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.
For centuries, humans have searched for crop plants that can survive and produce in spite of insect pests.
Knowingly or unknowingly, ancient farmers selected for pest resistance genes in their crops, sometimes by actions as simple as collecting seed from only the highest-yielding plants in their fields.
With the advent of genetic engineering, genes for insect resistance now can be moved into plants more quickly and deliberately.
Bt technology is only one example of ways genetic engineering may be used to develop insect-resistant crops now and in the future.
For centuries, humans have searched for crop plants that can survive and produce in spite of insect pests.
Knowingly or unknowingly, ancient farmers selected for pest resistance genes in their crops, sometimes by actions as simple as collecting seed from only the highest-yielding plants in their fields.
With the advent of genetic engineering, genes for insect resistance now can be moved into plants more quickly and deliberately.
Bt technology is only one example of ways genetic engineering may be used to develop insect-resistant crops now and in the future.
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
Answering the Call to Arms: Tools for assessing the anti-infective potential ...Cassandra Quave
This is a presentation delivered at the 16th Annual Conference on the Science of Botanicals and 5th Annual Interim American Society of Pharmacognosy Meeting from April 11-14, 2016 in Oxford, MS, USA.
Abstract:
Answering the Call to Arms: Tools for Assessing the Anti-infective Potential of Natural Products in a Time of Rising Antibiotic Resistance
Quave CL1,2
1 Center for the Study of Human Health, Emory University, 550 Asbury Circle, Candler Library 107, Atlanta, GA 30322 USA. 2 Department of Dermatology, Emory University School of Medicine, 615 Michael Street, Whitehead 105L, Atlanta, GA 30322 USA.
As antibiotic resistance continues to rise, the pool of viable anti-infective therapeutic options is becoming rapidly exhausted. New therapies are in high demand and natural products are a likely source of novel bioactive compounds to meet this need. In particular, botanical secondary metabolites represent a rich pool for antibiotic discovery efforts. Plants are often the primary ingredients used in traditional anti-infective therapies, and yet their activity and mechanisms of action are often poorly understood. Much of the antibacterial research on botanical extracts and essential oils has focused on growth inhibitory studies using outdated methods limited in their ability to obtain an accurate assessment of bioactivity. The emergence of new molecular and bioanalytical tools for drug discovery provides a unique opportunity for application to natural products research.
Using Staphylococcus aureus as a model, tools for anti-infective testing of plant extracts will be reviewed, specifically focusing on the merits and limitations of each method. Examples include standardized methods for examining activity for the inhibition of growth (e.g., MIC, MBC), virulence (e.g., quorum sensing and toxin quantification) and pathogenesis (e.g., biofilms and antibiotic synergy). Data from our recent discoveries of novel biofilm [1] and quorum sensing [2,3] inhibitors isolated from medicinal plants (Rubus ulmifolius, Castanea sativa and Schinus terebinthifolius) will be presented in the review of these tools.
Acknowledgements: This work was supported by a grant from the National Institutes of Health, National Center for Complementary and Integrative Health (R01 AT007052). The content is solely the responsibility of the authors and does not necessarily reflect the official views of NCCIH or NIH.
References: [1] Quave CL, Estévez-Carmona M, et al. (2012) PLoS ONE, 7(1): e28737. [2] Quave CL, Lyles JT, et al. (2015) PLoS ONE, 10(8): e0136486. [3] Quave CL, Horswill AR (2014) Frontiers in Microbiology, 5: 706.
Description of the major classes of antimicrobial drug, resistant mechanisms developed by bacteria to combat the action of antimicrobials, and the control measures needed to limit this horizontal gene transfer.
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
Answering the Call to Arms: Tools for assessing the anti-infective potential ...Cassandra Quave
This is a presentation delivered at the 16th Annual Conference on the Science of Botanicals and 5th Annual Interim American Society of Pharmacognosy Meeting from April 11-14, 2016 in Oxford, MS, USA.
Abstract:
Answering the Call to Arms: Tools for Assessing the Anti-infective Potential of Natural Products in a Time of Rising Antibiotic Resistance
Quave CL1,2
1 Center for the Study of Human Health, Emory University, 550 Asbury Circle, Candler Library 107, Atlanta, GA 30322 USA. 2 Department of Dermatology, Emory University School of Medicine, 615 Michael Street, Whitehead 105L, Atlanta, GA 30322 USA.
As antibiotic resistance continues to rise, the pool of viable anti-infective therapeutic options is becoming rapidly exhausted. New therapies are in high demand and natural products are a likely source of novel bioactive compounds to meet this need. In particular, botanical secondary metabolites represent a rich pool for antibiotic discovery efforts. Plants are often the primary ingredients used in traditional anti-infective therapies, and yet their activity and mechanisms of action are often poorly understood. Much of the antibacterial research on botanical extracts and essential oils has focused on growth inhibitory studies using outdated methods limited in their ability to obtain an accurate assessment of bioactivity. The emergence of new molecular and bioanalytical tools for drug discovery provides a unique opportunity for application to natural products research.
Using Staphylococcus aureus as a model, tools for anti-infective testing of plant extracts will be reviewed, specifically focusing on the merits and limitations of each method. Examples include standardized methods for examining activity for the inhibition of growth (e.g., MIC, MBC), virulence (e.g., quorum sensing and toxin quantification) and pathogenesis (e.g., biofilms and antibiotic synergy). Data from our recent discoveries of novel biofilm [1] and quorum sensing [2,3] inhibitors isolated from medicinal plants (Rubus ulmifolius, Castanea sativa and Schinus terebinthifolius) will be presented in the review of these tools.
Acknowledgements: This work was supported by a grant from the National Institutes of Health, National Center for Complementary and Integrative Health (R01 AT007052). The content is solely the responsibility of the authors and does not necessarily reflect the official views of NCCIH or NIH.
References: [1] Quave CL, Estévez-Carmona M, et al. (2012) PLoS ONE, 7(1): e28737. [2] Quave CL, Lyles JT, et al. (2015) PLoS ONE, 10(8): e0136486. [3] Quave CL, Horswill AR (2014) Frontiers in Microbiology, 5: 706.
Description of the major classes of antimicrobial drug, resistant mechanisms developed by bacteria to combat the action of antimicrobials, and the control measures needed to limit this horizontal gene transfer.
DOI:10.21276/ijlssr.2016.2.4.2
neoplastic progression through the action of viral oncoproteins, mainly E6 and E7.Cervical cancer remains the second
most common cancer in women worldwide with India as a major contributor to global burden with an annual incidence of
132,000 new cases and mortality rate of 74,000 deaths annually. In this study turmeric, neem, tulasi and ginger were
selected as natural anticancer drugs. The objective of the study was to analyze the anticancer property of turmeric
(Curcuma longa), neem (Azadirachta indica), tulasi (Occimum sanctum) and ginger (Zingiber officinale) on HeLa cells.
Turmeric, neem, tulasi and ginger capsules (Himalaya’s Company) were used and aqueous and methanolic extracts of the
turmeric, neem, tulasi and ginger were obtained using a soxhlet extraction. To check the efficacy of these drug MTT assay
was performed, that determines % viability and/or cytotoxicity. IC50 of aqueous turmeric, neem, tulasi and ginger extracts
in case of HeLa cells were 17.8, 22, 79.4, 27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger
extracts 17, 7.35, 75.24 and 16.1 respectively. To confirm apoptosis as the sole reason behind cell death
immunofluorescence based apoptosis assay was performed using TALI image based cytometer. The study has led to
postulate hypothesis that natural drugs e.g. turmeric, neem, tulasi and ginger are potent anti-cancer compound that are
capable of inhibiting the growth of immortal cells by apoptosis. Key-words- Cervical cancer, Human papillomavirus (HPV), Oncoproteins E6 and E7, Natural compounds, HeLa cell
line (adherent), Cell viability and MTT assay, Apoptosis assay
Cancer is the leading cause of death in worldwide whose treatments are costly and has different physical and emotional side effects. Parsporal inclusion proteins from B. thuringiensis having specific cytotoxic activity against specific cancer cells can be used for cancer treatment which might have relatively lower side effects and might be cost effective. We have reported in silico analysis that is physico-chemical characteristics, secondary structure, 3-D structure, motifs and phylogenetic relationship among these 19 parasporin proteins to characterize them and predict their cancer cell killing mechanism.
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVA...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a way to get native tPA protein sequence in subsequent purification steps
Evaluating the ability of anti-cancer drugs Etoposide and Staurosporine to in...Davient Bala
Cervical cancer is considered one of the most prevalent cancers affecting Singaporean women.
Although many novel chemotherapeutics have been developed recently, little has been done to
determine the efficiency of current anti-cancer agents working in combination. Here, we aimed to
evaluate the apoptosis induction efficiency of Etoposide and Staurosporine in HeLa cells. Cell cultures
were subjected to either 50 μM Etoposide, 10 nM Staurosporine or both for 24 hours prior visualization
under a fluorescence microscope. We found that Etoposide alone had an efficiency of 16.1% while
Staurosporine alone had 18.3%. However, the polytherapy achieved an efficiency of up to 33.6%,
which indicates an additive effect of both drugs to induce apoptosis. Our results demonstrate that
Etoposide and Staurosporine are both capable of inducing apoptosis in HeLa cells. Furthermore, it
reveals the potential of Etoposide-Staurosporine polytherapy to be a potent combinative treatment
option for cervical cancer patients resistant or sensitive to conventional anti-cancer agents.
Washington Global Health Alliance Discovery Series
Robert Sinden, PhD
July 28, 2008
'Understanding Malaria Development in the Mosquito, and its Pivotal Role in the Formulation of Effective Control Strategies'
Centre of innovation, Agricultural College and Research Institute,MaduraiSenthil Natesan
Establishment Central Instrumentation facility with the cost of 6.03 crore to take up multidisciplinary research project at AC&RI,Madurai. The analytical platform includes UP-HPLC for amino acid analysis, XRF for micronutrient analysis and GC-MS for metabolic profiling. The imaging facilities like upright, inverted and Florence microscope established for imaging pathogen & Insects. The molecular biology lab with real time PCR will help for the gene expression studies.
The agriculture sector employs nearly half of the workforce in the country. However, it contributes to 17.5% of the GDP (at current prices in 2015-16).Agriculture sector’s contribution has decreased from more than 50% of GDP in the 1950s to 15.4% in 2015-16 (at constant prices). This slides discuss about Indian agriculture status and problems and solutions.
paper presented during the National seminar on Challanges and Innovative approaches in Crop Improvement at AC&RI, Madurai. during December 16-17, 2014 .Germplasm conservation in Oil Palm by Dr P. Murugesan Indian Institute of Oil Palm Research
Improvement of Medicinal Plants: Challenges and Innovative ApproachesSenthil Natesan
Paper Presented during the National seminar on Challenges and Innovative approaches in crop improvement held at AC&RI, Madurai , TNAU by
Dr.P. Manivel, Directorate of Medicinal and Aromatic Plants Research, Boriavi-387310, Anand, Gujarat
Genomics platform for agriculture-CAT lectureSenthil Natesan
The popular lecture for the undergraduate students of agriculture to know about the application of biotechnology in agriculture science graduates. Some of the major break through inventions how it impact on agriculture research and development
Castor is an oilseed plant which is earning attention on researchers in recent days. Because of this, the gemplasms of ancient varieties were now recovered and grown in trial fields for getting genetically superior variety.
As a result, in Castor and Tapioca research station there a variety named YRCH (Yethapur Ricinus Communis Hybrid) with all desired traits which are essential for a plant both phenotypically and genetically was developed.
Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. Apart from seed oil vegetative tissue is potential source as bio mass for biofuel production, taking 15 tonnes per hectare as an average dry matter yield for a perennial grass, an oil content of 20– 25% by weight will produce about 3400 l of biodiesel (Heaton et al., 2004). There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID: DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway (Julian at al..2013). First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55 ( Andrianovet al 2010). Therefore Biotechnology has important and perhaps critical part to play in large-scale development of Biodiesel.
Vaccines have been revolutionary for the prevention of infectious diseases. Despite worldwide immunization of children against the six devastating diseases, 20% of infants are still left un-immunized; responsible for approximately two million unnecessary deaths every year, especially in the remote and impoverished parts of the globe. This is because of the constraints on vaccine production, distribution and delivery. One hundred percent coverage is desirable, because un-immunized populations in remote areas can spread infections and epidemics in the immunized safe areas, which have comparatively low herd immunity. For some infectious diseases, immunizations either do not exist or they are unreliable or very expensive. Immunization through DNA vaccines is an alternative but is an expensive approach, with disappointing immune response. Hence the search is on for cost-effective, easy-to-administer, easy-to-store, fail-safe and socio-culturally readily acceptable vaccines and their delivery systems. As Hippocrates said, Let thy food be thy medicine, scientists suggest that plants and plant viruses can be genetically engineered to produce vaccines against diseases such as dental caries; and life-threatening infections like diarrhea, AIDS, etc (Lal et al., 2007)
Cellular signal transduction pathways under abiotic stressSenthil Natesan
Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps (Knight & knight ,2001).Many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway (Miura and Furumoto,2013 ) . The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress (shi .et al ,2002). Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance (Nakashima and shinozaki, 2013) .ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response ( Mahajan,s and Tuteja, 2005) .
Genotyping by Sequencing is a robust,fast and cheap approach for high throughput marker discovery.It has applications in crop improvement programs by enhancing identification of superior genotypes.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Embracing GenAI - A Strategic ImperativePeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
1. Useful proteins from Bacillus thuringiensis
for non-agricultural applications
Subhada Pattanayak
2. OUTLINE
Bt for cancer treatment
Introduction Bt against protozoan diseases
Applications Mosquito control
Endoparasite control
Conclusion
Bacteriocins
Discussion
Enzyme production
3. Introduction
Bacillus thuringiensis- a gram positive,
spore forming bacteria produces
crystalline parasporal inclusions
Toxic to agriculturally and medically
important pests
Non-insecticidal Bt strains have
cytocidal activity
4. A conceptual diagram of the present and previous B. thuringiensis crystal
A conceptual diagram of the present and previous B. thuringiensis crystal
protein world.
protein world.
(Kitada et al., 2006)
6. Applications…
Bt proteins for cancer treatment
Scanning electron micrograph of HTLV-I virus (green) infecting a
human T-lymphocyte (yellow). Infection with this virus can stimulate
the T-cells to proliferate at an increased rate, causing a risk of
developing leukemia.
7. Brief history….
In 1970s, Prasad and Shethna had carried out research on the anti-tumour
effects of Bt.
Parasporal proteins of Bacillus thuringiensis serovar shandongiensis found
cytotoxic to human leukaemic T cells (Lee et al., 2000).
A soil isolate belonging to Bacillus thuringiensis serovar dakota (H15) was
found to produce non-cyt inclusion proteins that were highly cytotoxic against
human leukaemic T cells (MOLT-4) and moderately cytotoxic to human
cervical cancer cells (HeLa) (Kim et al., 2000).
8. Parasporins
“Parasporin” was first used by Mizuki et al to describe a
novel protein with a unique cytotoxicity in 2000.
Defined as Bt and related bacterial parasporal proteins
that are non-haemolytic but capable of preferentially
killing cancer cells
9. Classification of Parasporins
• In 2006, the Committee of Parasporin Classification and
Nomenclature was organized to construct a taxonomically sound
classification system based on the amino acid identity
• In the nomenclature scheme, the number and letter system B.
thuringiensis was adopted so that a novel parasporin protein is
assigned to a new class incorporating four ranks
• Currently, approximately 95, 78, and 45% sequence identities are
the borders of the four ranks.
10. The parasporins are broadly divided into 6 main classes
• Till date 19 parasporins discovered and placed on the list of
parasporins
• Mechanism of action of PS1, PS2, PS3 and PS4 has been
well studied and much less is known about PS5 and PS6.
11. List of parasporins ( till March 2012)
Name Cry No. Acc No. Authors & Year
PS1Aa1 Cry31Aa1 AB031065 Mizuki et al. 2000 A1190
PS1Aa2 Cry31Aa2 AY081052 Jung & Côté 2002 M15
PS1Aa3 Cry31Aa3 AB250922 Uemori et al. 2006 B195
PS1Aa4 Cry31Aa4 AB274826 Yasutake et al. 2006 Bt 79-25
PS1Aa5 Cry31Aa5 AB274827 Yasutake et al.2006 Bt 92-10
PS1Aa6 submitting AB375062 Nagamatsu et al.2010 CP78A, M019
PS1Ab1 Cry31Ab1 AB250923 Uemori et al. 2006 B195
PS1Ab2 Cry31Ab2 AB274825 Yasutake et al.2006 Bt 31-5
PS1Ac1 Cry31Ac1 AB276125 Yasutake et al.2006 Bt 87-29
PS1Ac2 Cry31Ac2 AB731600 Kuroda et al. 2012 B0462
PS1Ad1 submitting AB375062 Nagamatsu et al.2010 CP78B, M019
PS2Aa1 Cry46Aa1 AB099515 Ito & Kitada 2004 A1547
PS2Aa2 Cry46Aa2 AB454419 Ishikawa et al.2008 A1470
PS2Ab1 Cry46Ab1 (http://parasporin.fitc.pref.fukuoka.jp/index.html)
AB186914 Yamagiwa et al.2004 TK-E6
12. Characteristics
Differ in molecular weight and composition
Each PS has different cell specificity due to the presence
of different receptor, genome sequence, mode of action
and targeting specificities
(Wong,
2010)
13. Cytotoxicity spectra of parasporins
Cytotoxicity spectra of parasporins
The levels of cytotoxicity based on the EC50 values in cell proliferation assay graded as follows:
extremely high (++++), high (+++), moderate (++), low (+), and very low / non-toxic (–). NT: Not tested
(Ohba et al., 2009)
14. Cytocidal action and cell specificities
PS-1 has high toxicity against cancer cell lines like HeLa cells (cell line from
cervical cancer cells)
PS-2 shows toxicity against cell like MOLT-4 (Leukemic T cells), Jurkat (cell
line from T lymphocyte cells) and Hep G2 (Hepato cellular carcinoma-liver
tissue)
PS-3 has toxicity against Hep G2 and HL-60 (myeloid leukemia cells)
PS-4 shows toxicity against CACO-2 (epithelial colorectal adenocarcinoma
cells)
PS-5 – Unknown
PS-6 shows anticancer activity against human hepatocyte cancer cells and
cervical cancer cells (Wong et al., 2010)
15. Morphological changes caused by PSs
HeLa PS-1: Blebbing of the cells
HepG2 PS-2: Balloon shaped
HepG2 PS-3: Fragmenting
MOLT-4 PS-4: Detachment
2006
16. Parasporin 1
Exists as 81 kDa protein, Pro-PS-1
Upon activation, PS-1 exists as a 15 kDa and 56 kDa heterodimer.
Affects membrane permeability, calcium homeostasis
Several conclusions were drawn from the studies on PS1:
1) PS-1 cytotoxic against HeLa cells
2) PS-1 causes an increase in Ca2+ influx but the influx not related to Ca2+
channels and due to extracellular Ca2+
3) Heterotrimetric G-proteins or G-protein coupled receptors involved in
parasporin-1 induced Ca2+ influx
4) PS-1 not a pore forming toxin
5) Mode of cell death most likely apoptotic.
(Katayama et al., 2007)
18. Parasporin- 2
• 30kDa protein found to be a pore forming toxin which caused
increased permeability to the susceptible cells.
• PS-2 distributed at the cell periphery
• PS-2 oligomerised at the cell surface via binding to lipid rafts which
led to cell lysis and that glycosyl phosphatidyl inositol (GPI)-
anchored proteins involved in such cytocidal activity
(Kitada et al, 2009)
19. Detection of parasporin-2 in hepatocellular
carcinoma.
• Cancer and non-neoplastic tissues
incubated with parasporin-2, and analyzed
using an anti-parasporin-2 antibody.
• Extensive presence of parasporin-2 in
hepatocellular carcinoma cells but not in
non-neoplastic liver cells
• Parasporin-2 binds specifically to the
cancer cells.
Detection of parasporin-2 in colon
cancer cells.
• Sections of colon cancer tissues
treated with parasporin-2
• Columnar cancer and fibroblastic
cells indicated by arrows and
arrowheads, respectively.
• The toxins efficiently bound to the
cancer cells but not to the peripheral
fibroblastic cells.
(Kitada et al., 2006)
20. Parasporin-3
• PS-3 is a typical three-domain-type Cry protein
• PS-3 acts as a pore-forming toxin on the plasma membrane of
cancer cells and increases plasma membrane permeability of target
cells
(Wong et al., 2010)
21. Parasporin-4
Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus
thuringiensis strain A1470.
It exhibits high cytotoxicity against human cancer cell lines, CACO-
2, Sawano, and MOLT-4 cells
Exhibits strong cytotoxicity against several human tumor cell lines
when activated by protease treatment but does not exhibit
insecticidal or hemolytic activities
PS4 binds non-specifically to the plasma membrane and
oligomerizes to form pores only in target cells, inducing cell death.
(Okumura et al., 2011)
22. Cytopathic effect of parasporin-4 (PS4) against CACO-2 , HeLa , MOLT-4
Cells were observed by phase contrast microscopy before and 4 h after treatment with
4 μg/ml PS4.
(Okumura et al., 2011)
23. Cytopathic effects in MOLT-4 cells observed with a differential interference
microscope. PS4 was added at a final concentration of 2 μg/ml
(Okumura et al., 2011)
25. Erythrocytes of mice infected with
Plasmodium berghei
B. thuringiensis protein samples of 0.3 ml
each were injected into infected mice
through the tail vein on the 1st and 2nd day
after infection
26. Survival days of mice infected with Plasmodium berghei
after treatment with Bacillus thuringiensis crystal
proteins
Bt crystal proteins injection
prolonged the survival of the infected
mice.
The mean life for plasmodium-
infected mice was about 8.5 days.
For those injected with crystal
proteins from strains 007, 020, 021,
030, or 032, the mean life was
extended to 13.5–15 days
Crystal proteins could protect erythrocytes from Plasmodium attack.
This study suggested a novel way to control plasmodial infections and even malaria.
27. Leishmaniasis are caused by haemoflagellate protozoan which belongs to
genus Leishmania that infect vertebrate hosts through the bites of sand fly
females Phlebotomus spp.
28. Active non truncated core toxin of B.t. serovar thuringiensis (H14) 43 kDa
for their activity against Leishmania major promastigotes used.
The active protein fraction was bioassayed against L. major promastigotes
suspension (7×107 promastigotes /ml) in final concentrations ranging from
100 - 0.78 μg /ml aseptically in replicates.
The bioassay of protein fraction showed its LC50 is 4.95 μg/ml
29. Cytopathological changes in Leishmania major promastigotes started with
swelling (A) followed by changing from spindle shape to spheroid (B) berry
shaped cells (C) cytoplasmic proteins with a giant increase in size (D).
While the untreated promastigotes keep motile and alive in pairs (E&F)
31. Mosquitoes act worldwide as vectors transmitting disease causing viruses and
parasites such as malaria, yellow fever, dengue fever, filariasis, St. Louis
encephalitis and the West Nile virus between humans and animals (Tolle, 2009).
B. thuringiensis subsp. israelensis (Bti) has been extensively studied for
its specific and high toxicity to mosquito and black fly larvae since its discovery
in 1976.
The parasporal inclusion body of Bti consists of four major insecticidal crystal
proteins (Cry4Aa,Cry4Ba, Cry11Aa and Cyt1Aa) (Stein et al., 2006).
32. Although Bti and its toxins have been successfully commercialized for
mosquito control, screening programs have continued worldwide to identify
and characterize new mosquitocidal Bt isolates and toxin genes
The Bt S2160-1 strain was isolated from soil samples collected from
Southern China and found to have a comparable mosquitocidal activity to
Bti.
PCR-restriction fragment length polymorphism identification system was
developed and used in order to identify novel cry-type genes cry30Ea,
cry30Ga, cry50Ba and cry54Ba
(Zhang et al., 2012)
34. • Hookworms, whipworms (Ancylostoma duodenale, Necator
americanus, and Ancylostoma ceylanicum ) are major soil-transmitted
helminths (nematodes, roundworms) that parasitize humans, infecting 576–
740 million people globally and are the leading source of iron-deficient
anemia in endemic areas(Bethony et al., 2006).
• For mass drug administration against soil-transmitted helminths like
hookworms, the current drug of choice is albendazole.
• One promising group of alternative anthelmintics is roundworm- active
crystal proteins, in particular Cry5B, made by Bacillus thuringiensis
(Hu & Aroian, 2012)
35. • Cry5B and Cry21A have therapeutic activities against infections of
the roundworm Heligmosomoides polygyrus bakeri in mice.
• Cry5B shows highly therapeutic activity against Ancylostoma
ceylanicum infection in hamsters, a minor hookworm parasite of
humans
• Cry proteins show excellent combinatorial therapeutic properties
with nicotinic acetylcholine receptor (nAChR) agonists, one of the
two classes of compounds approved by the World Health
Organization for the treatment for intestinal roundworms in humans.
(Hu & Aroian, 2012)
36. Typical morphology (at 360 magnification) seen in the L4 Anterior intestine of nematodes fed with four toxic
plate assay after feeding nematode crystal proteins in E. coli. showing reduction of
species E. coli transformed with empty vector (Left), width of intestine at one position near the
vector plus nontoxic Cry protein insert (Center), or vector anterior.
plus toxic Cry protein insert
(Wei et al., 2003)
37. Efficacy of Cry proteins against roundworms
Efficacy of Cry proteins against roundworms
Parasitic roundworms. a A. ceylanicum b. Heligmosomoides polygyrus
bakeri (Hu & Aroian, 2012)
38. Glycolipids that bind Cry5B known as arthroseries glycolipids
are specific to roundworms (nematodes) but lacking in
mammals and vertebrates (Griffitts and Aroian , 2005).
Thus, Cry proteins like Cry5B are non-toxic to vertebrates due
to lack of the Cry5B arthroseries glycolipid receptors
Given their non-toxicity to humans and their broad spectrum
of nematicidal action, Cry proteins show great potential as
next-generation anthelmintics.
39. • Anthelminthic activities of purified recombinant Cry5B against the
hookworm parasite Ancylostoma ceylanicum, a bloodfeeding
gastrointestinal nematode for which humans are permissive hosts.
• Cry5B was found to be highly toxic to early stage hookworm larvae.
• Exposure of adult A. ceylanicum to Cry5B was also associated with
significant toxicity including a substantial reduction in egg excretion
by adult female worms.
40. Exposure to Cry5B impairs motility of
adult hookworms in culture.
Cry5B toxin reduces A. ceylanicum egg
excretion.
41. Cry5B treatment reduces
hookworm infection as measured
by weight gain and blood
haemoglobin
43. Bacteriocins ?
• Inhibitory peptides or proteins, produced by different groups of
bacteria, which have bactericidal effects on micro-organisms closely
related to the producer(Jack et al. 1995).
• Produced by bacteria as a defense mechanism in complex
environments.
44. Bacteriocins produced by Bacillus thuringiensis
Source Bacteriocin Mol wt(kDa) Activity
(Abriouel et al., 2011)
46. Proteases
• Proteases are essential for biological processes like cell cycle
regulation, cell growth and differentiation and sporulation.
• Bt is an excellent source of protease enzymes. (Brar et al., 2007)
Cellulases
Cellulases
Bacillus thuringiensis strains produced novel cellulases which could liberate
glucose from soluble cellulose, carboxymethyl cellulose (CMC), and insoluble
crystalline cellulose.
(Bisht, 2010)
47. Chitinases:
• Bacillus thuringiensis produces chitinases
• The presence of endochitinase and exochitinase genes was detected
via PCR screening of 16 B. thuringiensis isolates which showed also
an important chitinolytic activity on plates containing colloidal chitin
(Bisht, 2010).
as a major or unique carbon source
48. Autolysins
Endogenous peptidoglycan hydrolases that digest cell wall
peptidoglycans of the producer bacterium and of other bacteria
The characterisation of the autolytic phenotype of 112 B. thuringiensis
strains showed seven major proteins of molecular weights ranging
between 25 and 90 kDa which exhibited peptidoglycan hydrolase activity,
particularly at alkaline pH.
Several of these proteins retained lytic activity against other bacterial
species such as Micrococcus lysodeikticus, Listeria monocytogenes and
Staphylococcus aureus.
These are of great interest in field application of B. thuringiensis for
improving bacterial or insect biocontrol by coupling with other
antagonistic factors such as bacteriocins or chitinases
(Bisht, 2010)
Erythrocytes of mice infected with Plasmodium berghei. A, B Three days after inoculation with P. berghei. C, D Five days after inoculation. E, F Seven days after inoculation. A, C and E are samples from a control mouse; B, D and F show samples from a mouse treated with crystal proteins extracted from Bacillus thuringiensis strain 037. Arrows in A show examples of regular, infected, and lysed cells