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PDF file - 14.9MB, Loss of P53 expedited the kinetics of MEF cell reprogramming

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PDF file - 88K, Different from P53, P63 and P73 were not directly involved in iPSC generation.

PDF file - 415K, Normal stem cells can be cultured under temperatures ranged from 32C to 39C and normal iPSC can be formed from MEF cells under these temperatures.

Listeria monocytogenes is a Gram-positive, intracellular pathogen responsible for the highly fatal foodborne illness listeriosis. Establishing intracellular infections requires the coordinated expressions of a variety of virulence factors, such as the pore-forming toxin listeriolysin O (LLO), in response to various intra- and extracellular signals. For example, we previously reported that L. monocytogenes differentially modulated LLO production in response to exogenous propionate, a short chain fatty acid either used in salt form as a human food ingredient or produced endogenously by gut microbial fermentation. Therefore, propionate is likely a continuously present signal throughout the L. monocytogenes transmission and infection process. However, little is known about the role of propionate in modulating L. monocytogenes-host interactions. Here we investigated the impact of propionate treatment on L. monocytogenes intracellular infections using cell culture infection models. Propionate treatment was performed separately on L. monocytogenes or host cells before or during infections to better distinguish pathogen-versus-host responses to propionate. Intracellular CFU in RAW264.7 macrophages and plaque diameters in L-fibroblasts were measured as proxy for intracellular infection outcomes. Nitrite levels and cellular morphology were also measured to assess host responses to propionate. We found that propionate pretreatment of anaerobic, but not aerobic, L. monocytogenes significantly enhanced subsequent intracellular infections in both cell types and nitrite production by infected macrophages. Propionate treatment of uninfected macrophages significantly altered cell morphology, seen by longer cells and greater migration, and reduced nitrite concentration in activated macrophages. Treatment of macrophages with propionate prior to or during infections significantly inhibited intracellular growth of L. monocytogenes, including those pre-treated with propionate. These results showcased an opposing effect of propionate on L. monocytogenes intracellular infections and strongly support propionate as an important signaling molecule for both the pathogen and the host cell that can potentially alter the outcome of L. monocytogenes-host interactions.

Gold nanoparticles (AuNPs) bound with biomolecules have emerged as suitable biosensors exploiting unique surface chemistries and optical properties. Many efforts have focused on antibody bioconjugation to AuNPs resulting in a sensitive bioconjugate to detect specific types of bacteria. Unfortunately, bacteria thrive under various harsh environments, and an understanding of bioconjugate stability is needed. Here, we show a method for optimizing Listeria monocytogenes polyclonal antibodies bioconjugation mechanisms to AuNPs via covalent binding at different pH values, from 2 to 11, and 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid, NaOH, HCl conditions. By fitting Lorentz curves to the amide I and II regions, we analyze the stability of the antibody secondary structure. This shows an increase in the apparent breakdown of the antibody secondary structure during bioconjugation as pH decreases from 7.9 to 2. We find variable adsorption efficiency, measured as the percentage of antibody adsorbed to the AuNP surface, from 17 to 27% as pH increases from 2 to 6 before decreasing to 8 and 13% at pH 7.9 and 11, respectively. Transmission electron microscopy (TEM) analysis reveals discrepancies between size and morphological changes due to the corona layer assembly from antibody binding to single nanoparticles versus aggregation or cluster self-assembly into large aggregates. The corona layer formation size increases from 3.9 to 5.1 nm from pH 2 to 6, at pH 7.9, there is incomplete corona formation, whereas at pH 11, there is a corona layer formed of 6.4 nm. These results indicate that the covalent binding process was more efficient at lower pH values; however, aggregation and deactivation of the antibodies were observed. We demonstrate that optimum bioconjugation condition was determined at pH 6 and MES buffer-type by indicators of covalent bonding and stability of the antibody secondary structure using Fourier transform-infrared, the morphological characteristics and corona layer formation using TEM, and low wavelength shifts of ultraviolet–visible after bioconjugation.

Guiding analytes to the sensing area is an indispensable step in a sensing system. Most of the sensing systems apply a passive sensing method, which waits for the analytes to diffuse towards the sensor. However, passive sensing methods limit the detection of analytes to a picomolar range on micro/nanosensors for a practical time scale. Therefore, active sensing methods need to be used to improve the detection limit in which the analytes are forced to concentrate on the sensors. In this article, we have demonstrated the manufacturing of nanogap-rich structures for active chemical sensing. Nanogap-rich structures are manufactured from metallic nanoparticles through an optothermally generated microbubble (OGMB) which is a laser-induced micron-sized bubble. The OGMB induces a strong convective flow that helps to deposit metallic nanoparticles to form nanogap-rich structures on a solid surface. In addition, the OGMB is used to guide and concentrate analytes towards the nanogap-rich structures for the active sensing of analytes. An active sensing method can improve the detection limit of chemical substances by an order of magnitude compared to a passive sensing method. The microbubble assisted manufacturing of nanogap-rich structures together with an active analyte sensing method paves a new way for advanced chemical and bio-sensing applications.

Insect gut microbiomes consist of bacteria, fungi, and viruses that can act as mutualists to influence the health and fitness of their hosts. While much has been done to increase understanding of the effects of environmental factors that drive insect ecology, there is less understanding of the effects of environmental factors on these gut microbial communities. For example, the effect of environmental nutrients on most insect gut microbiomes is poorly defined. To address this knowledge gap, we investigated the relationship between environmental nutrients and the gut microbial communities in a small study of katydids (n = 13) of the orthopteran species Orchelimum vulgare collected from a costal prairie system. We sampled O. vulgare from unfertilized plots, as well as from plots fertilized with added nitrogen and phosphorus or sodium separately and in combination. We found significantly higher Shannon diversity for the gut bacterial communities in O. vulgare from plots fertilized with added sodium as compared to those collected from plots without added sodium. In contrast, diversity was significantly lower in the gut fungal communities of grasshoppers collected from plots with added nitrogen and phosphorus, as well as those with added sodium, in comparison to those with no added nutrients. There was also a strong positive correlation between the gut bacterial and gut fungal community diversity within each sample. Indicator group analysis for added sodium plots included several taxa with known salt-tolerant bacterial and fungal representatives. Therefore, despite the small sample number, these results highlight the potential for the gut bacterial and fungal constituents to respond differently to changes in environmental nutrient levels. Future studies with a larger sample size will help identify mechanistic determinants driving these changes. Based on our findings and the potential contribution of gut microbes to insect fitness and function, consideration of abiotic factors like soil nutrients along with characteristic gut microbial groups is necessary for better understanding and conservation of this important insect herbivore.

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Insect microbiomes play an important role in the health and fitness of insect hosts by contributing to nutrient absorption, immune health, and overall ecological fitness. As such, research interests in insect microbiomes have focused on agriculturally and industrially important organisms such as honey bees and termites. Orthopterans, on the other hand, have not been well explored for their resident microbial communities. Grasshoppers are an integral part of grassland ecosystems and provide important ecosystem services. Conversely, grasshoppers can be an agricultural pest requiring management with broad spectrum pesticides. However, little is known about the microbiomes of grasshoppers and their potential contribution to grasshopper biology. Here we examine the gut microbiome of six species of grasshoppers (n = 60) from a coastal tallgrass prairie ecosystem to gain a better understanding of the microbial communities present across the orthopteran order in this ecosystem. We found that there are bacterial phyla common to all six grasshopper species: Actinobacteria, Proteobacteria, Firmicutes, and to a lesser degree, Tenericutes. Although the grasshopper species shared a high relative abundance of these groups, there were notable shifts in dominant phyla depending on the grasshopper species. Moreover, measures of alpha diversity revealed a more diverse microbiome in males than females. Our observations support the hypothesis that there is a "core" group of bacterial families in these grasshopper species and factors such as trophic behaviors and the evolution of the host may contribute to the shifts in prevalence among these core microbial groups.

An ultrasensitive chemical and biosensing method has been demonstrated for active sensing of analytes. This method works based on an optothermal microbubble which is generated through laser heating of metallic nanoparticles solution.

Listeria monocytogenes is a human pathogen and a facultative anaerobe. To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures-a phenotype observed in wild type and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA. To explore potential regulatory mechanisms, we established that the addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.

This study used mice heterozygous for Tp53 mutations with different genetic backgrounds to investigate the genetic or stochastic factors that modify the penetrance of tumor formation by Tp53 mutation., p53 is the most frequently mutated gene in human cancers. Li-Fraumeni syndrome patients inheriting heterozygous p53 mutations often have a much-increased risk to develop cancer(s) at early ages. Recent studies suggest that some individuals inherited p53 mutations do not have the early onset or high frequency of cancers. These observations suggest that other genetic, environmental, immunological, epigenetic, or stochastic factors modify the penetrance of the cancerous mutant Tp53 phenotype. To test this possibility, this study explored dominant genetic modifiers of Tp53 mutations in heterozygous mice with different genetic backgrounds. Both genetic and stochastic effects upon tumor formation were observed in these mice. The genetic background of mice carrying Tp53 mutations has a strong influence upon the tissue type of the tumor produced and the number of tumors formed in a single mouse. The onset age of a tumor is correlated with the tissue type of that tumor, although identical tumor tissue types can occur at very different ages. These observations help to explain the great diversity of cancers in different Li-Fraumeni patients over lifetimes.

Propionate is a common food preservative and one of the major fermentation acids in the intestines. Therefore, exposure to propionate is frequent for foodborne pathogens and likely takes place under suboxic conditions. However, it is not clear whether the absence of oxygen affects how pathogens respond to propionate. Here, we investigated how propionate exposure affects Listeria monocytogenes growth and virulence factor production under aerobic or anaerobic conditions and showed that oxygen indeed plays a key role in modulating L. monocytogenes response to propionate. Under aerobic conditions, propionate supplementations had no effect on planktonic growth but resulted in decreased adherent growth. Under anaerobic conditions, propionate supplementations resulted in a pH-dependent inhibition of planktonic growth and increased adherent growth. Cultures grown with propionate accumulated higher levels of acetoin under aerobic conditions but lower levels of ethanol under both aerobic and anaerobic conditions. Metabolic perturbations by propionate were also evident by the increase in straight chain fatty acids. Finally, propionate supplementations resulted in increased listeriolyin O (LLO) production under anaerobic conditions but decreased LLO production under aerobic conditions. These results demonstrate for the first time that the presence or absence of oxygen plays a critical role in shaping L. monocytogenes responses to propionate.

The Kidney Donor Risk Index (KDRI) is a continuous measure of deceased donor kidney transplant failure risk that was derived in US patients based on 10 donor characteristics. In the United States, the KDRI is utilized to guide organ allocation and to inform clinical decisions regarding organ acceptance.To examine the application of the US-derived KDRI in a large Canadian province.All deceased donor kidney-only transplant recipients in British Columbia (BC) between 2005 and 2014.We examined the predictive performance of KDRI in BC transplant recipients and compared the overall performance of KDRI with donor age alone in predicting transplant failure (from all causes including death).Donors in BC (N = 785) were older but included no black donors and few Hepatitis C virus (HCV)-positive donors compared with the original derivation cohort of the KDRI in the United States. The KDRI was moderately predictive of transplant failure (Our findings suggest that the US-derived KDRI does not improve the prediction of kidney transplant failure compared with donor age alone in a Canadian cohort and highlight the need to determine the applicability of KDRI in different regions.L’indiceVérifier l’application de l’indice KDRI étasunien dans une grande province canadienne.Tous les receveurs d’une greffe rénale simple en Colombie-Britannique entre 2005 et 2014, et dont l’organe provenait d’un donneur décédé ont été inclus.Nous avons évalué la performance prédictive de l’indice KDRI chez les receveurs d’une greffe de rein. Nous avons aussi comparé la performance globale du KDRI pour la prédiction du rejet de la greffe (toutes causes confondues, y compris le décès) obtenue par rapport à une prédiction basée uniquement sur l’âge du donneur.Comparativement à la cohorte américaine d’où dérive l’indice KDRI, les donneurs britanno-colombiens (N = 785) étaient plus âgés, mais aucun d’eux n’était d’origine afro-américaine et quelques-uns seulement étaient porteurs du virus de l’hépatite C. L’indice KDRI s’est avéré moyennement fiable pour prédire le rejet de la greffe (statistique C = 0,63) et présentait une performance prédictive comparable à celle mesurée à partir de l’âge du donneur seulement (statistique C = 0,64).Nos constatations évoquent que, dans une cohorte de patients canadiens, le recours à l’indice KDRI ne permet pas de prédire plus précisément le risque de rejet de la greffe que l’âge du donneur seulement. Ainsi, nos constatations mettent en lumière la nécessité d’évaluer l’applicabilité de l’indice KDRI dans différentes régions.

Bacterial enteric pathogens are responsible for a tremendous amount of foodborne illnesses every year through the consumption of contaminated food products. During their transit from contaminated food sources to the host gastrointestinal tract, these pathogens are exposed and must adapt to fluctuating oxygen levels to successfully colonize the host and cause diseases. However, the majority of enteric infection research has been conducted under aerobic conditions. To raise awareness of the importance in understanding the impact of oxygen, or lack of oxygen, on enteric pathogenesis, we describe in this review the metabolic and physiological responses of nine bacterial enteric pathogens exposed to environments with different oxygen levels. We further discuss the effects of oxygen levels on virulence regulation to establish potential connections between metabolic adaptations and bacterial pathogenesis. While not providing an exhaustive list of all bacterial pathogens, we highlight key differences and similarities among nine facultative anaerobic and microaerobic pathogens in this review to argue for a more in-depth understanding of the diverse impact oxygen levels have on enteric pathogenesis.

Germline deletion of the p53 gene in mice gives rise to spontaneous thymic (T-cell) lymphomas. In this study, the p53 knockout mouse was employed as a model to study the mutational evolution of tumorigenesis. The clonality of the T-cell repertoire from p53 knockout and wild-type thymic cells was analyzed at various ages employing TCRβ sequencing. These data demonstrate that p53 knockout thymic lymphomas arose in an oligoclonal fashion, with tumors evolving dominant clones over time. Exon sequencing of tumor DNA revealed that all of the independently derived oligoclonal mouse tumors had a deletion in the Pten gene prior to the formation of the TCRβ rearrangement, produced early in development. This was followed in each independent clone of the thymic lymphoma by the amplification or overexpression of cyclin Ds and Cdk6. Alterations in the expression of Ikaros were common and blocked further development of CD-4/CD-8 T cells. While the frequency of point mutations in the genome of these lymphomas was one per megabase, there were a tremendous number of copy number variations producing the tumors’ driver mutations. The initial inherited loss of p53 functions appeared to delineate an order of genetic alterations selected for during the evolution of these thymic lymphomas.

The p53 protein ensures cellular fidelity by suppressing or killing cells under stresses that enhance the mutation rate. Evidence suggests that the p53 protein may also ensure the fidelity of the epigenome. In this study a group of drugs that alter the deoxycytosine methylation patterns in cellular DNA are shown to preferentially kill human and mouse cells that contain p53 mutations or deficiencies. These observations are extended to mice that contain p53 deficiencies or missense mutations in their genome, which are preferentially killed when compared to mice with a wild type p53 gene. This is also the case for human cancer cell xenografts containing p53 mutations, which preferentially are killed by these drugs when compared to similar tumors with wild type p53. The loss of p53 function enhances a synthetic lethality with drugs that block or alter the patterns of deoxycytidine methylation in the genome.

Among patients with neuroendocrine tumors, males are more likely than females to develop liver metastatic disease. We show that this male bias toward liver metastasis is also observed in a mouse model for pancreatic neuroendocrine tumorigenesis (PanNETs). Interestingly, primary tumors from mice with metastatic disease display hepatomimicry, expressing genes normally expressed in liver. Both male and female primary tumors from mice with metastatic disease show hepatomimicry, although one of the liver genes, complement C5, is only induced in males. In mice knocked out for the complement C5 gene, the frequency of liver metastasis dropped significantly; moreover, males and females lacking a gene for complement C5 developed liver metastasis at the same frequency. Liver metastasis was also prevented by PMX53, a small molecule antagonist of C5aR1/CD88, which is the receptor for complement C5a. Thus complement C5 is epistatic to metastasis and promotes gender bias of liver metastatic disease. Citation Format: Tanupriya Contractor, Chang Chan, Shinta Kobayashi, Richard Clausen, Yvonne Sun, Edaise da Silva, Evan Vosburgh, Arnold J. Levine, Laura Tang, Chris R. Harris. Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2015-3185

Loss of the tumor suppressor, p53, occurs in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is based on its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, genetic deletion of p53 in mice gives rise to spontaneous thymic (T-cell) lymphomas and sarcomas. In this study, we used the p53-knockout (KO) mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages and p53-KO thymic lymphomas using TCRβ sequencing, allowing us to determine the clonality of the T cell repertoire. These data suggest that p53-KO thymic lymphomas were mostly oligoclonal, with some tumors comprising of a dominant clone. We further analyzed tumors with dominant clones for genomic instability by exome sequencing. Sequencing analysis revealed that there were many non-synonymous mutations occurring in each tumor, but with no similarities between tumors. On the other hand, all of the murine tumors had deletion of Pten and amplification of Cdk6. This is in stark contrast to what is seen in most human T cell lymphoblastic leukemias, which have activating mutations in Notch1 and loss of Arf. Interestingly, loss of Pten not only occurred in all tumors, but also in most, if not all, clones in the tumor. This suggests that Pten loss is an early event driving T cell transformation in a p53-KO background. Using this data, we found the somatic mutation rate in the tumors to be 1000-fold greater than the germline mutation rate. Loss of p53 also caused chromosomal abnormalities as seen with increased copy number variations. Taken together, our work suggests that, with initial loss of p53, the path to T cell tumorigenesis is vastly different from that of human tumors. Without p53, the somatic genome increases in mutation rate and copy number variations, thus solidifying p53's role as guardian of the genome. Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Exome sequencing of murine p53-knockout T-cell lymphomas reveals Pten loss and Cdk6 amplification as key steps toward cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-80. doi:10.1158/1538-7445.AM2014-LB-80