The document summarizes Karan Sharma's masters thesis on engineering surfaces to support adhesion and growth of neural stem cells and hepatocytes. The goals were to develop xeno-free synthetic biomedical devices like nerve grafts and artificial livers. Methods included creating poly 4-vinylphenol and peptide coatings on polyacrylonitrile membranes to support long-term cell cultures. Results demonstrated adhesion and proliferation of neural stem cells and hepatocytes on the coatings and membranes. Future work involves testing different coating properties and constructing artificial tissues.
Peyer Labs Cloning a Fluorescent Gene BrochureTom Loughran
Cloning is the process of replicating DNA inside bacteria. Research scientists use cloning to begin experiments like genetically engineering mice and producing human insulin. Peyer Laboratory Systems presents their Cloning A Fluorescent Gene kit, which allows students to engage in complex experimental biology. The kit provides students with precision instruments, pre-packaged reagents and bacteria, and a full-color manual to guide them through amplifying, inserting, and expressing a green fluorescent protein gene across multiple lab periods.
2013 Merck Millipore Best Practices in Nucleic Acid Removal from Vaccine Proc...Frank Appel
The document discusses various methods for removing nucleic acids like DNA from vaccine manufacturing processes. It describes how density gradient centrifugation, depth filtration using Millistak+ filters, and nuclease treatment with Benzonase can each reduce DNA levels. It also outlines methods for removing residual Benzonase like chromatography using Fractogel resins and tangential flow filtration with Pellicon cassettes. The document provides an overview of regulatory guidelines for DNA limits and analytical techniques for detecting residual Benzonase.
Benzonase® endonuclease: use and clearance with a TFF step in a cell culture-...Frédéric Sengler
Benzonase® endonuclease is a powerful tool for degrading all
forms of (deoxy)ribonucleic acids (DNA/RNA) in cell culture
harvests to base pair (BP) lengths under 8 units. It is effective
over a wide range of conditions including temperature, pH, and
varying concentrations of Mg2+, detergents, chelating agents,
and monovalent cations. It is often employed in the production
of viral vaccines, completely digesting DNA and RNA to improve
clearance and reduce solution viscosity.
Removing Benzonase® endonuclease, a 60 kD dimer, from the
process stream post-treatment may be achieved with Tangential
Flow Filtration (TFF) with the appropriate molecular weight
cut-off membrane (MWCO), typically 300 kD. This process has
heretofore not been well described in the literature, so the
present study fills this gap pairing Benzonase® endonuclease
treatment of a DNA-spiked inactivated flu virus cell culture
harvest with Pellicon® 2 cassettes, for clearance of the digested
DNA and remaining Benzonase® endonuclease by diafiltration.
Personalized nanomedicine for the treatment of vascular hypertensionSusanta Kumar Rout
This study includes designing a nanomedical device for the treatment of vascular hypertension in polycystic kidney diseases (PKD) model through cilia targeting.
They generated and compared two different metal and polymer cilia-targeted nanoparticle drug delivery systems (DDS), i.e. gold (Au) and poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs)
The target is Dopamine-receptor type-5 (DRS) on primary cilia.
The drug-loaded is Fenoldopam (FD).
Stephen Friend Nature Genetics Colloquium 2012-03-24Sage Base
This document proposes using data intensive science to build models of disease within a shared computing environment or "commons". It notes that current disease models often oversimplify complex conditions. Five pilot projects are described that could leverage shared clinical and genomic data as well as model building to better represent diseases: 1) sharing comparator arm data from clinical trials, 2) a federated aging analysis project, 3) portable legal consent, 4) a Sage Congress modeling competition, and 5) the BRIDGE initiative for democratizing medical research. The document argues this approach could accelerate disease understanding and new therapy development.
This document discusses cytogenetic techniques used in biology. It begins by outlining the learning objectives which are to list cytogenetic techniques and understand cell culture, harvesting chromosomes, and karyotyping. It then describes various techniques including cell culture, harvesting and spreading chromosomes, karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The purpose of these techniques is to examine genetic components of cells including chromosomes to study genetic abnormalities.
This document discusses molecular biology techniques. It begins by outlining the learning objectives, which are to list techniques used in molecular biology and describe microscopy, centrifugation, extraction, electrophoresis, and chromatography. It then provides details on each technique, including their purposes and basic procedures. Microscopy is used to produce magnified images of cells and structures. Centrifugation separates substances by density. Extraction isolates molecules like DNA, RNA, and proteins. Electrophoresis separates charged substances using an electric field. Chromatography separates mixtures based on interactions with a solid or liquid medium.
Dna extraction from blood and forensic samplesCAS0609
This document provides instructions for extracting DNA from various forensic samples, including blood, absorbing substrates like cloth or paper, and non-absorbing substrates like metal or plastic. The summary is as follows:
1) Precautions must be taken when handling forensic samples to prevent contamination, including working in a dedicated clean room, using dedicated equipment and reagents, frequent changing of gloves and cleaning of surfaces.
2) DNA can be extracted from blood samples by lysing red blood cells, then purifying the DNA through phenol-chloroform extraction and ethanol precipitation.
3) For absorbing substrates like cloth or paper, a small piece is cut and placed in lysis buffer for extraction. For non-absorbing substrates,
Peyer Labs Cloning a Fluorescent Gene BrochureTom Loughran
Cloning is the process of replicating DNA inside bacteria. Research scientists use cloning to begin experiments like genetically engineering mice and producing human insulin. Peyer Laboratory Systems presents their Cloning A Fluorescent Gene kit, which allows students to engage in complex experimental biology. The kit provides students with precision instruments, pre-packaged reagents and bacteria, and a full-color manual to guide them through amplifying, inserting, and expressing a green fluorescent protein gene across multiple lab periods.
2013 Merck Millipore Best Practices in Nucleic Acid Removal from Vaccine Proc...Frank Appel
The document discusses various methods for removing nucleic acids like DNA from vaccine manufacturing processes. It describes how density gradient centrifugation, depth filtration using Millistak+ filters, and nuclease treatment with Benzonase can each reduce DNA levels. It also outlines methods for removing residual Benzonase like chromatography using Fractogel resins and tangential flow filtration with Pellicon cassettes. The document provides an overview of regulatory guidelines for DNA limits and analytical techniques for detecting residual Benzonase.
Benzonase® endonuclease: use and clearance with a TFF step in a cell culture-...Frédéric Sengler
Benzonase® endonuclease is a powerful tool for degrading all
forms of (deoxy)ribonucleic acids (DNA/RNA) in cell culture
harvests to base pair (BP) lengths under 8 units. It is effective
over a wide range of conditions including temperature, pH, and
varying concentrations of Mg2+, detergents, chelating agents,
and monovalent cations. It is often employed in the production
of viral vaccines, completely digesting DNA and RNA to improve
clearance and reduce solution viscosity.
Removing Benzonase® endonuclease, a 60 kD dimer, from the
process stream post-treatment may be achieved with Tangential
Flow Filtration (TFF) with the appropriate molecular weight
cut-off membrane (MWCO), typically 300 kD. This process has
heretofore not been well described in the literature, so the
present study fills this gap pairing Benzonase® endonuclease
treatment of a DNA-spiked inactivated flu virus cell culture
harvest with Pellicon® 2 cassettes, for clearance of the digested
DNA and remaining Benzonase® endonuclease by diafiltration.
Personalized nanomedicine for the treatment of vascular hypertensionSusanta Kumar Rout
This study includes designing a nanomedical device for the treatment of vascular hypertension in polycystic kidney diseases (PKD) model through cilia targeting.
They generated and compared two different metal and polymer cilia-targeted nanoparticle drug delivery systems (DDS), i.e. gold (Au) and poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs)
The target is Dopamine-receptor type-5 (DRS) on primary cilia.
The drug-loaded is Fenoldopam (FD).
Stephen Friend Nature Genetics Colloquium 2012-03-24Sage Base
This document proposes using data intensive science to build models of disease within a shared computing environment or "commons". It notes that current disease models often oversimplify complex conditions. Five pilot projects are described that could leverage shared clinical and genomic data as well as model building to better represent diseases: 1) sharing comparator arm data from clinical trials, 2) a federated aging analysis project, 3) portable legal consent, 4) a Sage Congress modeling competition, and 5) the BRIDGE initiative for democratizing medical research. The document argues this approach could accelerate disease understanding and new therapy development.
This document discusses cytogenetic techniques used in biology. It begins by outlining the learning objectives which are to list cytogenetic techniques and understand cell culture, harvesting chromosomes, and karyotyping. It then describes various techniques including cell culture, harvesting and spreading chromosomes, karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The purpose of these techniques is to examine genetic components of cells including chromosomes to study genetic abnormalities.
This document discusses molecular biology techniques. It begins by outlining the learning objectives, which are to list techniques used in molecular biology and describe microscopy, centrifugation, extraction, electrophoresis, and chromatography. It then provides details on each technique, including their purposes and basic procedures. Microscopy is used to produce magnified images of cells and structures. Centrifugation separates substances by density. Extraction isolates molecules like DNA, RNA, and proteins. Electrophoresis separates charged substances using an electric field. Chromatography separates mixtures based on interactions with a solid or liquid medium.
Dna extraction from blood and forensic samplesCAS0609
This document provides instructions for extracting DNA from various forensic samples, including blood, absorbing substrates like cloth or paper, and non-absorbing substrates like metal or plastic. The summary is as follows:
1) Precautions must be taken when handling forensic samples to prevent contamination, including working in a dedicated clean room, using dedicated equipment and reagents, frequent changing of gloves and cleaning of surfaces.
2) DNA can be extracted from blood samples by lysing red blood cells, then purifying the DNA through phenol-chloroform extraction and ethanol precipitation.
3) For absorbing substrates like cloth or paper, a small piece is cut and placed in lysis buffer for extraction. For non-absorbing substrates,
This document discusses different methods of DNA isolation including the Maxwell 16 Plant DNA Kit method, spin column method, and CTAB method. It explains the structure of DNA and importance of DNA extraction for uses such as genetic testing, body identification, and analysis of forensic evidence. DNA can be stored long term at temperatures of -20°C, -80°C, -196°C or dried at room temperature. Isolated DNA has applications in understanding plant individuality, plant modification, forensic analysis, and solving historical puzzles.
This document provides information about a two-day conference on next-generation sequencing taking place on September 17-18, 2012 in London. The conference will feature talks from industry leaders on topics such as sequencing technologies, data analysis and interpretation, antibiotic drug discovery, and applications in disease research. Attendees can gain insights into RNA sequencing, variant detection, antimicrobial discovery, and epigenetics. Interactive workshops on RNA sequencing and bacterial genome analysis will also be offered on the third day. Registration discounts are available before May 31st and June 29th.
This document provides an overview of molecular detection techniques used in food quality control. It discusses how chemistry alone cannot solve all detection problems and that molecular biology methods like PCR, RFLP, and sequencing are better alternatives as they are more accurate, rapid and cost-effective. It describes several common molecular detection methods and their applications in detecting food pathogens, adulterants, allergens and GM ingredients. The document emphasizes that molecular methods can identify microbes at the strain level and detect viable cells, but may not be able to find non-authorized GMOs due to lack of molecular information.
This document describes several methods for isolating and purifying DNA, RNA, and bacteriophages from plant and bacterial cells. For DNA isolation from plant cells, the method involves freezing and grinding plant tissue, lysing the cells with CTAB buffer, purifying the DNA with chloroform, precipitating it with isopropanol, washing it, and eluting the purified DNA. For plasmid and bacteriophage DNA isolation from bacterial cells, several techniques are described that separate DNA based on size or conformation differences, such as alkaline lysis and CsCl gradient centrifugation. RNA isolation methods include organic extraction to separate RNA from other cell components, as well as direct lysis methods.
Benzonase is an endonuclease enzyme that can effectively degrade DNA and RNA within 4 minutes. It is recognized for its ability to reduce the size and infectivity of residual DNA in vaccines. Treatment with Benzonase followed by filtration or chromatography can reduce DNA levels in vaccines such as those produced in Vero cells. By digesting cellular DNA, Benzonase prevents the formation of virus-DNA complexes during purification of vaccines such as AAV vaccines.
This document provides a protocol for extracting ancient DNA from bones and teeth. The method aims to maximize DNA recovery while minimizing co-extraction of PCR inhibitors. Key steps include extracting DNA from bone powder using an EDTA-proteinase K buffer, then purifying the DNA by binding it to silica in the presence of guanidinium thiocyanate. All steps are done at room temperature to reduce DNA degradation. The protocol yields DNA extracts within 2 days and has advantages over other methods such as being quick, scalable, simple to implement, and efficiently removing PCR inhibitors.
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
Differentiation & Activity Of Human Pre-Osteoclasts On Chitosan-Ashish Sh...as747
1. The document studied the effects of chitosan on human pre-osteoclast differentiation and activity. Pre-osteoclasts were cultured on cementek, cementek/chitosan, and PMMA pellets for 7 days.
2. Live/dead staining showed high viability on all materials. TRACP staining showed large, positive multinucleated osteoclasts on cementek but few on cementek/chitosan. Scanning electron microscopy and gene expression analysis were also performed.
3. The results suggest that chitosan may inhibit pre-osteoclast differentiation and reduce osteoclast activity, which could impact bone remodeling. Further analysis of chitosan's
The document summarizes a study that imaged biological samples using atomic force microscopy (AFM) in dry and liquid conditions. The objectives were to image metaphase chromosomes from normal male lymphocytes and cervical tumor cells under different AFM modes and probe types, and to evaluate the imaging effects. The results showed that while contact mode in liquid increased resolution, high resolution was also achieved using intermittent contact mode in dry conditions with a high stiffness probe. Repeated height measurements showed nominal statistical differences, indicating no apparent sample damage from this method. The study demonstrated the potential of AFM for biological micro- and nano-materials characterization.
Overcoming challenges of host cell DNA removal in vaccine manufacturingDr. Priyabrata Pattnaik
Regulatory agencies require residual host cell DNA in vaccines to be extremely low, typically below 10 pg/dose. Various methods are used to remove DNA during vaccine manufacturing, including nuclease treatment, adsorptive depth filtration, chromatography, and tangential flow filtration. Nuclease treatment with Benzonase is widely used to digest DNA but the nuclease then needs to be removed using techniques like anion exchange chromatography, gel filtration, or ultrafiltration with diafiltration to achieve over 99% clearance.
Eastern blotting is a technique used to detect post-translational modifications (PTMs) of proteins, especially carbohydrate epitopes. It involves separating proteins by gel electrophoresis, transferring them to a membrane, then using probes like antibodies to detect PTMs. It is an extension of western blotting developed in 1979 by Towbin. The method involves separation, transfer to a membrane, addition of primary then secondary antibodies, and confirmation of the molecule of interest via autoradiography. It can detect PTMs in disease studies and compare modifications between bacterial species.
The document summarizes research aiming to clone the gene for Serine Carboxypeptidase Y (CPD-Y) from Saccharomyces cerevisiae into E. coli. The methodology involved growing E. coli cells containing the CPD-Y gene construct, purifying the plasmid DNA, digesting it with EcoR1, and visualizing and quantifying the DNA using gel electrophoresis and an Agilent bioanalyzer. Results showed improvements in techniques and achievement of single peaks and clear DNA bands, suggesting the CPD-Y gene was present in the construct. Future work will involve confirming clones and sequencing the DNA.
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.
The document provides an analysis of the effect of different pretreatment methods in combination with organosolv delignification on the enzymatic hydrolysability of three feedstocks. It examines the composition of untreated biomass, the impact of different pretreatments on composition, and lignin characterization. The goal is to compare delignification ability, enzymatic hydrolysability, and establish correlations between lignin structure and delignification potential for different feedstocks and pretreatment combinations.
This document summarizes a research project investigating essential genes and β-lactam resistance mechanisms in Klebsiella pneumoniae. The student conducted transposon mutagenesis and next-generation sequencing to identify 374 essential genes under laboratory conditions, including 50 hypothetical genes with no homologs in other bacteria. Certain transposon insertions were associated with resistance to clinically relevant β-lactams like cefotaxime and meropenem. Specifically, insertions in ramR, bamB and hupA conferred resistance, with hupA not previously known to be involved in β-lactam resistance. The results provide new antibiotic targets and resistance genes warranting further study to address the growing problem of multidrug
This dissertation examines the bacterial etiology of wound infections and the antibiotic susceptibility patterns of isolates from patients visiting B and B Hospital in Nepal. Cultures were taken from 1164 wound samples over one year. Common gram-positive isolates included Staphylococcus aureus (92.46%) and gram-negative isolates included E. coli (28.1%), Pseudomonas spp. (30.91%). Antibiotics like amikacin and vancomycin were effective against most isolates. The study concludes that wound infections are commonly caused by resistant bacteria, so alternative antibiotics need to be used for treatment.
This document outlines a study on the identification and characterization of antimicrobial resistance and genetic traits of zoonotic Klebsiella pneumoniae isolates from a dairy farm in Laguna, Philippines. The study aims to determine the prevalence of K. pneumoniae in mastitic and bulk tank milk, and characterize the antibiotic resistance patterns and mechanisms. Isolates will be collected from cow milk and human workers from the dairy farm and tested for antibiotic susceptibility. Genes conferring antimicrobial resistance and virulence factors will be identified using molecular techniques. Results will provide data on antimicrobial resistance genes in K. pneumoniae from animals and humans to inform industry and policy.
This document provides an overview of urinary tract infections (UTIs). It discusses the terminology, classification, epidemiology, etiology, pathogenesis, risk factors, clinical presentation, diagnosis, and treatment of UTIs. UTIs can affect different parts of the urinary tract and are classified as uncomplicated or complicated depending on underlying conditions. Escherichia coli is the most common cause. Diagnosis involves urinalysis, urine culture, and imaging tests. Treatment depends on the site and severity of infection, and commonly involves short courses of antibiotics like trimethoprim-sulfamethoxazole or fluoroquinolones.
This study demonstrated a novel natural transformation mechanism in Actinobacillus actinomycetemcomitans (A.a.) that is independent of uptake signal sequences and the Tfox gene. The study showed that A.a. could be transformed with genomic and plasmid DNA present in microvesicles secreted into the growth medium of donor cells. This transformation occurred both in the presence and absence of components normally required for natural transformation in A.a. The results suggest outer membrane adhesion and fusion of donor microvesicles with recipient cells allows DNA delivery and homologous recombination. This novel mechanism could provide an easier method for genetically transforming A.a. compared to conventional techniques.
Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering
Discover Therapeutic Aptamers For Vegf165 And EgfrJessica Myers
The document discusses discovering therapeutic aptamers for VEGF165 and EGFR through in vitro selection. Aptamers are ssDNA or RNA oligonucleotides that bind targets with high selectivity and specificity due to their well-defined tertiary structures. They have advantages over antibodies such as not requiring animals or cell culture for selection. The author aims to select aptamers for VEGF165 and EGFR to potentially use as therapeutic agents.
Presentation 18: Problems other than AHPND in EMS ponds, including the micros...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
This document discusses different methods of DNA isolation including the Maxwell 16 Plant DNA Kit method, spin column method, and CTAB method. It explains the structure of DNA and importance of DNA extraction for uses such as genetic testing, body identification, and analysis of forensic evidence. DNA can be stored long term at temperatures of -20°C, -80°C, -196°C or dried at room temperature. Isolated DNA has applications in understanding plant individuality, plant modification, forensic analysis, and solving historical puzzles.
This document provides information about a two-day conference on next-generation sequencing taking place on September 17-18, 2012 in London. The conference will feature talks from industry leaders on topics such as sequencing technologies, data analysis and interpretation, antibiotic drug discovery, and applications in disease research. Attendees can gain insights into RNA sequencing, variant detection, antimicrobial discovery, and epigenetics. Interactive workshops on RNA sequencing and bacterial genome analysis will also be offered on the third day. Registration discounts are available before May 31st and June 29th.
This document provides an overview of molecular detection techniques used in food quality control. It discusses how chemistry alone cannot solve all detection problems and that molecular biology methods like PCR, RFLP, and sequencing are better alternatives as they are more accurate, rapid and cost-effective. It describes several common molecular detection methods and their applications in detecting food pathogens, adulterants, allergens and GM ingredients. The document emphasizes that molecular methods can identify microbes at the strain level and detect viable cells, but may not be able to find non-authorized GMOs due to lack of molecular information.
This document describes several methods for isolating and purifying DNA, RNA, and bacteriophages from plant and bacterial cells. For DNA isolation from plant cells, the method involves freezing and grinding plant tissue, lysing the cells with CTAB buffer, purifying the DNA with chloroform, precipitating it with isopropanol, washing it, and eluting the purified DNA. For plasmid and bacteriophage DNA isolation from bacterial cells, several techniques are described that separate DNA based on size or conformation differences, such as alkaline lysis and CsCl gradient centrifugation. RNA isolation methods include organic extraction to separate RNA from other cell components, as well as direct lysis methods.
Benzonase is an endonuclease enzyme that can effectively degrade DNA and RNA within 4 minutes. It is recognized for its ability to reduce the size and infectivity of residual DNA in vaccines. Treatment with Benzonase followed by filtration or chromatography can reduce DNA levels in vaccines such as those produced in Vero cells. By digesting cellular DNA, Benzonase prevents the formation of virus-DNA complexes during purification of vaccines such as AAV vaccines.
This document provides a protocol for extracting ancient DNA from bones and teeth. The method aims to maximize DNA recovery while minimizing co-extraction of PCR inhibitors. Key steps include extracting DNA from bone powder using an EDTA-proteinase K buffer, then purifying the DNA by binding it to silica in the presence of guanidinium thiocyanate. All steps are done at room temperature to reduce DNA degradation. The protocol yields DNA extracts within 2 days and has advantages over other methods such as being quick, scalable, simple to implement, and efficiently removing PCR inhibitors.
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
Differentiation & Activity Of Human Pre-Osteoclasts On Chitosan-Ashish Sh...as747
1. The document studied the effects of chitosan on human pre-osteoclast differentiation and activity. Pre-osteoclasts were cultured on cementek, cementek/chitosan, and PMMA pellets for 7 days.
2. Live/dead staining showed high viability on all materials. TRACP staining showed large, positive multinucleated osteoclasts on cementek but few on cementek/chitosan. Scanning electron microscopy and gene expression analysis were also performed.
3. The results suggest that chitosan may inhibit pre-osteoclast differentiation and reduce osteoclast activity, which could impact bone remodeling. Further analysis of chitosan's
The document summarizes a study that imaged biological samples using atomic force microscopy (AFM) in dry and liquid conditions. The objectives were to image metaphase chromosomes from normal male lymphocytes and cervical tumor cells under different AFM modes and probe types, and to evaluate the imaging effects. The results showed that while contact mode in liquid increased resolution, high resolution was also achieved using intermittent contact mode in dry conditions with a high stiffness probe. Repeated height measurements showed nominal statistical differences, indicating no apparent sample damage from this method. The study demonstrated the potential of AFM for biological micro- and nano-materials characterization.
Overcoming challenges of host cell DNA removal in vaccine manufacturingDr. Priyabrata Pattnaik
Regulatory agencies require residual host cell DNA in vaccines to be extremely low, typically below 10 pg/dose. Various methods are used to remove DNA during vaccine manufacturing, including nuclease treatment, adsorptive depth filtration, chromatography, and tangential flow filtration. Nuclease treatment with Benzonase is widely used to digest DNA but the nuclease then needs to be removed using techniques like anion exchange chromatography, gel filtration, or ultrafiltration with diafiltration to achieve over 99% clearance.
Eastern blotting is a technique used to detect post-translational modifications (PTMs) of proteins, especially carbohydrate epitopes. It involves separating proteins by gel electrophoresis, transferring them to a membrane, then using probes like antibodies to detect PTMs. It is an extension of western blotting developed in 1979 by Towbin. The method involves separation, transfer to a membrane, addition of primary then secondary antibodies, and confirmation of the molecule of interest via autoradiography. It can detect PTMs in disease studies and compare modifications between bacterial species.
The document summarizes research aiming to clone the gene for Serine Carboxypeptidase Y (CPD-Y) from Saccharomyces cerevisiae into E. coli. The methodology involved growing E. coli cells containing the CPD-Y gene construct, purifying the plasmid DNA, digesting it with EcoR1, and visualizing and quantifying the DNA using gel electrophoresis and an Agilent bioanalyzer. Results showed improvements in techniques and achievement of single peaks and clear DNA bands, suggesting the CPD-Y gene was present in the construct. Future work will involve confirming clones and sequencing the DNA.
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.
The document provides an analysis of the effect of different pretreatment methods in combination with organosolv delignification on the enzymatic hydrolysability of three feedstocks. It examines the composition of untreated biomass, the impact of different pretreatments on composition, and lignin characterization. The goal is to compare delignification ability, enzymatic hydrolysability, and establish correlations between lignin structure and delignification potential for different feedstocks and pretreatment combinations.
This document summarizes a research project investigating essential genes and β-lactam resistance mechanisms in Klebsiella pneumoniae. The student conducted transposon mutagenesis and next-generation sequencing to identify 374 essential genes under laboratory conditions, including 50 hypothetical genes with no homologs in other bacteria. Certain transposon insertions were associated with resistance to clinically relevant β-lactams like cefotaxime and meropenem. Specifically, insertions in ramR, bamB and hupA conferred resistance, with hupA not previously known to be involved in β-lactam resistance. The results provide new antibiotic targets and resistance genes warranting further study to address the growing problem of multidrug
This dissertation examines the bacterial etiology of wound infections and the antibiotic susceptibility patterns of isolates from patients visiting B and B Hospital in Nepal. Cultures were taken from 1164 wound samples over one year. Common gram-positive isolates included Staphylococcus aureus (92.46%) and gram-negative isolates included E. coli (28.1%), Pseudomonas spp. (30.91%). Antibiotics like amikacin and vancomycin were effective against most isolates. The study concludes that wound infections are commonly caused by resistant bacteria, so alternative antibiotics need to be used for treatment.
This document outlines a study on the identification and characterization of antimicrobial resistance and genetic traits of zoonotic Klebsiella pneumoniae isolates from a dairy farm in Laguna, Philippines. The study aims to determine the prevalence of K. pneumoniae in mastitic and bulk tank milk, and characterize the antibiotic resistance patterns and mechanisms. Isolates will be collected from cow milk and human workers from the dairy farm and tested for antibiotic susceptibility. Genes conferring antimicrobial resistance and virulence factors will be identified using molecular techniques. Results will provide data on antimicrobial resistance genes in K. pneumoniae from animals and humans to inform industry and policy.
This document provides an overview of urinary tract infections (UTIs). It discusses the terminology, classification, epidemiology, etiology, pathogenesis, risk factors, clinical presentation, diagnosis, and treatment of UTIs. UTIs can affect different parts of the urinary tract and are classified as uncomplicated or complicated depending on underlying conditions. Escherichia coli is the most common cause. Diagnosis involves urinalysis, urine culture, and imaging tests. Treatment depends on the site and severity of infection, and commonly involves short courses of antibiotics like trimethoprim-sulfamethoxazole or fluoroquinolones.
This study demonstrated a novel natural transformation mechanism in Actinobacillus actinomycetemcomitans (A.a.) that is independent of uptake signal sequences and the Tfox gene. The study showed that A.a. could be transformed with genomic and plasmid DNA present in microvesicles secreted into the growth medium of donor cells. This transformation occurred both in the presence and absence of components normally required for natural transformation in A.a. The results suggest outer membrane adhesion and fusion of donor microvesicles with recipient cells allows DNA delivery and homologous recombination. This novel mechanism could provide an easier method for genetically transforming A.a. compared to conventional techniques.
Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering
Discover Therapeutic Aptamers For Vegf165 And EgfrJessica Myers
The document discusses discovering therapeutic aptamers for VEGF165 and EGFR through in vitro selection. Aptamers are ssDNA or RNA oligonucleotides that bind targets with high selectivity and specificity due to their well-defined tertiary structures. They have advantages over antibodies such as not requiring animals or cell culture for selection. The author aims to select aptamers for VEGF165 and EGFR to potentially use as therapeutic agents.
Presentation 18: Problems other than AHPND in EMS ponds, including the micros...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
This document summarizes the education and research experience of Jaeho Lee. He earned a B.S. in Environmental Horticulture from the University of Seoul and an M.S. in Entomology from Seoul National University. For his graduate thesis, Lee developed a film-assisted honeybee egg collection system to improve honeybee genome editing techniques. He then used these techniques to successfully knockout the nAChR alpha6 gene in honeybees, proving the concept of creating pesticide-resistant honeybees. Lee has also published research on head louse adhesion proteins and acetylcholine esterase paralogs in bedbugs and honeybees.
PPT in Biotechnology
Biotechnology provides powerful tools for the sustainable development of aquaculture, fisheries, as well as the food industry. Increased public demand for seafood and decreasing natural marine habitats have encouraged scientists to study ways that biotechnology can increase the production of marine food products, and making aquaculture as a growing field of animal research. Biotechnology allows scientists to identify and combine traits in fish and shellfish to increase productivity and improve quality. Scientists are investigating genes that will increase production of natural fish growth factors as well as the natural defense compounds marine organisms use to fight microbial infections. Modern biotechnology is already making important contributions and poses significant challenges to aquaculture and fisheries development. It perceives that modern biotechnologies should be used as adjuncts to and not as substitutes for conventional technologies in solving problems, and that their application should be need-driven rather than technology-driven.
The use of modern biotechnology to enhance production of aquatic species holds great potential not only to meet demand but also to improve aquaculture. Genetic modification and biotechnology also holds tremendous potential to improve the quality and quantity of fish reared in aquaculture. There is a growing demand for aquaculture; biotechnology can help to meet this demand. As with all biotech-enhanced foods, aquaculture will be strictly regulated before approved for market. Biotech aquaculture also offers environmental benefits. When appropriately integrated with other technologies for the production of food, agricultural products and services, biotechnology can be of significant assistance in meeting the needs of an expanding and increasingly urbanized population in the next millennium. Successful development and application of biotechnology are possible only when a broad research and knowledge base in the biology, variation, breeding, agronomy, physiology, pathology, biochemistry and genetics of the manipulated organism exists. Benefits offered by the new technologies cannot be fulfilled without a continued commitment to basic research. Biotechnological programmes must be fully integrated into a research background and cannot be taken out of context if they are to succeed.
Mayekar et al., 2021
Nanoparticles have various applications in modern separation science techniques. They can be used in liquid chromatography, gas chromatography, capillary electrophoresis, microchip electrophoresis, and ion chromatography. Nanoparticles are relatively easy to synthesize and functionalize, and have large surface area to volume ratios ideal for separations. Common nanoparticles used include gold nanoparticles, silica nanoparticles, and magnetic nanoparticles. They have been shown to improve separation efficiency, selectivity, and resolution compared to conventional separation methods. However, while successful in research, nanoparticle-based separations have not been widely adopted in industrial settings.
This document describes a study that aimed to develop a fully decellularized bovine caudal intervertebral disc scaffold. Researchers extracted bovine caudal discs from C4-C5 to C6-C7 and subjected them to a decellularization process involving freezing, ultrasonication, and exposure to a decellularization solution. Tissue samples from the nucleus pulposus and annulus fibrosis were then analyzed to compare glycosaminoglycan and DNA content between decellularized and fresh discs. Histological analysis and DNA gel electrophoresis were also performed to evaluate the decellularization process. The results were meant to determine if a fully decellularized bovine caudal IVD scaffold could be developed.
Blindness and cardiovascular disease are targeted areas for stem cell therapy. For blindness, clinical trials are ongoing using human embryonic stem cell-derived retinal pigment epithelial cells to treat age-related macular degeneration and Stargardt's disease. Perivascular progenitor cells may also help diseases like diabetic retinopathy. For cardiovascular applications, stem cell-derived cardiomyocytes and brown fat cells are being studied. Preclinical studies show stem cell treatments improve vascular permeability and function in disease models.
RFLP (Restriction Fragment Length Polymorphism) is a molecular marker technique based on detecting length differences in DNA fragments after restriction enzyme digestion and gel electrophoresis. Polymorphisms arise from mutations that create or remove restriction sites. RFLP has applications in fingerprinting, mapping, and phylogenetic/population studies. The technique involves isolating DNA, restriction digestion, gel electrophoresis, Southern blotting to transfer DNA to a membrane, hybridizing with a labeled probe, washing to remove unhybridized probe, and detecting polymorphisms via autoradiography. It is useful but labor intensive, requiring large DNA quantities. Automation is difficult due to the gel-based nature of early steps.
“N2MO provides cutting-edge ex vivo insect based screening models for predicting ADMET properties of chemical substances in the early drug discovery phase. Insect models represents excellent model systems due to biological active test situation, combined with fast reproducibility with no need for resynthesizing compound and at same time the model produces data that are reflecting mammalian ADMET properties”
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
This document describes research into the microbial synthesis of platinum nanoparticles using Saccharomyces boulardii and evaluation of the anticancer activity of the synthesized platinum nanoparticles. Key findings include:
1) Platinum nanoparticles were successfully synthesized using the cell free extract of S. boulardii when reacted with chloroplatinic acid.
2) Various parameters like metal salt concentration, temperature, cellmass concentration, pH, and reaction time were optimized to control the yield and properties of the synthesized nanoparticles.
3) The synthesized platinum nanoparticles showed anticancer activity against A431 and MCF-7 cell lines with IC50 values between 57-100 μg/ml, indicating potential for use as an antic
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
Immunohistochemistry (IHC) is a technique used in pathology to determine the origins, prognosis, and treatment of tumors by detecting antigens in cells of a tissue section. It is important for accurate and consistent IHC results that laboratories follow rigorous quality control procedures and standards for tissue fixation, processing, sectioning, and staining. Key considerations include using the appropriate fixation time and temperature, optimizing antigen retrieval, selecting the proper primary antibody and detection system, and including appropriate controls. Delays in fixation or suboptimal fixation can compromise antigenicity and affect IHC results.
The document discusses the formulation and characterization of cubosomes for controlled drug delivery. Cubosomes are nanostructured liquid crystalline particles that can encapsulate drugs. The study formulated cubosomes using lipids and block copolymers to encapsulate dextromethorphan for controlled release. Various formulations were tested in vitro and using cryo-TEM. The results showed that cubosomes can provide controlled release of drugs and balance structure, charge and viscosity is important to achieve this.
Target enrichment enables researchers to focus their next generation sequencing (NGS) efforts on regions of interest, allowing them to obtain more sequencing data relevant to their study. In-solution target capture is a method of enrichment using oligonucleotide probes directed to specific regions within a genome. Target capture can be used to enrich multiple samples simultaneously, reducing the cost per sample, while using individually synthesized probes allows researchers to construct gene panels that can be optimized over time.
Expanding Your Research Capabilities Using Targeted NGS
Thesis_Karan Sharma_final
1. Engineering Surfaces to Support Neural Stem Cells (hNSC’s) and Hepatocytes Adhesion and Growth.
By: Karan Sharma
August 9th, 2016
Advisor: Dr. Xuejun Wen M.D., Ph.D.
Committee Members: Dr. Daniel Conway, Dr. B. Frank Gupton
Masters Thesis Presentation
All Rights Reserved
2. The Plan
• Introduction
• Purpose of the thesis
• Goals
• Methods
• Results
• Conclusion
• Future Work
• Acknowledgements
3. • Need to develop xeno-free and pure synthetic
biomedical devices
– Nerve grafts: current using decellularized nerve grafts
from cadavers (possible immune response)
– Artificial livers: easier FDA approval.
• Wen lab has developed reproducible protocol to
induce human induced pluripotent stem cells into
functional mature hepatocytes.
• Limited cell attachment on hollow fibers.
Introduction
5. • Most in vitro studies are conducted in 2D – 3D
culture settings.
• Human Neural Stem Cells (hNSCs)
• Isolated in the early 1990’s
• Generate mainly cells for nervous system.
• Differentiate into astrocytes, oligodendrocytes and
neurons
Introduction
6. Introduction (conti.)
• Hepatocyte
• Model hepatocyte cell line: Liver Hepatocellular
Carcinoma Cells (HepG2)
• Epithelial morphology
• Chosen for their application and robust nature
7. Introduction (conti.)
• Looped peptides (W-945 and 947 peptides)
• Pure synthetic (xeno-free, non-bio-derived)
• Application here was to coat an artificial substrate
• Replacement of laminin and Matrigel®
Conventional peptides
Our looped peptides
Cell binding site
Cell Binding site
Day 1 Day 5 Day 10
Commercial
Peptide
Novel
Peptide
Looped
Peptides
(W-945, 947)
Conventional
peptides
Human neural stem cells
8. Introduction (conti.)
• Poly 4-vinylphenol (P4VP)
• A polymeric substance similar to polystyrene
• Artificial synthetic commercially available
• Used mainly with electronics in the past
• Application here was to make an artificial substrate
• Replacement of laminin and Matrigel®
• Molecular weight Ranges from 11,000 to 25,000
• Effective ability to create a hydrophilic surface
• Observed to create an attractive surface for cell adhesion
and growth
9. Introduction (conti.)
• Polyacrylonitrile (PAN) Membrane
• Synthetic, semi-crystalline organic polymer
• Thermoplastic, porous membrane, thermally stable,
commercially available, resistant to most organic
solvents
• Used for separation and purification processes
• Used currently in dialyzers
• Originally have hydrophobic surface
11. Goals
• To develop different artificial substrates to support
cell adhesion and growth.
• Possible applications:
– Pure synthetic artificial nerve grafts
– Artificial Livers
• To develop biocompatible polymeric flat
membrane.
• Use the coatings and membrane to conduct cell
culture experiments
12. Methods
• To develop Poly 4-vinylphenol (P4VP)
Coating(s) protocol
• To fabricate Polyacrylonitrile (PAN) Flat
Membrane
• Imaging cultures with different conditions
(Microscope, Immunofluorescence, SEM)
• Metabolic Testing
13. Methods: Poly 4-vinylphenol (P4VP)
• P4VP coating preparation (MW: 11,000 – 25,000)
• P4VP powders were dissolved in different
Ethanol%
• Sterilized using 0.22 µm syringe filter
• 0.0625, 0.125, 0.25, 0.5 and 1% of P4VP
concentrations
• Incubated for 4 hours minimum to be a working
coating for cell culture at 37°C under 5% CO2.
14. Methods: Peptide Coating
• Fabricated in our lab
• Artificial synthetic coating developed for use with
hNSCs
• Uses a PAVAS as a precursor coating (0.01%
PVAVS in PBS)
• Peptide W-945 & W-947 (0.04mg.mL peptide
mixture)
• Incubation time of about 2-4 hours at 37°C under
5% CO2.
15. Methods: Polyacrylonitrile (PAN) Hollow
Fiber (HF) Flat Membrane
• PAN power and N, N-Dimethylformamide (DMF-
anhydrous 99.8%)
• Put on shaker for 24 hours to complete dissolve
• Originally made solution was 15% reduced to 12%
• To attach to culture dish it was spin coated at
150RPMS, 30 seconds.
• Dissolved in Nano-pure water for 120 seconds
• Sterilized by 100% Ethanol for 4 hours minimum
16. Methods: Imaging cultures
• Many difficulties were faced for the purposes of
imaging.
• Good images for normal cell cultures through
microscope, confocal and SEM.
• Imaging cultures on membrane only through
SEM.
17. Methods: Imaging cultures
• Immunofluorescence
• Strict protocol was developed to fit needs for
different coatings and membrane.
• Stained with DAPI (4’, 6-diamidino-2-phenylindole )
and Alexa Fluor® 546 phalloidin (Actin)
18. Methods: Imaging cultures
• Scanning Electron Microscope
• As Immunofluorescence did not work with
membrane conditions
• Serial dehydration was conducted on fixed cells
• Super Critical Drying was carried out to maintain
cell morphology
• Sputter coating of Platinum and Gold
19. Methods: Metabolic Testing
• Made for testing cell expression and
proliferation
• 1:9 dye – media ratio.
• Light sensitive
• Inncubation time of 4 hours at 37°C under 5%
CO2.
• Inserted into to a 96 well plate with 9 samples
for every condition
20. Results
• Most studies were conducted in 2D culture setting
• Successful long term peptides and P4VP artificial
coating studies (hNSCs & HepG2 cell lines)
• Successful long term Membrane and artificial
coating cell studies (HepG2)
21. Results P4VP Coating (hNSCs)
hNSC cell line A,B: 50% 0.5EtOH P4VP, C: 75%0.5% EtOH P4VP, D: 75%1% EtOH P4VP, E,F: 100%0.5% EtOH P4VP,
G,H: 100%1% EtOH P4VP all in a 6 well plate
22. Results hNSCs P4VP Coating Study
hNSC Long term P4VPcoatings study where A-D is 75% EtOH Day 4: A1-A2 (0.25% P4VP), A3-A4 (0.5% P4VP), A5
(1% P4VP), A6 (matrigel) Day 7: B1 (0.25% P4VP), B2-B3 (0.5% P4VP), B4 (1% P4VP), B5 (laminin). Day 9: C1-C2
(0.25% P4VP), C3-C4 (0.5% P4VP), C5 (laminin). Day 12: D1-D2 (0.25% P4VP), D3-D4 (0.5% P4VP), D5(1%
P4VP), D6 (laminin).
23. Results hNSCs P4VP Coating Study
hNSC Long term P4VPcoatings study where E-H is 100% EtOH. Day 4: E1-E2 (0.25% P4VP), E4 (matrigel), E3
(laminin). Day 7: F1-F2 (0.25% P4VP), F3 (0.5% P4VP),F4-F5 (1% P4VP), F6 (matrigel). Day 9: G1-G2 (0.25% P4VP),
G3 (0.5% P4VP), G4-G5 (1% P4VP) G6 (laminin). Day 12: H1 (0.25% P4VP), H2-H3 (1% P4VP), H4 (matrigel).
24. Results HepG2 P4VP Coating Study
HepG2 Liver Cell cultures for a long term study on 75% 1% (EtOH, P4VP) coatings where A-
H is days (2, 5, 9, 14, 18, 26, 36, and 43)
25. Results HepG2 P4VP Coating Study
HepG2 Liver Cell cultures for a long term study on 100% 1% (EtOH, P4VP) coatings where A-
D is days (2, 5, 9 and 14)
26. Results HepG2 P4VP Coating Study
HepG2 Liver Cell cultures for a long term study on 100% 1% (EtOH, P4VP) coatings where E-
H is days (18, 26, 36 and 43)
27. Immunofluorescence Characterization of HepG2 Cells
Images of HepG2 taken through a confocal microscope where the stains are DAPI Nuclear Counterstains (blue), Actin (Red)
and Pink (DAPI + Actin).Where A1-D1 (1 week), A2-D2 (2 weeks) A is 75%1% EtOH P4VP coating, B is 100%1% EtOH
P4VP coating, C is control cells and D is Peptide coating.
28. Immunofluorescence Characterization of HepG2 Cells
Images of HepG2 taken through a confocal microscope where the stains are DAPI Nuclear Counterstains (blue), Actin (Red)
and Pink (DAPI + Actin). A3-D3 (4 weeks) and A4-D4 (5 weeks). A is 75%1% EtOH P4VP coating, B is 100%1% EtOH
P4VP coating, C is control cells and D is Peptide coating.
29. Immunofluorescence Characterization of
HepG2 Cells on PAN membrane
Images of HepG2 cells on a flat
Polyacrylonitrile (PAN) hollow fiber
membrane where A1-A5 is 1-5 weeks
taken through a confocal microscope
where the stains are DAPI Nuclear
Counterstains (blue), Actin (Red) and
Pink (DAPI + Actin).
30. HepG2 Cell culture (control)
Images taken through an SEM: Images of a HepG2 cell culture grown in a normal condition (control). A-G
are different magnifications of different regions of the culture taken at 15keV. A (300X, 100µm), B (800X,
50 µm), C (1kX, 50 µm), D (2kX, 20 µm), E (5kX, 10 µm), F (8kX, 5 µm) and G (20kX, 2 µm).
31. HepG2 Cell culture on Peptide coating
Images taken through an SEM: Images of a HepG2 cell culture grown in a peptide coating. A-F are different
magnifications of different regions of the culture taken at 15keV. A (300X, 100µm), B (900X, 50 µm), C (1kX,
50 µm), D (2kX, 20 µm), E (5kX, 10 µm), F (10kX, 5 µm).
32. HepG2 Cell culture on PAN membrane
Images of different regions taken through an SEM (15keV) of a HepG2 cell culture grown on a PAN flat hollow fiber
membrane. A-B (300X, 100 µm), C, G, K (500X, 1KX, 2.5KX), D, E, F, H, I, J (500X, 800X, 100KX, 2.5KX, 5KX, 10KX) and
L (20keV, 2KX, 10 µm).
33. HepG2 Cell culture on PAN membrane with Peptide coating
Images of different regions taken through an SEM (15keV) of a HepG2 cell culture grown on a PAN flat hollow fiber
membrane with a peptide coating. A-B (300X, 100 µm), C-D (400X, 100 µm), E (40X, 1mm), F (70X, 500 µm), G (500X, 100
µm), H (1KX, 50 µm), I (2KX, 20 µm), J (7KX, 5 µm), K (10KX, 5 µm), L (20KX, 2 µm).
34. Metabolic Assay Results
No Membrane
(control)
Membrane Peptide Membrane Peptide 100% 1% P4VP
100% 1% P4VP
Mem
100% 0.5% P4VP
100% 0.5% P4VP
Mem
Day 2 80% 57% 57% 55% 48% 30% 48% 45%
Day 4 87% 64% 69% 81% 49% 32% 57% 49%
Day 8 93% 62% 65% 93% 84% 45% 63% 54%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
%REDUCED
HepG2 Cell Growth Over Time
A cell viability test was conducted on all 8 conditions with HepG2 cell cultures to metabolically determine cell expression
and proliferation.
35. Conclusion
• Excellent adhesion and proliferation of HepG2
cell culture on Peptide (W-945) & P4VP coating
• Increased growth with PAN membrane with
most on Peptide (W-945) & 100% 0.5% EtOH
P4VP coating
• Membrane is observed to be biocompatible.
36. Future Work
• Test different molecular weight of P4VP coating
• Test with stem cell derived hepatocytes or primary
liver cells and neural cells
• Develop hollow fiber tubular membranes of
different morphologies and structures, identify
optimal membranes to support neural cells and
liver cells
• Construct artificial livers and nerve grafts
38. Acknowledgements
• I would like to thank my mentor and advisor Dr.
Wen for all the experience, support and knowledge
he has given me.
• My committee members Dr. Conway and Dr.
Gupton.
• My lab coworkers Dr. Vasudha Surampudi, Dr.
Pettinato, Dr. Bo Xue, Dr. Xiomei Li, Jessica
Forrester, Debbie Campbell, Chenyang Jiang and
the many interns
• My family and friends for all their support.