dried and transformed into coarse powder. The seed core was defatted and concentrated using methanol in a soxhlet apparatus. The methanolic extract obtained from seeds is subjected to High Performance Liquid Chromatography [HPLC] by direct reverse phase method for isolation of Nimbolide using Molecular Docking of Nimbolide through Molecular Operatin Environment [MOE] 2008 software. This declared that Nimbolide has capability of binding 12.3% to a Glycine 131 undergoing polar interactions. Nimbolide was found to interact with Cyclin E, was redeemed from the Protein Data Bank [PDB code; 1W98]. It was observed that Nimbolide showed an satisfactory increased life span (54.32 and 65.16), decreased the viable cells (52.3±1.5 and 32.5±1.3), and tumor volume (3.2±0.03 and 2.4±0.04).
Development of Nimbolide from Seeds of Azadirachta Indica Extract Assisted by...IOSRJPBS
The recent studies on Nimbolide, a robust cervical anticancer agent was identified for the separation of Nimbolide from Azadirachta indica berry collected from Ranga Reddy- Dist, the seeds were initially isolated, dried and transformed into coarse powder. The seed core was defatted and concentrated using methanol in a soxhlet apparatus. The methanolic extract obtained from seeds is subjected to High Performance Liquid Chromatography [HPLC] by direct reverse phase method for isolation of Nimbolide using Molecular Docking of Nimbolide through Molecular Operatin Environment [MOE] 2008 software. This declared that Nimbolide has capability of binding 12.3% to a Glycine 131 undergoing polar interactions. Nimbolide was found to interact with Cyclin E, was redeemed from the Protein Data Bank [PDB code; 1W98]. It was observed that Nimbolide showed an satisfactory increased life span (54.32 and 65.16), decreased the viable cells (52.3±1.5 and 32.5±1.3), and tumor volume (3.2±0.03 and 2.4±0.04).
MRI rat organ Assessment under recurrent Interferon administrationtheijes
This study aims to assess serial and transverse quantitative Magnetic Resonance Imaging (MRI) in four rat groups using different doses of a formulation based on the combinations of IFN alpha 2b and gamma. Axial and coronal T1, T2 and Diffusion MRI images have been performed in order to follow up morphological and tissue texture changes in the rat brain, cerebellum, spinal cord and kidney. As a result, no morphological changes have been observed during 28 days in any of the four groups including the placebo. Yet, doses until 15 times were bigger than the therapeutic dose. This MRI studies is robust and complementary evidence that the pharmaceutical formulation mixing in the same vial IFNs alpha2b and gamma is safe. For the first time, results of a longitudinal MRI study in rats based on the effects of this pharmacological combination are reported.
insecticidal effects of eudesmanes from pluchea sagittalisIJEAB
Eudesmanoids play an important role in the protection of plants against herbivores. Pluchea sagittalis (Lamarck) Cabrera (Asteraceae) is widespread in tropical South America and contains compounds that provide protection against phytophagous insects. In the present work we isolated seven sesquiterpenoids with eudesmane skeletons that were evaluated for their insecticidal activities against Spodoptera frugiperda and Ceratitis capitata, pests that cause serious damage to crops in the Argentine northwest. The Eudesmanes were incorporated at different concentrations to the diet of Spodoptera frugiperda. In the choice test, larval feeding behavior was altered. The eudesmanes 1, 5 and 7 showed the highest activity with feeding election indexes (FEI) of 50, 50, and 72 %, respectively at 200 µg/g of diet. When tested for insecticidal activity using neonate larvae with the no-choice artificial diet bioassays, eudesmane 1 was the most toxic in the larval stage (LD50 177.80 mg/g of diet). Compounds 5 lowered the percentage of adult emergence and produced the most malformations (72%) compared with control. Drastic effects were observed in the oviposition deterrence activity against C. capitata. The maximum oviposition deterrence (87 %) was recorded with eudesmane 5 at dose 30 µg/cm2 of artificial fruit. Finally, eudesmanes 6 and 7 showed significant larval and pupal mortality against the first generation larvae of viable eggs oviposited by females fed with the treated diet (100 µg / g artificial diet).
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future.
stimulatory effect of the magnetic treatment on the germination of cereal seedsIJEAB
The main objective of this study is to determine the effects of 125 mT magnetic treatment on the germination of different cereals seeds. This objective has a practical application in agriculture science: early growth of seeds. Germination tests were carried out under laboratory conditions by exposing seeds to magnetic field for different times. For each treatment the number of germinated seeds was counted to determine the time necessary to achieve the final maximum percentage of germinated seeds; rate of germination was assessed by determining the mean germination time (MGT) and parameters T1, T10, T25, T50, T75, T90, time required to germinate 1 - 90 percent of seeds. An increase in the percentage and rate of germination of seeds as positive response to magnetic field treatment in rice, wheat, maize and barley seeds have been found for all treatments applied. The mean germination time and parameters were reduced for all the magnetic treatments applied. Most significant differences were obtained for time of exposure of chronically exposure and 24 hours. External magnetic fields enhance seed vigor by influencing the biochemical processes by stimulating activity of proteins and enzymes. Numerous studies suggested that magnetic field increases ions uptake and consequently improves nutrition value.
Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Ela...Premier Publishers
Chlorophyll mutations provide one of the most dependable indices for evaluating genetic effects of mutagenic treatments and have been reported in various pulse crops by several workers. The present investigation was undertaken to study the effects of gamma irradiation in inducing chlorophyll mutations and as well investigate chlorophyll content after gamma radiation at 10 Gy in M1, M2, M2M1 populations of oil palm seedlings. Chlorophyll mutations were observed 2 MAP and chlorophyll content was determined using Chlorophyll content meter (SPAD 502) at 4, 6, 8, 10, 12 months after planting (MAP). Results revealed six chlorophyll mutants namely xantha, chlorina, chimerical leaves, maculata, striata, and viridis. Significant differences were observed for chlorophyll content at 4, 6, 8 MAP. Results showed that at 10 Gy of gamma irradiation, chlorophyll mutations could be induced and chlorophyll content reduces at the initial stages of plant growth after gamma irradiation (4-8 MAP) and later becomes normalized (10-12 MAP).
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Personalized Medicine and the Omics Revolution by Professor Mike SnyderThe Hive
Personalized medicine is expected to benefit from the combination of genomic information with the global monitoring of molecular components and physiological states. To ascertain whether this can be achieved, we determined the whole genome sequence of an individual at high accuracy and performed an integrated Personal Omics Profiling (iPOP) analysis, combining genomic, transcriptomic, proteomic, metabolomic, and autoantibodyomic information, over a 38-month period that included healthy and two virally infected states. Our iPOP analysis of blood components revealed extensive, dynamic and broad changes in diverse molecular components and biological pathways across healthy and disease conditions. Importantly, genomic information was also used to estimate medical risks, including Type 2 Diabetes, whose onset was observed during the course of our study. Our study demonstrates that longitudinal personal omics profiling can relate genomic information to global functional omics activity for physiological and medical interpretation of healthy and disease states.
Meet the speaker, Professor Michael Snyder (Stanford):
Michael Snyder is the Stanford Ascherman Professor, Chair of Genetics and the Director of the Center of Genomics and Personalized Medicine. He received his Ph.D. from the California Institute of Technology and postdoctoral training at Stanford University. He is a leader in the field of functional genomics and proteomics, and one of the major participants of the ENCODE project. His laboratory study was the first to perform a large-scale functional genomics project in any organism, and has launched many technologies in genomics and proteomics. These including the development of proteome chips, high resolution tiling arrays for the entire human genome, methods for global mapping of transcription factor binding sites (ChIP-chip now replaced by ChIP-seq), paired end sequencing for mapping of structural variation in eukaryotes, de novo genome sequencing of genomes using high throughput technologies and RNA-Seq. These technologies have been used for characterizing genomes, proteomes and regulatory networks. Seminal findings from the Snyder laboratory include; the discovery that much more of the human genome is transcribed and contains regulatory information than was previously appreciated, and a high diversity of transcription factor binding occurs both between and within species. He has also combined different state-of–the-art omics technologies to perform the first longitudinal detailed integrative personal omics profile (iPOP) of person and used this to assess disease risk and monitor disease states for personalized medicine. He is a co-founder of several biotechnology companies including; Protometrix (now part of Life Technologies), Affomix (now part of Illumina), Excelix, and Personalis, and he presently serves on the board of a number of companies.
In Vivo Precision Genetic Change of Soybean Δ9-Stearoyl (18:0)-ACP Desaturase...IIJSRJournal
Altering genes in their native environment is a powerful tool for biologists and breeders to study gene function and to genetically modify or redesign plant metabolism toward production of specific higher –value products. Even though gene targeting has been widely applied in organisms such as yeast and mammals, its efficiency in plants still is not high enough for routine application. The strategy used in this work consists of using ssDNA oligonucleotide–directed gene targeting to generate a site-specific base conversion or amino acid conversion in the soybean Δ9-stearoyl (18:0)-ACP desaturase and ALS (acetolactate synthase) genes to make the former specific to (16:0)-ACP (in order to produce 16:1) and the latter to make it resistant to a sulfonurea herbicide (for selection). In the same manner, yeast Saccharomyces cerevisiae was used as a model to test the approach since advantages of using such a model were well recognized. Though there were reports of success and reproducibility of such an approach in certain agronomical crops where most targeted genes for repair were transient plasmid genes or episomal genes (Gamper, 2000), this was the first time such a strategy was applied to soybean. The approach was not a success with the soybean; however, positive results were recorded with the yeast model.
Bifidobacterium strain that helps reduce body fatBiopolis_SL
Bifidobacterium animalis subsp. lactis strain CECT 8145 is able to reduce body fat content and improve metabolic syndrome biomarkers. Here, we report the draft genome sequence of this strain, which may provide insights into its safety status and functional role.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
MRI rat organ Assessment under recurrent Interferon administrationtheijes
This study aims to assess serial and transverse quantitative Magnetic Resonance Imaging (MRI) in four rat groups using different doses of a formulation based on the combinations of IFN alpha 2b and gamma. Axial and coronal T1, T2 and Diffusion MRI images have been performed in order to follow up morphological and tissue texture changes in the rat brain, cerebellum, spinal cord and kidney. As a result, no morphological changes have been observed during 28 days in any of the four groups including the placebo. Yet, doses until 15 times were bigger than the therapeutic dose. This MRI studies is robust and complementary evidence that the pharmaceutical formulation mixing in the same vial IFNs alpha2b and gamma is safe. For the first time, results of a longitudinal MRI study in rats based on the effects of this pharmacological combination are reported.
insecticidal effects of eudesmanes from pluchea sagittalisIJEAB
Eudesmanoids play an important role in the protection of plants against herbivores. Pluchea sagittalis (Lamarck) Cabrera (Asteraceae) is widespread in tropical South America and contains compounds that provide protection against phytophagous insects. In the present work we isolated seven sesquiterpenoids with eudesmane skeletons that were evaluated for their insecticidal activities against Spodoptera frugiperda and Ceratitis capitata, pests that cause serious damage to crops in the Argentine northwest. The Eudesmanes were incorporated at different concentrations to the diet of Spodoptera frugiperda. In the choice test, larval feeding behavior was altered. The eudesmanes 1, 5 and 7 showed the highest activity with feeding election indexes (FEI) of 50, 50, and 72 %, respectively at 200 µg/g of diet. When tested for insecticidal activity using neonate larvae with the no-choice artificial diet bioassays, eudesmane 1 was the most toxic in the larval stage (LD50 177.80 mg/g of diet). Compounds 5 lowered the percentage of adult emergence and produced the most malformations (72%) compared with control. Drastic effects were observed in the oviposition deterrence activity against C. capitata. The maximum oviposition deterrence (87 %) was recorded with eudesmane 5 at dose 30 µg/cm2 of artificial fruit. Finally, eudesmanes 6 and 7 showed significant larval and pupal mortality against the first generation larvae of viable eggs oviposited by females fed with the treated diet (100 µg / g artificial diet).
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future.
stimulatory effect of the magnetic treatment on the germination of cereal seedsIJEAB
The main objective of this study is to determine the effects of 125 mT magnetic treatment on the germination of different cereals seeds. This objective has a practical application in agriculture science: early growth of seeds. Germination tests were carried out under laboratory conditions by exposing seeds to magnetic field for different times. For each treatment the number of germinated seeds was counted to determine the time necessary to achieve the final maximum percentage of germinated seeds; rate of germination was assessed by determining the mean germination time (MGT) and parameters T1, T10, T25, T50, T75, T90, time required to germinate 1 - 90 percent of seeds. An increase in the percentage and rate of germination of seeds as positive response to magnetic field treatment in rice, wheat, maize and barley seeds have been found for all treatments applied. The mean germination time and parameters were reduced for all the magnetic treatments applied. Most significant differences were obtained for time of exposure of chronically exposure and 24 hours. External magnetic fields enhance seed vigor by influencing the biochemical processes by stimulating activity of proteins and enzymes. Numerous studies suggested that magnetic field increases ions uptake and consequently improves nutrition value.
Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Ela...Premier Publishers
Chlorophyll mutations provide one of the most dependable indices for evaluating genetic effects of mutagenic treatments and have been reported in various pulse crops by several workers. The present investigation was undertaken to study the effects of gamma irradiation in inducing chlorophyll mutations and as well investigate chlorophyll content after gamma radiation at 10 Gy in M1, M2, M2M1 populations of oil palm seedlings. Chlorophyll mutations were observed 2 MAP and chlorophyll content was determined using Chlorophyll content meter (SPAD 502) at 4, 6, 8, 10, 12 months after planting (MAP). Results revealed six chlorophyll mutants namely xantha, chlorina, chimerical leaves, maculata, striata, and viridis. Significant differences were observed for chlorophyll content at 4, 6, 8 MAP. Results showed that at 10 Gy of gamma irradiation, chlorophyll mutations could be induced and chlorophyll content reduces at the initial stages of plant growth after gamma irradiation (4-8 MAP) and later becomes normalized (10-12 MAP).
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
Personalized Medicine and the Omics Revolution by Professor Mike SnyderThe Hive
Personalized medicine is expected to benefit from the combination of genomic information with the global monitoring of molecular components and physiological states. To ascertain whether this can be achieved, we determined the whole genome sequence of an individual at high accuracy and performed an integrated Personal Omics Profiling (iPOP) analysis, combining genomic, transcriptomic, proteomic, metabolomic, and autoantibodyomic information, over a 38-month period that included healthy and two virally infected states. Our iPOP analysis of blood components revealed extensive, dynamic and broad changes in diverse molecular components and biological pathways across healthy and disease conditions. Importantly, genomic information was also used to estimate medical risks, including Type 2 Diabetes, whose onset was observed during the course of our study. Our study demonstrates that longitudinal personal omics profiling can relate genomic information to global functional omics activity for physiological and medical interpretation of healthy and disease states.
Meet the speaker, Professor Michael Snyder (Stanford):
Michael Snyder is the Stanford Ascherman Professor, Chair of Genetics and the Director of the Center of Genomics and Personalized Medicine. He received his Ph.D. from the California Institute of Technology and postdoctoral training at Stanford University. He is a leader in the field of functional genomics and proteomics, and one of the major participants of the ENCODE project. His laboratory study was the first to perform a large-scale functional genomics project in any organism, and has launched many technologies in genomics and proteomics. These including the development of proteome chips, high resolution tiling arrays for the entire human genome, methods for global mapping of transcription factor binding sites (ChIP-chip now replaced by ChIP-seq), paired end sequencing for mapping of structural variation in eukaryotes, de novo genome sequencing of genomes using high throughput technologies and RNA-Seq. These technologies have been used for characterizing genomes, proteomes and regulatory networks. Seminal findings from the Snyder laboratory include; the discovery that much more of the human genome is transcribed and contains regulatory information than was previously appreciated, and a high diversity of transcription factor binding occurs both between and within species. He has also combined different state-of–the-art omics technologies to perform the first longitudinal detailed integrative personal omics profile (iPOP) of person and used this to assess disease risk and monitor disease states for personalized medicine. He is a co-founder of several biotechnology companies including; Protometrix (now part of Life Technologies), Affomix (now part of Illumina), Excelix, and Personalis, and he presently serves on the board of a number of companies.
In Vivo Precision Genetic Change of Soybean Δ9-Stearoyl (18:0)-ACP Desaturase...IIJSRJournal
Altering genes in their native environment is a powerful tool for biologists and breeders to study gene function and to genetically modify or redesign plant metabolism toward production of specific higher –value products. Even though gene targeting has been widely applied in organisms such as yeast and mammals, its efficiency in plants still is not high enough for routine application. The strategy used in this work consists of using ssDNA oligonucleotide–directed gene targeting to generate a site-specific base conversion or amino acid conversion in the soybean Δ9-stearoyl (18:0)-ACP desaturase and ALS (acetolactate synthase) genes to make the former specific to (16:0)-ACP (in order to produce 16:1) and the latter to make it resistant to a sulfonurea herbicide (for selection). In the same manner, yeast Saccharomyces cerevisiae was used as a model to test the approach since advantages of using such a model were well recognized. Though there were reports of success and reproducibility of such an approach in certain agronomical crops where most targeted genes for repair were transient plasmid genes or episomal genes (Gamper, 2000), this was the first time such a strategy was applied to soybean. The approach was not a success with the soybean; however, positive results were recorded with the yeast model.
Bifidobacterium strain that helps reduce body fatBiopolis_SL
Bifidobacterium animalis subsp. lactis strain CECT 8145 is able to reduce body fat content and improve metabolic syndrome biomarkers. Here, we report the draft genome sequence of this strain, which may provide insights into its safety status and functional role.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
The current slide focuses on different screening models for neurodegenerative diseases along with a brief description of the diseases where the slides are to the points and brief with detailed evaluation.
The Radiosensitivity Effect of Hydroxyurea on HT29 Cell Lineiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Formulation and Evaluation of Moxifloxacin Loaded Alginate Chitosan Nanoparti...
2011 Mayo Clinic Drosophila CIPN poster
1. Mitochondrial dynamics and behavioral defects in Taxol and Cisplatin treated
Drosophila melanogaster larvae: A new model for chemotherapy induced
peripheral neuropathy
Figure 3. D. melanogaster larvae behavioral assays. A. The righting
assay measures larval motor control (adapted from Mudher, 2004). B.
The heating assay measures larval sensory reception. The protocol was
adapted from Oswald, et al., 2011.
A.Righting Assay
dorsal
ventral
B. Heating Assay
Chemotherapy induced peripheral neuropathy (CIPN) affects ~ 40
percent of patients receiving chemotherapy treatment causing its
premature termination and sometimes irreversible nerve damage 1,2.
Lack of the model organisms, preferentially with intact nervous system,
to study the CIPN mechanisms prevents from the development of
efficient therapeutic strategies. Changes in neuronal mitochondria
have been observed in many neurodegenerative disorders3. Therefore,
the overall goals of this project are (1) to develop a novel model in D.
melanogaster larvae to study CIPN, (2) to establish whether treatment
with the chemotherapy drugs Taxol and Cisplatin induces changes in
mitochondrial dynamics and function, and (3) examine whether
pretreatment with the drug, code name CP2, that enhances
mitochondrial function could alleviate the effect of chemotherapy
drugs. Since chemotherapy treatments in people are delivered on a
regular schedule, drugs that have a protective effect on peripheral
nerves could potentially eliminate CIPN.
Introduction and Objectives
ã 2012 Mayo Foundation for Medical Education and Research
Steven Forsythe*, Kiley Schmidt*, Georgiy Yudintsev*, Jewel Podratz, Anthony Windebank, Eugenia Trushina
Department of Neurology
Mayo Clinic, Rochester, MN
Figure 2. Growth, treatment, and analysis schedules. A. The
A.Development cycle development cycle of D.
melanogaster. B.
Protocol A is the initially designed
experiment schedule. C. Protocol B was
adapted to include time for full
development and the
possibility of pre-treatment with CP2.
B,C. For both protocols the
concentration curve for Cisplatin was
0uM, 1uM, 5uM, 10uM, 25uM, 50uM,
for Taxol, 0uM, 0.5uM, 0.8uM, 1uM,
10uM, 30uM.
Flagg, 1979
B. Protocol A C. Protocol B
Day 1: Add adult flies to grape Day 1: Add adult flies to grape
plate apparatus. plate apparatus
Day 2: Remove adult flies. Remove Day 2: Remove adult flies. Remove
plate containing eggs from plate containing eggs from
apparatus. apparatus.
Day 3: Transfer 1st instar larvae to Day 3: Transfer 1st instar larvae to
drug treated blue food. clean blue food or CP2
pre-treated blue food.
Day 4,5: Incubate at 25°C
Day 6: Harvest early 3rd instar larvae. Day 4: Incubate at 25°C
Perform behavioral tests and Day 5: Treat 2nd instar larvae with
confocal imaging. Cisplatin or Taxol food.
Day 7+: Measure percent survival.
Day 6,7: Incubate at 25°C
Day 8: Perform behavioral tests and
confocal imaging.
Day 9+: Measure percent survival
Protocol Overview
• Breed w; +/+; D42-Gal 4, UAS-mitoGFP/TM6 Tb adult D.
melanogaster
• Treat larvae with various concentrations of Cisplatin or Taxol
• Perform behavioral assays on 3rd instar larvae to determine motor
and sensory axon dysfunction
• Dissect 3rd instar larvae to display intact nervous system
• Image motor axons using confocal microscopy
• Analyze mitochondria dynamics
Methods
measure time
Figure 4. Survival measurement A.
A. Developed protocol
to measure lethality
for each drug treatment.
B. Data recorded for Taxol and
Cisplatin percent survival.
B.
Taxol Survival Curve Cisplatin Survival Curve
1. National Cancer Institute. 2010. Chemotherapy-induced peripheral neuropathy. NCI Cancer Bulletin 7(4).
2. Marchettini P, Lacerenza M, Mauri E, Marangoni C. 2006. Painful peripheral neuropathies. Current Neuropharmacology 4(3): 175-181.
3. Filosto M, Scarpelli M, Cotelli MS, Vielmi V, Todeschini A, Gregorelli V, Tonin P, Tomelleri G, Padovani A. 2011. The role of mitochondria in
neurodegenerative diseases. Journal of Neurology 258(10): 1763-1774.
4. Flagg RO. 1979. The Carolina Drosophila manual. Carolina Biological Supply Co.
5. Mudher A, Shepherd D, Newman TA, Mildren P, Jukes JP, Squire A, Mears A, Berg S, MacKay D, Asuni AA, Bhat R, Lovestone S. 2004. GSK-3 inhibition
reverses axonal transport defects and behavioural phenotypes in Drosophila. Molecules Psychiatry 9: 522-530
6. Oswald M, Rymarczyk B, Chatters A, Sweeney S. 2011. A novel thermosensitive escape behavior in Drosophila larvae. Fly 5(4): 304-306.
References
•A successful protocol for using D. melanogaster for the study of CIPN was
developed. This included the treatment schedule, the behavioral and survival
assay, modified larval dissection and preparation methods, and confocal
imaging and analysis techniques.
•Preliminary data has been collected for the behavioral assays and survival
tests. The results display the trend that as the concentration of drug is
increased, the motor and sensory function is decreased and lethality is
increased.
•Kymographs were prepared to observe mitochondrial dynamics.
Conclusion
Figure 1. Fluorescent image of D. melanogaster nervous system. The
ventral ganglion (VG) is labeled on the left (anterior end). Motor neuron
axons extend posteriorly from VG with GFP tagged mitochondria.
0
20
40
60
80
100
0 0.5 0.8 1 10 30
PercentSurvival
Taxol Concentration (uM)
0
20
40
60
80
100
0 0.5 0.8 1 10 30
SecondstoRight
Taxol Concentration (uM)
0
20
40
60
80
100
28 29 30 31 32
NumberofLarvae
Temperature (C)
0
2
4
6
8
10
12
0 1 5 10 25 50
NumberofLarvae
Cisplatin Concentration (ug/ml)
Larva Nonreacted
Larva Reacted
0
20
40
60
80
100
0 1 5 10 25 50
PercentSurvival
Cisplatin Concentration (ug/ml)
•Replicate experiments to increase numbers
•Refine techniques
•Perform CP2 pre-treatment experiments and analyze for recovered phenotype
•Analyze numerical data from confocal videos
•Analyze mitochondrial function, fission and fusion
•Link behavioral data to mitochondrial dynamics and function
Future Directions
Figure 5. D. melanogaster segmental nerve confocal imaging.
Dissected larval
preparations were placed
On a glass slide in
hemolymph like solution
(HL6 buffer) at room
Temperature and
coverslipped. Distal region
of the 8th segmental nerve
was bleached for 15 sec
at 100% power of 488 nm
Laser and then imaged for
600 frames with 1 sec.
interval using 60x water
objective. Anterograde
and retrograde movements
of mitochondria were
tracked from frame to frame.
Figure 6. Change in mitochondrial dynamics upon treatment with
Cisplatin and Taxol. Kymographs were generated for Cisplatin 1.0
µg/ml, 5.0 µg/ml, 10.0 µg/ml and Taxol 0.8 µM, 1.0 µM, 10 µM treated 3rd
instar larvae versus untreated 3rd instar larvae. X-axis represents the
length of the region of interest (distal part of segmental nerve 8).
Y-axis represents the time (1 sec/pixel). From these treatments we have
observed that both retrograde and anterograde fluxes decrease in
treated flies, with most prominent
changes in mitochondrial transport
seen in 5.0 µg/ml of Cisplatin where no
movement of the mitochondria is seen.
This work was supported by funding through UREP and SURF programs, as well as
the Department of Molecular and Cellular biology at the University of Illinois Urbana-
Champaign. We also would like to give special thanks to Jewel Podratz, Swathi
Devireddy and Dr. Hollenbeck, Dr. Eugenia Trushina, and Han Lee for all their help
and guidance in the project and Mayo Clinic Microscopy Core facility.
Acknowledgments