The research aimed to understand how to better control mosquito populations using Bacillus thuringiensis Cry19Aa toxin. Experiments were conducted from 1/11/16 to 5/1/16 under a PhD student to determine the toxicity of Cry19Aa against Anopheles gambiae cell lines (Ag55). Preliminary results showed trypsin-activated Cry19Aa was cytotoxic to Ag55 cells in a time- and concentration-dependent manner, reducing cell viability to zero after 24 hours. This suggests Cry19Aa could be an effective toxin for mosquito control.
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, usi...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater. Moreover there were reports existing on genetic manipulation of koicarp by application of the heat shock. Hence the present study was made to contribute a protocol for induction of tetraploidy by heat shock in the koicarp.Induction of tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five females and milt from five males ok Koicarp were pooled to ensure the required quantity and quality of gametes for fertilization. After insemination the eggs were divided into three batches each experiment based on the post fertilization viz., 25min, 27min and 30min after insemination. Batches of eggs held in plastic containers were exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min. One batch of the eggs without heat shock treatment was used as control. After treatments, eggs were immediately transferred to incubation troughs. Tetraploidy was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large proportion of the heat shocked embryos displayed morphological abnormalities such as short and curved tail, destroyed yolksac, deformed vertebral column and malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times greater than those of the diploid RBC nucleus.Given that koicarp are such a useful model for other areas of research, perhaps further studies on the induction of tetraploidy in this species will lead to a better understanding of polyploidy induction and the establishment of tetraploid lines of koicarp and other species as well.
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1(1): 013-019.
Full Text:
http://janimalsciences.com/documents/AS0006.pdf
Induction of tetraploidy in an ornamental fish koicarp Cyprinus carpio L, usi...researchanimalsciences
Koicarp is potentially an important cultured ornamental fish in freshwater. Moreover there were reports existing on genetic manipulation of koicarp by application of the heat shock. Hence the present study was made to contribute a protocol for induction of tetraploidy by heat shock in the koicarp.Induction of tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five females and milt from five males ok Koicarp were pooled to ensure the required quantity and quality of gametes for fertilization. After insemination the eggs were divided into three batches each experiment based on the post fertilization viz., 25min, 27min and 30min after insemination. Batches of eggs held in plastic containers were exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min. One batch of the eggs without heat shock treatment was used as control. After treatments, eggs were immediately transferred to incubation troughs. Tetraploidy was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large proportion of the heat shocked embryos displayed morphological abnormalities such as short and curved tail, destroyed yolksac, deformed vertebral column and malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times greater than those of the diploid RBC nucleus.Given that koicarp are such a useful model for other areas of research, perhaps further studies on the induction of tetraploidy in this species will lead to a better understanding of polyploidy induction and the establishment of tetraploid lines of koicarp and other species as well.
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1(1): 013-019.
Full Text:
http://janimalsciences.com/documents/AS0006.pdf
Bacillus thuringiensis, an aerobic, Gram positive, spore forming bacterium produces unique proteinaceous crystalline parasporal inclusions during sporulation which have insecticidal properties. Besides being widely used as an insecticide in agriculture, Bt has been found to be useful in several fields like medicine, endoparasite control, bacteriocin production as well as enzyme production. Parasporin, a new category of bacterial parasporal protein capable of discriminately killing the cancer cells have been discovered. There are six classes of parasporins having different mode of action and cell specificities against cancer and tumor cells (Ohba et al., 2009).Bt proteins have also been used successfully to suppress the population levels of medically important Dipteran pests like mosquitoes by use of mosquitocidal strains that produce Cry proteins (Zhang et al., 2012) as well as potential therapeutic agent against protozoan disease Leishmaniases (El-Sadawy et al., 2008). Crystal proteins, like Cry5B from Bacillus thuringiensis are found to be safe to vertebrates and have been shown to have efficacy against intestinal hookworm parasites (Capello et al., 2006). Thus the multifarious applications of Bacillus thuringiensis have made it a microbe to reckon with and further study its genome for future developments.
The development and commercialization of insect-resistant transgenic Bt crops expressing Cry toxins revolutionized the history of agriculture. At the end of 2010, an estimated 26.3 million hectares of land were planted with crops containing the Bt gene (James 2011). Bt cotton has reduced the use of traditional insecticides by 207,900,000 lbs of active ingredient of insecticide (Brookes and Barfoot, 2006).
Resistance is a genetic change in the insect pest — that allows it to avoid harm from Bt toxins. The high and consistent levels of ICP production in the Bt plants make them much less favorable for the development of resistance. Insect Resistance Management is of great importance because of the threat insect resistance poses to the future use of Bt plant-incorporated protectants and is said to be the key to sustainable use of the genetically modified Bt crops. The US EPA usually requires a “buffer zone,” or a structured refuge of 20% non-Bt crops that is planted in close proximity to the Bt crops.
First documented case of insect resistance to Bt cotton came in 2008, when Tabashnik and coworkers found field-evolved Bt toxin resistance in bollworm, Helicoverpa zea (Boddie), in the United States. Field-Evolved Resistance to Bt Maize by Western Corn Rootworm (Gassmann, 2011) displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays.
Expanded use of transgenic crops for insect control will likely include more varieties with combinations of two or more Bt toxins (pyramiding), novel Bt toxins such as VIP, modified Bt toxins that have been genetically engineered to kill insects resistant to standard Bt toxins. Transgenic plants that control insects via RNA interference are also under development.
Increasing use of transgenic crops in developing nations is likely, with a broadening range of genetically modified crops and target insect pests .Incorporating enhanced understanding of observed patterns of field-evolved resistance into future resistance management strategies can help to minimize the drawbacks and maximize the benefits of current and future generations of transgenic crops.
Bacillus thuringiensis, an aerobic, Gram positive, spore forming bacterium produces unique proteinaceous crystalline parasporal inclusions during sporulation which have insecticidal properties. Besides being widely used as an insecticide in agriculture, Bt has been found to be useful in several fields like medicine, endoparasite control, bacteriocin production as well as enzyme production. Parasporin, a new category of bacterial parasporal protein capable of discriminately killing the cancer cells have been discovered. There are six classes of parasporins having different mode of action and cell specificities against cancer and tumor cells (Ohba et al., 2009).Bt proteins have also been used successfully to suppress the population levels of medically important Dipteran pests like mosquitoes by use of mosquitocidal strains that produce Cry proteins (Zhang et al., 2012) as well as potential therapeutic agent against protozoan disease Leishmaniases (El-Sadawy et al., 2008). Crystal proteins, like Cry5B from Bacillus thuringiensis are found to be safe to vertebrates and have been shown to have efficacy against intestinal hookworm parasites (Capello et al., 2006). Thus the multifarious applications of Bacillus thuringiensis have made it a microbe to reckon with and further study its genome for future developments.
The development and commercialization of insect-resistant transgenic Bt crops expressing Cry toxins revolutionized the history of agriculture. At the end of 2010, an estimated 26.3 million hectares of land were planted with crops containing the Bt gene (James 2011). Bt cotton has reduced the use of traditional insecticides by 207,900,000 lbs of active ingredient of insecticide (Brookes and Barfoot, 2006).
Resistance is a genetic change in the insect pest — that allows it to avoid harm from Bt toxins. The high and consistent levels of ICP production in the Bt plants make them much less favorable for the development of resistance. Insect Resistance Management is of great importance because of the threat insect resistance poses to the future use of Bt plant-incorporated protectants and is said to be the key to sustainable use of the genetically modified Bt crops. The US EPA usually requires a “buffer zone,” or a structured refuge of 20% non-Bt crops that is planted in close proximity to the Bt crops.
First documented case of insect resistance to Bt cotton came in 2008, when Tabashnik and coworkers found field-evolved Bt toxin resistance in bollworm, Helicoverpa zea (Boddie), in the United States. Field-Evolved Resistance to Bt Maize by Western Corn Rootworm (Gassmann, 2011) displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays.
Expanded use of transgenic crops for insect control will likely include more varieties with combinations of two or more Bt toxins (pyramiding), novel Bt toxins such as VIP, modified Bt toxins that have been genetically engineered to kill insects resistant to standard Bt toxins. Transgenic plants that control insects via RNA interference are also under development.
Increasing use of transgenic crops in developing nations is likely, with a broadening range of genetically modified crops and target insect pests .Incorporating enhanced understanding of observed patterns of field-evolved resistance into future resistance management strategies can help to minimize the drawbacks and maximize the benefits of current and future generations of transgenic crops.
Keratin 19 is a member of the keratin family. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins.
Keratin 19 is a type I keratin. The type I cytokeratins consist of acidic proteins which are arranged in pairs of heterotypic keratin chains. Unlike its related family members, this smallest known acidic cytokeratin is not paired with a basic cytokeratin in epithelial cells. It is specifically found in the periderm, the transiently superficial layer that envelops the developing epidermis.
Anti-Cytokeratin 19 -http://www.stjohnslabs.com/cytokeratin-19-antibody-p-91962
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination. Binds to DNA and to DNA ligase IV (LIG4). The LIG4-XRCC4 complex is responsible for the NHEJ ligation step, and XRCC4 enhances the joining activity of LIG4. Binding of the LIG4-XRCC4 complex to DNA ends is dependent on the assembly of the DNA-dependent protein kinase complex DNA-PK to these DNA ends.
Anti-XRCC4-http://www.stjohnslabs.com/xrcc4-antibody-p-98623
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Presentation based on a research article published in the journal scientific reports in 2017, entitled as "A Novel Pathogen Capturing Device for Removal and Detection"
Type I keratin involved in the formation and maintenance of various skin appendages, specifically in determining shape and orientation of hair (By similarity). Required for the correct growth of hair follicles, in particular for the persistence of the anagen (growth) state (By similarity). Modulates the function of TNF-alpha in the specific context of hair cycling. Regulates protein synthesis and epithelial cell growth through binding to the adapter protein SFN and by stimulating Akt/mTOR pathway (By similarity). Involved in tissue repair. May be a marker of basal cell differentiation in complex epithelia and therefore indicative of a certain type of epithelial "stem cells". Acts as a promoter of epithelial proliferation by acting a regulator of immune response in skin: promotes Th1/Th17-dominated immune environment contributing to the development of basaloid skin tumors (By similarity). May act as an autoantigen in the immunopathogenesis of psoriasis, with certain peptide regions being a major target for autoreactive T-cells and hence causing their proliferation.
Anti-CK17-http://www.stjohnslabs.com/ck17-antibody-p-98618
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
In vitro mutagenesis of Cymbidium La bell “Anna Belle” by γ-rays irradiation ...IJEAB
The optimum media for multiplication of protocorm like bodies (PLBs) and shoot buds of Cymbidium La bell “Anna Belle” were studied in order to prepare the in vitro samples for irradiation. The values of LD50 (lethal dose of 50% samples) of PLBs, shoot buds and plantlets of tested Cymbidium after cultivation of 4 months were also determined about 35.0, 41.0 and 83.1 Gy, respectively. The addition of oligochitosan played as an very important trigger for promotion on the generation of shoot bud from PLBs after irradiation. The in vitro variations have been generated by γ-rays irradiation of PLBs with doses in range of 20 - 50 Gy. The highest mutant frequency (3.83‰) of C. La bell was found by the irradiation of PLB samples at 30 Gy. The different properties of obtained in vitro variations compared to wild types were found to be chlorophyll, short leaves, long leaves, and violet pericardium variations. The genetic relationships among generated variant lines in M1V4 and wild type were analyzed using RAPD techniques.
CD23, also known as Fc epsilon RII, or FcεRII, is the "low-affinity" receptor for IgE, an antibody isotype involved in allergy and resistance to parasites, and is important in regulation of IgE levels. Unlike many of the antibody receptors, CD23 is a C-type lectin. It is found on mature B cells, activated macrophages, eosinophils, follicular dendritic cells, and platelets.
There are two forms of CD23: CD23a and CD23b. CD23a is present on follicular B cells, whereas CD23b requires IL-4 to be expressed on T-cells, monocytes, Langerhans cells, eosinophils, and macrophages. CD23 is known to have a role of transportation in antibody feedback regulation. Antigens which enter the blood stream can be captured by antigen specific IgE antibodies. The IgE immune complexes that are formed bind to CD23 molecules on B cells, and are transported to the B cell follicles of the spleen. The antigen is then transferred from CD23+ B cells to CD11c+ antigen presenting cells. The CD11c+ cells in turn present the antigen to CD4+ T cells, which can lead to an enhanced antibody response.
Anti-CD23 -http://www.stjohnslabs.com/cd23-antibody-p-98586
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Receptor for the Fc region of IgG. Binds complexed or aggregated IgG and also monomeric IgG. Mediates antibody-dependent cellular cytotoxicity (ADCC) and other antibody-dependent responses, such as phagocytosis.
Anti-CD16-http://www.stjohnslabs.com/cd16-antibody-p-98627
Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation
Pharmacological activity of the methanolic extract of sea urchins against esc...Innspub Net
This study elucidated the pharmacological potential of sea urchins using methanol as extracting medium. The antibacterial potential was evaluated using the paper disc method and zone of inhibition against Escherichia coli and Staphylococcus aureus was measured. Antioxidant properties of sea urchins were evaluated using DPPH radical scavenging assay. Three species of sea urchin randomly collected along the intertidal zone of Diguisit, Baler Aurora were identified using diagnostic keys by the National Museum of the Philippines and they were identified as follows; Echinothrix diadema, Echinometra mathaei, and Echinometra oblonga. E. diadema recorded the highest diameter zone of inhibition against E. coli and S. aureus after 24 hours of incubation with 11.03 ± 1.75mm and 13.52 ± 1.13mm respectively while E. mathaei only inhibited S. aureus with zone of inhibition of 9.27 ± 2.06mm in 24 hours of incubation as well. As the zone of inhibition prolongs, the zone of inhibition decreases as observed in 48 hours of incubation. E. oblonga did not show inhibitoy effect, however it recorded the highest radical scavenging activity with 64.46% among the three species of sea urchins. This was followed by E. mathaei (51.52%) and E. diadema (37.38%). All collected species manifested antioxidant potential. Based on the results, the collected species of sea urchins has a pharmacological potential.
Similar to Investigating role of Cry19Aa toxin on Specific Membrane proteins (20)
Pharmacological activity of the methanolic extract of sea urchins against esc...
Investigating role of Cry19Aa toxin on Specific Membrane proteins
1. Pg.1
Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell line
Dr Adang’s Lab
Research done from 1/11/16 to 5/1/16
Supervison under Ruchir Mishra P.H.D student
Mihir Panchal
2. Pg.2
Determining the toxicity of Cry19Aa against Ag55(anopheles gambiae) cell lines
Summary:
The overall purpose of this laboratory research conducted from 1/11/16 to
5/1/16 in Dr. Adang’s molecular lab was to understand how to better control
mosquito populations in regions susceptible to diseases in which mosquitoes are a
vector. The use of Bacillus thuringiensis Cry19Aa toxin was central to our research.
Working with a doctoral student (Ruchir Mishra), our research project focused on
the affects of the BT Cry19Aa toxin on the cell membrane proteins of different
species of mosquitoes (Culex, and Anopheles), Cry19Aa is also especially effective
against Culex strains resistant to Cry4Aa, Cry4Ba, Cry11Aa toxins, investigating this
resistance and its effectively to mosquito cells was key to our research.
In order to conduct our research we used Ag55 cell lines. Ag55 cell lines have
been generally considered relatively easy to study Cry interaction and cellular
responses to those interactions. Thus we used Ag55 cells to carry out assays to
figure out the toxicity of Cry19Aa to Ag55 cell and larval cell lines. Preliminary
results showed that trypsin present in activated Cry19Aa was cytotoxic to Ag55 cells
and these results can give insights to other more efficient methods for mosquito
control.
Introduction:
Mosquito control has been central to human civilization for over millennia,
many civilizations have tried to control mosquitoes and the various disease they are
vectors for such as malaria, Chickungunya, Dengue, West Nile virus and various
other emerging diseases such as the Zika Virus. The most common form of mosquito
control is sanitation and insecticides (Teixeira, 2012).
3. Pg.3
The ability of diseases vectored by mosquitoes to disrupt life and increase
morbidity, especially in the developing world, where Malaria kills nearly 660,000
people every year (Nadjm,2012) led us to research more effective toxins against
different types of mosquitoes. We decided to use Bacillus thuringiensis, a toxin of the
bacterial origin that targets the larval stages of certain mosquitoes. A key advantage
to using B. thuringiensis toxins is they affect few non target species, which makes
them much more environmentally friendly (Roh,2009). Bt(Bacillus thuringiensis)
toxins incorporate various different Cry and Cyt toxins, the crystal aggregation
which these toxins form contains at least four major toxic compounds. Cry and Cyt
toxins both are pore-forming toxins that lyse midgut epithelial cells by inserting into
a target cell membrane and forming pores.
Our research focused on Cry19Aa, a toxin found in the pBtoxis plasmid which
is isolated from B.thuringiensis subsp. Jegathesan . Our research targeted Cry19Aa
because it is known to be toxic to Anopheles and Culex but not to Aedes and it is also
toxic to Culex strains resistant to Cry4Aa, Cry4ba, and Cry11Aa, showing no cross-
resistance. The lack of resistance suggests a mechanism of action distinct from other
Bti toxins and Cry11Ba. This lack of resistance makes studying and investigating
Cry19Aa and its affectivity compared to other toxins of importance for developing
better ways of controlling mosquito-vectored diseases.
Understanding the efficiency and rate at which the Cry19Aa protein kills the
Ag55 cells, a cell line of Anopheles gambiae, can lead to further research to
genetically develop even more effective toxins against mosquito cells.
4. Pg.4
Experimental Methods:
In order to perform our experiment, we first grew a cell culture model conducive
for investigating Cry toxin action. The cell cultures we used and found effective were
the Ag55 cell lines. Ag55 cell lines were previously effective for investigating
malarial plasmodium research. After growing 1x10^6 Ag55 cell lines, we prepared
activated Cry19Aa using trypsin. Additional details and order of the experiment is
provided below:
Culturing Cry19Aa toxin:
A single colony was seed cultured in 5ml LB supplemented with 10ul erythromycin
(10mg/ml in ethanol filtered and stored at -20 degrees Celsius) to make a final
concentration of 20ug/ml, shake at 250-350rpm at 30 degrees Celsius overnight.
First we prepared a medium to grow the toxin, we added 2g/L Peptone, 5g/L Yeast
extract, 12.54g/L K2HPO4(Dibasic), 2.31g/L KH2PO4 (Monobasic), we also filter
sterilized and added 5 ml 20% glucose + 1.23% MgSO4 , 1 ml 7.5% Cacl-2H2O
,1 ml 0.15 MnSO4- 7H2O and 1ml 0.014% FeSO4- 7H2O to the Erlenmeyer flask.
and then add 2ml Erythromycin stock (10mg/ml), Shake at 250-300 rpm at 30
degrees Celsius. After one day, we added 1L of sodium phosphate solution
[(Na2HPO4 8g/L (Na2HPO4-12H2O 20.18g) + NaH2PO4 5g/L (NaH2PO4-H2O 5.75g)]
into the growing medium. After this, we cultured the toxin for another two days
until complete sporulation. Then we centrifuged the solution having sporulated
Cry19Aa at 5000rpm for 20 minutes. Then we discarded the supernatant. Next, we
purified the Cry19Aa.
5. Pg.5
Purification of Cry19Aa:
Resuspended the pellet in 80ml of CWI(0.1M NaCl, 2%Triton X-100, 20mM Bis-Tris
pH 6.5) and sonicated it for 3 miniutes twice. Then Centrifuged and re-suspended
the pellet in 80ml of CWI. Then we repeated the above step with CWI twice and with
ultra pure H2O once. Then once again repeated the above step with CWII (1M Nacl)
twice and with ultra pure water once. After that we re-suspended the pellet in 5ml
of ultra pure H2O.
Separation of spores and crystals:
To further separate spores from crystals, we used a discontinues NaBr gradient. We
prepared a 30-60% NaBr gradient, after the gradient formed in the tubes we
overlayed the pellet on the gradient. Then we ultra centrifuged the gradient at
20,000 rpm for 1 hours and 15 minutes. This caused a layer of crystals to float up to
the top; we then extracted the crystals and washed them twice with ultra pure
water.
Solubilization and trypsin activation of Cry19Aa crystals:
To solubilize the cry19aa crystals, we solubilized the crystals in carbonate
buffer(Na2CO3/NaHCO3,buffer, pH 11.5) and 10mM DTT. We solubilized the crystals
at 37OC overnight. After solubilization, we added trypsin in 1:10(weight by weight
ratio).
Anion exchange chromatography was performed using Buffer A= 50 mM Carbonate
buffer + 100mM NaCl(pH 11.50) and Buffer B= 50mM Carbonate buffer + 1 M
Nacl(pH 11.50) and this separated the activated cry19Aa from other protein
6. Pg.6
degrading toxin proteins. Then we carried out a concentration course and time
course assay.
To carry out the Cry19Aa concentration course and time course quantitate assay on
Ag55 cells to measure toxicity:
We seeded the 1x10^6 cells in the 7 wells of the 12 wells in the cell culture
plate.
The cells were treated with different concentrations of trypsin activated
Cry19Aa.
Live and dead cells were counted after 16 hours of incubation for
concentration course assay @ 2,4,6,8,24 hours for time course assay with
trypsin activated Cry19Aa using Trypan blue and Hemocytometer(an
instrument for visual counting of number of cells). Cells were counted from 4
corners. Each corner square having 16 small squares.
We then calculated the percentage of cells viable. (% cells viable/total
number of cells)x100.
We then repeated the experiment 3 and 2 times for time course and
concentration course respectively.
This part of the experiment helped us measure the toxicity and affectivity of
Cry19Aa to Ag55 cells.
Results:
The size of the activated Cry19Aa toxin was approximately 42 kDa. This is
confirmed below in the gel-electrophoresis. Lane 1 is the marker while lane 2 is the
solubilized Cry19Aa. While lane 3 is trypsin activated Cry19Aa.
7. Pg.7
The approximate band length of trypsin activated Cry19Aa was 42 kDa. This was
obtained using SDS PAGE electrophoresis. The results of the toxicity of trypsin
activated Cry19Aa on the Ag55 cell lines is shown below. We obtained these results
using an inverted phase contrast microscope.
Lane 1 2 3
97.4
66.2
45
31
21.5
kDA
8. Pg.8
The results show rapid toxicity to Ag55 cell lines by 8 minutes. Using these pictures,
we were able to carry out a concentration and time course assay. The results of the
time and concentration course are also shown below.
9. Pg.9
Viability of cells was checked at 16 hrs. after incubation with activated Cry19Aa
using trypan blue.
Trypan Blue Ag55 cell viability time course assay. Blue: Control (0.8 nM Carbonate
buffer pH 9.6).
Red: 0.8 nM Cry19Aa. Readings were taken at 2 hrs , 4 hrs , 6 hrs , 8 hrs & 24 hrs.
10. Pg.10
Discussion:
Our results were consistent in showing that the trypsin activated Cry19Aa
toxin was indeed cytotoxic to the Anopheles gambiae cell lines. The time course and
concentration course assay showed that the percentage of viable cells with
increasing Cry19Aa concentration decreased significantly with larger Cry19Aa
concentrations. The time course assay also showed that after 24 hours of exposure
to Cry19Aa, the cell viability decreased to zero. These results show that Cry19Aa is
an efficient toxin that could be used to effectively control major mosquito vectored
diseases around the world by eliminating mosquitoes rapidly from the area. Further
research on the Cry19Aa toxin and its mechanism on cell membrane proteins of the
Ag 55 cell lines could give insights into developing or discovering even more
efficient toxins for mosquito control.
11. Pg.11
References
Nadjm, B. (2012). Malaria: An update for physicians. PubMed. Retrieved April 27,
2016, from http://www.ncbi.nlm.nih.gov/pubmed/22632637
Roh et al.2009. Mutagenic analysis of putative domain II and surface residues in
mosquitocidal Bacillus thuringiensis Cry19Aa toxin. FEMS Microbiol
Lett. 295(2):156-63.
Teixeira Correa et al. 2012.Cytotoxicity Analysis of three Bacillus thuringiensis
subsp. Israelensis δ-Endotoxins towards Insect and Mammalian Cells. PLoS ONE
7(9): e46121