R-3 Repository :: Browsing by Author "Bennett, George"

Browsing by Author "Bennett, George"

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Construction of recombinant molecules containing RNA processing signals and binding of human snRNPs to some nucleic acids
(1984) Nees, David; Berget, Susan M.; Palmer, Graham; Bennett, George
The restriction endonuclease Pst I cleavage site map of Ad2 was generated. Several recombinant molecules were formed, containing Pst I-generated fragments of Ad2 DNA, inserted into the E. coli vector pBR322. These molecules are designed to be useful for the study of RNA processing because they contain signals for splicing and polyadenylation. A filter binding assay was developed to measure the affinity of snRNPs for nucleic acids. The of U1 snRNPs was determined to be on the order of 1 to 11 M for RNA with or without a donor splice junction and for denatured DNA. U2 snRNP also bound RNA containing a donor splice junction with a KD of 1 M. The dissociation of U1 snRNP from denatured DNA was followed and the remainder of the snRNPs dissociated in less than one minute and fifty percent dissociated with a t1/2 of 2 minutes.
Development of Recombinase Polymerase Amplification (RPA) Assays to Diagnose Infectious Diseases
(2015-06-24) Crannell, Zachary; Richards-Kortum, Rebecca Rae; Bennett, George; Tkaczyk, Tomasz S; White, Arthur C
This thesis describes the development of Recombinase Polymerase Amplification (RPA) assays that can be used to improve access to diarrheal diagnostics and thereby reduce the number of preventable deaths that occur each year due to persistent diarrhea. In low-resource settings (LRS), where the majority of the almost 1.5 million annual diarrheal deaths occur, a major obstacle to receiving life-saving treatment is the inability to identify the specific cause of diarrhea. Diagnosis in LRS is usually done via stool smear microscopy, which fails to identify the cause of diarrhea up to half of the time. The widely considered gold standard diagnostic method is Polymerase Chain Reaction (PCR), which detects trace amounts of pathogen DNA from stool samples. While highly sensitive, PCR requires highly trained technicians and access to expensive thermal cycling equipment, restricting its use to centralized reference laboratories. The RPA diagnostics presented here amplifies trace amounts of pathogen DNA (much like PCR), but unlike PCR do not require the use of expensive thermal cycling equipment and can function at low temperatures, alleviating the need for any external heating equipment. RPA-based diagnostics and sample preparation protocols that are appropriate for low resource settings were developed to detect Cryptosporidium, Giardia, and Entamoeba, three of the leading causes of diarrhea. The three diagnostic assays were individually characterized on the benchtop where they demonstrated limits-of-detection and specificities comparable to the gold standard of PCR. The assays were further characterized in field studies using clinical samples where they demonstrated sensitivity and specificity nearly equivalent to that of the gold standard PCR. The three individual assays were then integrated into a multiplexed test designed to simultaneously amplify and detect DNA from Cryptosporidium, Giardia, and Entamoeba. This test was also characterized on the benchtop and in pre-clinical studies. All of the assays presented here are read using lateral flow strips that can easily be used in the field. This work demonstrates for the first time that multiplex RPA results can be read with lateral flow strips. By modifying the DNA primers, this diagnostic platform could be adapted to diagnose a broad variety of infectious diseases.
Isolation and characterization of human small nuclear ribonucleoproteins
(1984) Kinlaw, Claire S.; Berget, Susan M.; Matthews, Kathleen S.; Beckingham, Kathleen M.; Bennett, George; Kellems, Rodney E.
Human small nuclear ribonucleoproteins (snRNPs) containing U snRNAs have been fractionated into three RNA-specific populations, and snRNPs containing U1 and U2 snRNAs have been isolated~by biochemical methods. U1 and U2 snRNPs remained immunoprecipitable by Systemic Lupus Erythematosus antibodies during isolation, and purified snRNPs contained polypeptides of the same molecular weights as those defined by immunoprécipitation of crude extracts. The polypeptide components of U1 and U2 snRNPs have been compared by two-dimensional gel electrophoresis and immunobinding. U1 and U2 snRNPs contained both unique and common polypeptides. Purified U1 snRNPs contained U1 RNA and 1 snRNP polypeptides of molecular weights 67, (P67), 3, (P3), 23, (P23F), 21,5 (P22), 17,5 (P18), 2 at 12,3 (P12F and P12S), 1,2 (P1), 9,1 (P9), and 8,5 (P8). Purified U2 snRNPs contained U2 RNA and 9 snRNP polypeptides including the common polypeptides P23F, P22, P12F, P12S, P1, P9, and P8 as well as two U2 specific polypeptides of 23, (P23S) and 27, (P27) daltons. Five of the common polypeptides, including P23F, P22, P12F, P12S, and P9, were basic and may be important in snRNP assembly. One common polypeptide, P1 was acidic, and the remaining common polypeptide, P8, was neutral. Two of the U1 specific polypeptides, P3 and P18, as well as the two U2 specific polypeptides, P27 and P23S, were neutral. The third Ul specific polypeptide, P67, was slightly basic. Two of the common polypeptides, P23F and P22, were present in nonstoichiometric amounts and were recognized by a monoclonal anti-Sm antibody. Only one of them is present in rodent cells (Conner et al., 1982). Thus, in human cells, P23F and P22 may be two variants of the same polypeptide, with each snRNP complex containing either P23F or P22. P12F is also recognized by the same monoclonal antibody, suggesting it may be related to P23F and P22. Because Ul and U2 snRNPs contain common and unique polypeptides, it is suggested that they serve similar but distinct functions in. vivo. Ul has been implicated in hnRNA splicing (Lerner et al., 198; Rogers and Wall, 198). U2 may be involved in another aspect of hnRNA processing.
Making C4+ products in bacteria
(2017-05-16) Bennett, George; Thakker, Chandresh; Rice University; United States Patent and Trademark Office
Methods of making C4+ hydrocarbon feedstocks using anaerobic microbes are described.
Nitric Oxide: The Missing Link in Omentum-Induced Metabolic reprogramming of ovarian cancers
(2015-11-24) Salimian Rizi, Bahar; Nagrath, Deepak; Zygourakis, Kyriacos; Bennett, George; Klopp, Ann H
A novel metabolic regulatory mechanism of ovarian cancer by omentum adipose-derived stroma cells (O-ASCs) has been discovered. O-ASCs induce survival, migration, and chemoresistance of ovarian cancer cells. However, the underpinning mechanism behind the metabolic modulation was not understood. Here, O-ASCs are shown to promote nitric oxide (NO) homeostasis in ovarian cancers by generating the pool of arginine. Ovarian cancer cells benefit from tumor microenvironment’s elements and expand their growth. In turn, cancer cells modify the elements’ fate to further take advantage of nutrients and resources. A unique combinatory drug treatment is proposed to target O-ASCs-induced chemoresistance of ovarian cancer cells.
Reduced activity of ubiCA in E. coli
(2017-02-07) San, Ka-yiu; Bennett, George; Rice University; United States Patent and Trademark Office
Production of products by engineered bacteria is increased by regulating cellular respiration. Cellular respiration is controlled by reducing electron transfer enzyme activity. Some examples of electron transfer enzymes include NADH dehydrogenases, Succinate dehydrogenases, ubiquinone synthesis, cytochrome O, and cytochrome D. In one example, deletion of UbiCA prevents respiration. Respiration can the be controlled by addition of ubiquinone or expression of ubiCA.
The effect of an oral or intravenous nucleotide-free diet on selected enzyme activities in purine metabolism in rats
(1982) Snyder, Sandra Lynn; Rudolph, Frederick B.; Berget, Susan M.; Schroepfer, George J.; Bennett, George; Storck, Roger L.
Interrelationships between purine metabolism and immunity, cancer, and the diet have been considered. In studying trends of metabolic changes which occur in response to changes in the purine content of the diet, it has been hypothesized that when purines are lacking from the diet, there is a general shift from a catabolic to an anabolic state. In the present investigation, the activities of selected enzymes on purine metabolism in rats were studied with respect to an oral or intravenous nucleotide-free diet compared to the activities in rats fed normal chow. The intravenous nucleotide-free diet caused a decrease in purine nucleoside phosphorylase activity, an increase in adenine phosphoribosyl transferase, and no change in the activity of hypoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. The orally fed nucleotide-free diet caused a decrease in the activity of purine nucleoside phosphorylase and xanthine oxidase and increase in pancreatic ribonucléase and no change in adenine phosphoribosyl transferase or hyphoxanthine-guanine phosphoribosyl transferase, with respect to a normal control diet. These observations support the predicted shift from catabolism to anabolism. They also augment the growing awareness of the interrelationships between diet and cancer as well as diet and the immune response.
Understanding and modulating electron transfer through ferredoxins
(2020-04-23) Campbell, Ian; Silberg, Jonathon; Bennett, George
Ferredoxins are a ubiquitous family of protein electron carriers that support electron transfer in a massive number of pathways across the tree of life. As central and ancient distributors of redox equivalents, they are advantageous targets for controlling electron flow and for studying the evolution of electron flux in cells. Herein, I describe my efforts to catalogue ferredoxins and their natural substitute, flavodoxins, from over 7000 organisms to understand the usage of protein electron carriers throughout the three domains. I apply my findings to study the distribution of cyanophage ferredoxins, focusing on the ferredoxin from a phage that infects the world’s most abundant phototroph. I demonstrate this viral Fd is unusually unstable yet presents a midpoint potential similar to bacterial homologs and can transfer electron to host oxidoreductases. When this viral Fd is recombined with a thermostable homolog, I am able to generate chimeric Fds with variable heat tolerances and potentials shifted by up to 50 mV that are capable of conducting electron transfer in vivo. Additionally, I will describe my efforts to reconstruct missing links in the evolution of [4Fe-4S] ferredoxins, demonstrating that symmetric ferredoxins and fragmented ferredoxins are capable of supporting electron transfer. These results expand our knowledge of natural ferredoxins and elucidate the design rules we might use to make synthetic variants.
When function is biological: Discerning how silver nanoparticle structure dictates antimicrobial activity
(Cell Press, 2022) Zhang, Qingbo; Hu, Yue; Masterson, Caitlin M.; Jang, Wonhee; Xiao, Zhen; Bohloul, Arash; Garcia-Rojas, Daniel; Puppala, Hema L.; Bennett, George; Colvin, Vicki L.
Silver nanomaterials have potent antibacterial properties that are the foundation for their wide commercial use as well as for concerns about their unintended environmental impact. The nanoparticles themselves are relatively biologically inert but they can undergo oxidative dissolution yielding toxic silver ions. A quantitative relationship between silver material structure and dissolution, and thus antimicrobial activity, has yet to be established. Here, this dissolution process and associated biological activity is characterized using uniform nanoparticles with variable dimension, shape, and surface chemistry. From this, a phenomenological model emerges that quantitatively relates material structure to both silver dissolution and microbial toxicity. Shape has the most profound influence on antibacterial activity, and surprisingly, surface coatings the least. These results illustrate how material structure may be optimized for antimicrobial properties and suggest strategies for minimizing silver nanoparticle effects on microbes.

Browsing by Author "Bennett, George"

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