Normal Colony Research Articles

Evaluating the effectiveness of real-time feedback on the bedside hand hygiene behaviors of nursing students.

Traditional hand hygiene teaching methods lack long-term effectiveness. A longitudinal, within-subject design explored the influence of real-time hand microbe feedback and a critical-thinking decision exercise on nursing student hand hygiene behaviors. In three community hospitals, the students' (n = 68) hand swabs were tested for normal flora, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus at three time points. Students completed the Partnering to Heal (PTH) online exercise on hospital-acquired infection prevention decisions. Normal flora colony counts decreased across the semester and MRSA-positive cultures increased in frequency and colony counts. MRSA-positive cultures were not associated with caring for patients in isolation precautions. Significantly higher colony counts were noted in the students who completed the PTH than those who did not complete the PTH. This study explores innovative pedagogy bringing the nonvisible microbial risk to the consciousness of nursing students in an attempt to change hand hygiene behaviors.

Disruption of the pdhB pyruvate dehydrogenase [corrected] gene affects colony morphology, in vitro growth and cell invasiveness of Mycoplasma agalactiae.

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

Colony foraging allocation is finely tuned to food distance and sweetness even close to a bee colony

AbstractSocial bee colonies can allocate their foraging resources over a large spatial scale, but how they allocate foraging on a small scale near the colony is unclear and can have implications for understanding colony decision‐making and the pollination services provided. Using a mass‐foraging stingless bee, Scaptotrigona pectoralis (Dalla Torre) (Hymenoptera: Apidae: Meliponini), we show that colonies will forage near their nests and allocate their foraging labor on a very fine spatial scale at an array of food sources placed close to the colony. We counted the foragers that a colony allocated to each of nine feeders containing 1.0, 1.5, or 2.0 M sucrose solution [31, 43, and 55% sucrose (wt/wt), respectively] at distances of 10, 15, and 20 m from the nest. A significantly greater number of foragers (2.6–5.3 fold greater) visited feeders placed 10 vs. 20 m away from the colony. Foraging allocation also corresponded to food quality. At the 10‐m feeders, 4.9‐fold more foragers visited 2.0 M as compared to 1.0 M sucrose feeders. Colony forager allocation thus responded to both differences in food distance and quality even when the travel cost was negligible compared to normal colony foraging distances (10 m vs. an estimated 800–1 710 m). For a nearby floral patch, this could result in unequal floral visitation and pollination.

Differential Tumor Prevalence of Geriatric Rats that had been Exposed Prenatally to One of Two Complex, Physiologically- Patterned Magnetic Fields

A total of 165 male and female rats that had been exposed during their entire prenatal development to either one of two complex, patterned magnetic fields or to reference (colony) conditions were monitored for emergence of “spontaneous” body tumours until old age (two years after birth). The two patterns were a frequency modulated (FM) field that has been shown to slow malignant cell but not normal cell growth in culture and a complex-sequenced (CSF) field. It was composed of 8 components with 6 of these components containing 8 subcomponents presented for 200 ms for 10 s every 40 s. Significantly greater proportion (33%) of the old rats that had been exposed prenatally to the complex-sequenced field displayed tumors (primarily mammary adenocarcinoma and fibrocarcinoma varieties). Most of these tumors occurred in rats that had been exposed to 30 to ~600 nT strengths but not less than 30 nT or greater than ~600 nT intensities. In comparison 4% of the rats exposed to the single, repeated frequency-modulated field exhibited tumors. The prevalence for normal background colony room conditions was 2%. These data suggest that exposure during prenatal development to weak intensity magnetic fields whose temporal patterns were designed to be convergent with the timing of base sequencing could increase the probability of the development of cancer during the later stages of life. However the frequency-modulated magnetic field pattern that suppress the growth of human breast cells in culture and elicits strong analgesia in rodents produced no significant increase in emergence of tumors in later life compared to non-treated rats.https://maltcasino.fun/https://pinbahis.fun/https://onwin.direct/https://130betebet.com/https://detectivestripes.com/

The Pharmacological Inhibition of KDM1A Displays Preclinical Efficacy in AML and MDS By Inducing Myelomonocytic Differentiation

Background: Histone methylation is one of the major systems of epigenetics and reversibly regulated by lysine (K) specific methyltransferases (KMTs) and demethylases (KDMs). Dysregulation of KMTs such as EZH2 and MLL play a key role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) while the precise function of KDMs remains unclear.KDM1A is the first reported histone demethylase, which mainly catalyzes demethylation of mono- and di-methylated lysine 4 of histone 3 (H3K4me1 and me2 respectively). According to recent reports, the inhibition of KDM1A either alone or in combination of all-trans retinoic acid is effective for AML expressing MLL-AF9 or for several types of AML respectively, suggesting KDM1A could be a therapeutic target of AML. However, other reports argue that KDM1A is essential in hematopoiesis, raising concern that KDM1A-targeted therapy could lead to severe hematological toxicity. Here, we try to clarify what types of leukemia can be ameliorated by the pharmacological inhibition of KDM1A with a possible therapeutic window and what are functional and molecular mechanisms utilizing highly selective KDM1A inhibitors we have newly designed.Results: First we validated the effect of our novel inhibitors on murine leukemia cells harboring MLL-AF9. In accordance with a previous report, our novel KDM1A inhibitors suppressed cell proliferation, diminished clonogenic capacity and induced G1-S cell cycle arrest and myelomonocytic differentiation but not apoptosis in quite low concentration that clonogenicity of normal murine bone marrow cells was spared. Next we examined the effect of these drugs on diverse types of human myeloid leukemia cells and found that our drugs were particularly effective in erythroid leukemia cells (HEL), megakaryocytic leukemia cells (CMK11-5), and a blastic subline from a MDS patient with complex karyotype (MDS-L). MDS-L cells were changed phenotypically and morphologically towards myelomonocytic differentiation such as the increase of CD11b expression level and the induction of neutrophil-like cells. HEL and CMK cells which are negative for myelomonocytic markers also gained CD11b expression and decreased erythroid markers such as CD235a and CD71. These data suggest that the inhibition of KDM1A induces myeloid differentiation across various types of leukemia.To investigate an underlying molecular basis for the cell fate conversion in HEL and myeloid differentiation in MDS-L by the inhibition of KDM1A, we performed gene expression profiling and analyzed a change of gene signatures. The expression pattern of transcriptional factors was changed from the erythroid signature (e.g. GATA1 and TAL1) to the myeloid signature (e.g. SPI1 and CEBPA) in HEL. Gene Set Enrichment Analysis (GSEA) showed that the myeloid differentiation-associated gene signature was positively enriched and the leukemia stem cell-associated gene signature was negatively enriched in both HEL and MDS-L.Finally, we investigated the effect of our KDM1A inhibitors on primary human samples such as AML with MLL-AF9 and MDS in the phase of overt leukemia (MDS/AML) with complex karyotype. The colony formation capacity was clearly impaired in relatively low concentration that normal colonies were spared. We transplanted primary MDS/AML cells with complex karyotype to immunodeficient mice and treated with a KDM1A inhibitor or vehicles after confirming the engraftment. In one MDS/AML case, all mice treated with vehicles (n=4) died of anemia and the increase of human leukemia cells within four months while 3 of 4 mice treated with a KDM1A inhibitor have survived for more than six months. Also in another case, both of two vehicle-treated mice died while all drug-treated mice had survived for more than six months. At day 200 after transplantation, we sacrificed all survived mice treated with a KDM1A inhibitor and found that human blasts were displaced from the bone marrow of treated mice. Those data suggest that our KDM1A inhibitor is effective in vivo and have a possibility for the clinical application.Conclusion: Our study suggests that KDM1A involves with myeloid differentiation and the leukemia stem cell signature and that the pharmacological inhibition of KDM1A by highly selective inhibitors is a promising way to ameliorate AML with poor prognosis such as erythroleukemia and MDS/AML with complex karyotype, without impairing normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3196: Antitumor effect of a Compound A, a derivative of a naturally occurring rotenoid deguelin, by inhibition of ATP binding to heat shock protein 90

Abstract Despite several decades of intensive efforts to develop anti-cancer drugs and therapeutic strategies, lung cancer is still one of the main causes of cancer-related deaths worldwide. Deguelin, a natural plant product, has shown anti-cancer activities in skin, colon, breast and non-small cell lung (NSCLC) cancers. However, the potential side effects of deguelin could be an obstacle to its use in clinic. In the current study, we aimed to develop a derivative of deguelin with reduced potential side effects. We have synthesized a novel deguelin analog, compound A, and evaluated its effects on cancer and normal bronchial epithelial cell proliferation, survival, and colony formation by the MTT, FACS, and/or anchorage-dependent and -independent colony forming assays. Mechanisms underlying anti-tumor effects of compound A were investigated by Western blot, and RT-PCR. Treatment with compound A inhibited cancer cell proliferation and colony formation, promoted cell cycle arrest and induced apoptosis. Compound A also suppressed the expression of HIF-1α protein and its target gene, VEGF, in NSCLC cells. Administration of compound A to NSCLC xenograft-bearing mice significantly decreased tumor growth by inhibiting tumor angiogenesis and by inducing apoptosis. In its action mechanism, compound A was involved in binding to the ATP-binding pocket of HSP90, leading to the suppression of HSP90 function. These collective results suggest that a novel deguelin derivative, compound A, has a strong potential as an anti-cancer drug. Citation Format: Su-Chan Lee, Ju-Sung Lee, Seung-Yeob Hyun, Hoon Choi, Hongchan An, Kyu-Won Kim, Young-Ger Suh, Ho-Young Lee. Antitumor effect of a Compound A, a derivative of a naturally occurring rotenoid deguelin, by inhibition of ATP binding to heat shock protein 90. [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 3196. doi:10.1158/1538-7445.AM2014-3196

The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation

The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.

Triclosan-Induced Aminoglycoside-Tolerant Listeria monocytogenes Isolates Can Appear as Small-Colony Variants

Exposure of the human food-borne pathogen Listeria monocytogenes to sublethal concentrations of triclosan can cause resistance to several aminoglycosides. Aminoglycoside-resistant isolates exhibit two colony morphologies: normal-size and pinpoint colonies. The purposes of the present study were to characterize the small colonies of L. monocytogenes and to determine if specific genetic changes could explain the triclosan-induced aminoglycoside resistance in both pinpoint and normal-size isolates. Isolates from the pinpoint colonies grew poorly under aerated conditions, but growth was restored by addition of antibiotics. Pinpoint isolates had decreased hemolytic activity under stagnant conditions and a changed spectrum of carbohydrate utilization compared to the wild type and isolates from normal-size colonies. Genome sequence comparison revealed that all seven pinpoint isolates had a mutation in a heme gene, and addition of heme caused the pinpoint isolates to revert to normal colony size. Triclosan-induced gentamicin-resistant isolates had mutations in several different genes, and it cannot be directly concluded how the different mutations caused gentamicin resistance. However, since many of the mutations affected proteins involved in respiration, it seems likely that the mutations affected the active transport of the antibiotic and thereby caused resistance by decreasing the amount of aminoglycoside that enters the bacterial cell. Our study emphasizes that triclosan likely has more targets than just fabI and that exposure to triclosan can cause resistance to antibiotics that enters the cell via active transport. Further studies are needed to elucidate if L. monocytogenes pinpoint isolates could have any clinical impact, e.g., in persistent infections.

Systems oncology: A new paradigm in cancer research.

Systems oncology: A new paradigm in cancer research.

Pluripotent Fibrosis Initiating Mesenchymal Stromal Cell Linked To Cytopenias In LGL Leukemia Through Genetic Reprogramming

BackgroundLarge Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. [Display omitted] MethodsPrimary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. ResultsSupervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p<0.001) and the proliferative capacity of the LGL MSCs was severely diminished. Exogenous FGFb, but not LIF, restored LGLL MSC proliferation comparable to normal MSCs (Fig. 1E), normalized their spindle-shaped morphology, and maintained MSC plasticity. Interestingly, treatment with exogenous FGFb normalized collagen deposition (Fig. 1F) and restored hematopoietic supporting function. ConclusionSTAT-activated LGL leukemia cells, like myeloproliferative neoplasms, share a propensity for the development of bone marrow fibrosis. These results indicate that MSCs from LGL leukemia are pluripotent, but are re-programmed to deposit excessive amounts of collagen. Restoration of FGFb maintains the pluripotency of these MSCs while reversing the fibrosis phenotype. Our data suggests that FGFb-cultured MSCs may be used to reverse cytopenias in LGL leukemia. Disclosures:No relevant conflicts of interest to declare.

Disease-Specific Induced Pluripotent Stem Cells Recapitulate The Pathophysiology Of Familial Platelet Disorder With Predisposition To Acute Myelogenous Leukemia

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. About 30 pedigrees of FPD/AML have been reported in the literature, when the first FPD/AML pedigree was reported. Heterozygous inherited RUNX1 mutations as the cause of this disorder was confirmed. Mutations of RUNX1 observed in FPD/AML are heterogeneous and tend to be specific to individual families. An individual mutation is thought to cause a functional loss of the RUNX1 protein with an individual severity, accounting for the variable phenotypes of FPD/AML among pedigrees. Mutations that cause haploinsufficiency are most frequent, though some mutations are predicted to result in dominant-negative effects. Indeed, the ability to gain more insight into the pathogenesis of FPD itself or leukemia progression of FPD/AML has been hampered by small patient numbers and the heterogeneity of the disease. Human induced pluripotent stem cells (iPSCs) derived from somatic cells hold promise to develop research models, especially for rare inherited and acquired diseases. Therefore, we obtained samples from a 45-year old female FPD/AML patient, who had inherited RUNX1 R174X mutation. She was diagnosed as acute myeloid leukemia at the age of 41 and received matched unrelated bone marrow transplantation, and was in remission for three years. Using primary dermal fibroblasts, we established induced pluripotent stem cells with heterozygous RUNX1 R174X mutation (FPD-iPSCs), by two-rounds of infection with pMX retrovirus encoding Oct4, Sox2, Klf4 and c-Myc. The morphology of FPD-iPSCs looked like embryonic stem cells. The endogenous stem cell genes were expressed. And the RUNX1 mutation derived from the primary patient cells was confirmed in the FPD-iPSCs. If iPSCs which harbor genomic abnormalities specific for the diseases are established, we can differentiate them and obtain the genetically abnormal disease cells continuously. To investigate the differentiation capacity of FPD-iPSCs for hematopoietic lineages, we first differentiated FPD-iPSCs into hematopoietic progenitor cells (HPCs) within the ‘unique sac-like structures', which were called iPS-sacs, by co-culture in the presence of VEGF with C3H10T1/2 cells, a murine stromal cell line. After 15 days of culture, FPD-iPSCs produced the almost normal number of hematopoietic progenitors. It is an interesting observation because primitive hematopoiesis is impaired in murine RUNX1 deficient models, while adult hematopoietic stem cells/progenitors rather increase in number. CD34+/CD43+ hematopoietic progenitor cells (FPD-HPCs) propagated in the iPS-sacs were collected and analyzed about hematopoietic lineage differentiation capacity. First, colony forming capacity of FPD-HPCs was measured using CD34+/CD43+ cells by semisolid culture supplemented with SCF, GM-CSF, IL-3, G-CSF, and EPO. FPD-HPCs showed normal erythroid and myeloid colony forming capacities. On the other hand, megakaryocytic colony forming assay revealed that FPD-HPCs formed a significantly lower number of CD41+ CFU-Mk colonies than HPCs from normal iPSCs in culture supplemented with IL-3, IL-6 and TPO. Also, we analyzed the in vitro megakaryocyte differentiation of the FPD-HPCs on C3H10T1/2 cells with SCF, MGDF (megakaryocyte growth and differentiation factor) and heparin. Cytospin showed CD41a+/CD42b+ mature megakaryocytes from FPD-HPCs were morphologically normal. However, the ratio of CD41a+/CD42b+ megakarocytes to CD41a+/CD42b- immature megakaryocytes were reduced, showing that RUNX1 R174X blocks megakaryocytic maturation. We conclude that FPD-HPCs derived from FPD-iPSCs recapitulate the pathophysiology of FPD/AML on a cellular level. FPD-iPSCs would be an attractive tool to elucidate the pathogenesis of this disease, including leukemic transformation caused by additional somatic mutations. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

Abstract B47: Characterization of carcinoma-associated fibroblasts from head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 350,000 deaths annually. Metastasis remains a major cause of mortality for HNSCC patients. Elucidating the mechanisms leading to invasion and spread of tumor cells is required to find new therapeutic approaches for controlling this disease. The stroma of HNSCCs consists of several non-tumor cell types, of which carcinoma-associated fibroblasts (CAFs) are prominent. We hypothesize that CAFs facilitate the invasion and migration of tumor cells leading to metastasis. Primary cultures of CAFs and their normal adjacent fibroblast (NAF) counterparts were established from 12 matched patient tissue samples using selective conditions. Each resulting cell line was confirmed to be vimentin positive and pan-cytokeratin negative by IHC staining. Characterization of the 12 pairs was performed by microarray analysis, high throughput flow cytometry cell surface marker profiling and intracellular flow cytometry for α-smooth muscle actin (αSMA) expression, a marker of fibroblast activation. The level of α-SMA expression was consistently low in the NAFs but varied widely in the CAFs where lines displayed 5-40% positive cells. Microarray gene expression analysis indentified over 500 differentially expressed genes between CAFs and NAFs. Extracellular matrix proteins (e.g. FN1 &amp; COL11A1) and cell-cell or cell-matrix adhesion proteins (e.g. ITGA11 &amp; ITGB5) were the most highly up-regulated genes in the CAFs, while immuno-modulatory receptors (e.g. IL6R, PTGER4, TGFβR, IL1R) were down-regulated. Co-injection of CAFs with SCC4 tongue carcinoma cells into immuno-compromised mice, resulted in decreased latency of tumor development and a reduced number of cells required to initiate a tumor – 1000 cells with CAF, compared to 10,000 SCC4 cells alone. In vitro co-culture of SCC4 and SCC9 tumor cells in several conditions including CAF and NAF-derived extracellular matrix (ECM), CAF and NAF-conditioned media (CM), and live CAF and NAF feeder layers, resulted in different growth phenotypes. Cells grown on ECM gave rise to tightly packed colonies with defined edges. Cells grown in CM were more scattered with some loose colonies. Cells grown on NAFs formed normal colonies, however, cells grown on matched CAFs took on a migratory phenotype, forming lines of cells along the length of the fibroblasts. Transwell migration assays were also performed where matched CAF and NAF or CAF-CM and NAF-CM were placed in the bottom chamber and SCC4 were seeded in the upper chamber. CAFs and NAFs induced more migration over the control conditions of no cells and un-conditioned media. CAFs tended to induce more cells to migrate compared to NAFs; though in one pair, the NAFs promoted more migration. Interestingly, gene expression data from this pair indicate that the expression of HGF, a potent migration inducer, is almost twice as high in the NAFs versus the CAFs. These results show that CAFs are able to enhance tumor formation and facilitate tumor cell migration. Further experiments using primary patient tumor cells are necessary to confirm this effect. Laser-capture microdissection of tumor and stromal cells, as well as cell sorting of these cell compartments from patient tissue is underway. Microarrays will be performed to identify a mechanism of interaction between these cell populations and will be integrated with our current data. Citation Format: Keira Pereira, Christina Karamboulas, Elzbieta Hyatt, Joshua Paterson, Laurie Ailles. Characterization of carcinoma-associated fibroblasts from head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B47.

Staphylococcus aureus but not Listeria monocytogenes adapt to triclosan and adaptation correlates with increased fabI expression and agr deficiency

BackgroundThe ability of pathogens to adapt to the widely used biocide, triclosan, varies substantially. The purpose of the study was to examine bacterial adaptation over an extended period of time to low increments of triclosan concentrations. Focus was two human pathogens, S. aureus and L. monocytogenes that previously have displayed inherent high and low adaptability, respectively.ResultsThree strains of L. monocytogenes and two strains of S. aureus including the community-acquired USA300 were exposed to increasing, sub-lethal concentrations of triclosan in triclosan-containing agar gradients. Following 25 days of exposure on agar plates to sub-lethal concentrations of triclosan with a twofold concentration increase every second day, minimum inhibitory concentration (MIC) for S. aureus increased from 0.125 (8325–4) and 0.0625 (USA 300) mg/L to 4 mg/L. The MIC of all three L. monocytogenes strains was initially 4 mg/L and remained unaltered by the exposure. The adapted S. aureus isolates retained normal colony size but displayed increased expression of fabI encoding an essential enzyme in bacterial fatty acid synthesis. Also, they displayed decreased or no expression of the virulence associated agrC of the agr quorum sensing system. While most adapted strains of USA300 carried mutations in fabI, none of the adapted strains of 8325–4 did.ConclusionsAdaptability to triclosan varies substantially between Gram positive human pathogens. S. aureus displayed an intrinsically lower MIC for triclosan compared to L. monocytogenes but was easily adapted leading to the same MIC as L. monocytogenes. Even though all adapted S. aureus strains over-expressed fabI and eliminated expression of the agr quorum sensing system, adaptation in USA300 involved fabI mutations whereas this was not the case for 8325–4. Thus, adaptation to triclosan by S. aureus appears to involve multiple genetic pathways.

Queen control of a key life-history event in a eusocial insect

In eusocial insects, inclusive fitness theory predicts potential queen-worker conflict over the timing of events in colony life history. Whether queens or workers control the timing of these events is poorly understood. In the bumble-bee Bombus terrestris, queens exhibit a 'switch point' in which they switch from laying diploid eggs yielding females (workers and new queens) to laying haploid eggs yielding males. By rearing foundress queens whose worker offspring were removed as pupae and sexing their eggs using microsatellite genotyping, we found that queens kept in the complete absence of adult workers still exhibit a switch point. Moreover, the timing of their switch points relative to the start of egg-laying did not differ significantly from that of queens allowed to produce normal colonies. The finding that bumble-bee queens can express the switch point in the absence of workers experimentally demonstrates queen control of a key life-history event in eusocial insects. In addition, we found no evidence that workers affect the timing of the switch point either directly or indirectly via providing cues to queens, suggesting that workers do not fully express their interests in queen-worker conflicts over colony life history.

Novel aspects of understanding molecular working mechanisms of salivary glands of worker honeybees (Apis mellifera) investigated by proteomics and phosphoproteomics

Honeybee salivary glands (SGs) are important exocrine glands. However, the molecular basis of how SGs fulfill their biological duty is still elusive. Proteomics and phosphoproteomics of cephalic SG (HSG) and thoracic SG (TSG) were compared between normal and single-cohort honeybee colonies. Of 113 and 64 differentially regulated proteins and phosphoproteins, 86 and 33 were identified, respectively. The SGs require a wide spectrum of proteins to support their multifaceted functions and ensure normal social management of the colony. Changes of protein expression and phosphoproteins are key role players. The HSG triggers labor transition from in-hive work to foraging activities via the regulation of juvenile hormone and ethyl oleate levels. The stronger expression of proteins involved in carbohydrate and energy metabolism, protein folding, protein metabolism, cellular homeostasis and cytoskeleton in TSG, supports the gland to efficiently enhance honey processing by synthesis and secretion of saliva into nectar. The age structure of the colony is vital for increased production efficiency. This data reveals the molecular underpinning of SGs to accomplish their biological missions and provides new knowledge for the beekeeping industry for enhancing the management and production efficiency of the colony and honey quality through manipulation of potential target proteins. This study comprehensively analyses the characteristic of the proteome and phosphoproteome of honeybee salivary glands (SGs) between normal and single-cohort honeybee colonies. The SGs need a wide spectrum of proteins to support their multifaceted functions and ensure normal social management of the colony. The cephalic SG triggers labor transition from in-hive work to foraging behavior via the regulation of juvenile hormone and ethyl oleate titer. The thoracic SG stronger expressed of proteins related to carbohydrate and energy metabolism, protein folding, protein metabolism, cellular homeostasis and cytoskeleton to support the gland to efficiently enhance honey processing by synthesis and secretion of saliva into nectar. This data reveals the molecular underpinning of SGs to accomplish their biological missions and provides new knowledge for the beekeeping industry for enhancing the maintenance and production efficiency of the colony and honey quality through manipulation of potential target proteins.

Egg wax from the cattle tick Rhipicephalus (Boophilus) microplus inhibits Pseudomonas aeruginosa biofilm

Rhipicephalus (Boophilus) microplus is constantly challenged during its life cycle by microorganisms present in their hosts or in the environment. Tick eggs may be especially vulnerable to environmental conditions because they are exposed to a rich and diverse microflora in the soil. Despite being oviposited in such hostile sites, tick eggs remain viable, suggesting that the egg surface has defense mechanisms against opportunistic and/or pathogenic organisms. R. microplus engorged females deposit a superficial wax layer onto their eggs during oviposition. This egg wax is essential for preventing desiccation as well as acting as a barrier against attack by microorganisms. In this study, we report the detection of anti-biofilm activity of R. microplus egg wax against Pseudomonas aeruginosa PA14. Genes involved in the functions of production and maintenance of the biofilm extracellular matrix, pelA and cdrA, respectively, were markedly downregulated by a tick egg-wax extract. Moreover, this extract strongly inhibited fliC gene expression. Instead of a compact extracellular matrix, P. aeruginosa PA14 treated with egg-wax extract produces a fragile one. Also, the colony morphology of cells treated with egg-wax extract appears much paler and brownish, instead of the bright purple characteristic of normal colonies. Swarming motility was also inhibited by treatment with the egg-wax extract. The inhibition of P. aeruginosa biofilm does not seem to depend on inhibition of the quorum sensing system since mRNA levels of the 3 regulators of this system were not inhibited by egg-wax extract.

Colony status of Asian giant honeybee, Apis dorsata Fabricius in Southern Karnataka, India

The field survey conducted to collect more than twenty parameters of Apis dorsata colonies during different seasons at different districts of southern Karnataka revealed interesting results. Altogether, 2,407 normal colonies (comb with live bees) were recorded at various habitats of southern Karnataka. Of all, Mysore district has recorded highest (1,560) colonies followed by Chamarajanagar (544) and Kodagu (303) districts. Among the seasons, winter has recorded highest (839) colonies followed by rainy (807) and summer (761) seasons.Apis dorsata selected 20 tree species to nest 1,646 colonies at an elevation ranged from five to 80 feet. About 580 colonies nested on human built structures at the height of eight to 75 feet and 181 colonies were found on rock cliffs at 10 to 30 m elevation. The colony density was more (3.64) in Mysore district followed by Chamarajanagar and Kodagu districts. Accordingly, the abundance and percent nesting frequency were also varied considerably. Further, the colony aggregates, aggregates density per 52 meter area, abundance and percent frequency was 1.57, 3.19 and 134.07 respectively on trees, rock cliffs and human built structures at Chamarajanagar, Kodagu and Mysore districts. The morphometrics indicated considerable variations. A. dorsata produced multifloral honey (10,831.5 kg) and good quantity of beeswax (1,444.2 Kg) every year. The floral source included 687 flowering plants which belong to 223 familes, supplied continuous nectar flow during most of the seasons with little variations. Apis dorsata honey quality was moderate with below detection limit of pesticide residues. Continuous interference from various predators, pests and parasites along with man-made activities have stressed severely A. dorsata colony population which resulted in the colony decline considerably. Thus, there is a dire need to conserve A. dorsata population under natural conditions in this part of the state. Key words: Colony status, Apis dorsata, southern Karnataka, India.

Pacemaker lead infection and related bacteraemia caused by normal and small colony variant phenotypes of Bacillus licheniformis

Here, we report what we believe to be the first case of bacteraemia with small colony variants of Bacillus licheniformis related to a pacemaker lead infection by B. licheniformis displaying the normal phenotype. Arbitrarily primed PCR analysis showed a clonal strain. The infection was cured after the removal of the infected device.

Rapid adaptation to mammalian sociality via sexually selected traits.

BackgroundLaboratory studies show that the components of sexual selection (e.g., mate choice and intrasexual competition) can profoundly affect the development and fitness of offspring. Less is known, however, about the total effects of sexual selection on offspring in normal social conditions. Complex social networks, such as dominance hierarchies, regulate the opportunity for mating success, and are often missing from laboratory studies. Social selection is an extended view of sexual selection that incorporates competition during sexual and nonsexual interactions, and predicts complex evolutionary dynamics. Whether social selection improves or constrains offspring fitness is controversial.ResultsTo identify fitness consequences of social selection, wild-derived mice that had bred under laboratory conditions for eight generations were re-introduced to naturalistic competition in enclosures for three consecutive generations (promiscuous line). In parallel, a control lineage bred in cages under random mate assignment (monogamous line). A direct competition experiment using second-generation animals revealed that promiscuous line males had greater reproductive success than monogamous line males (particularly during extra-territorial matings), in spite of higher mortality and equivalent success in social dominance and sperm competition. There were no major female fitness effects (though promiscuous line females had fewer litters than monogamous line females). This result suggested that selection primarily acted upon a sexually attractive male phenotype in the promiscuous line, a hypothesis we confirmed in female odor and mating preference trials.ConclusionsWe present novel evidence for the strength of sexual selection under normal social conditions, and show rapid male adaptation driven largely by sexual trait expression, with tradeoffs in survivorship and female fecundity. Re-introducing wild-derived mice to competition quickly uncovers sexually selected phenotypes otherwise lost in normal colony breeding.

CD123-Specific Chimeric Antigen Receptor Redirected T Cells Exhibit Potent Cytolytic Activity and Multiple Effector Functions Against Acute Myeloid Leukemia without Altering Normal Hematopoietic Colony Formation in Vitro

AbstractAbstract 950Current treatment regimes for acute myeloid leukemia (AML) achieve complete remissions in only a subset of individuals and most adult patients will relapse within 5-years, emphasizing the need for novel treatment alternatives. One such therapy may be the administration of T cells engineered to express chimeric antigen receptors (CARs) specific for AML-associated antigens. CARs are typically composed of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3ζ signaling domain and may contain one or more costimulatory endodomains. When expressed in T cells, CARs redirect T cell specificity to surface antigens on target cells in an MHC-independent manner. The interleukin 3 receptor alpha chain (IL3Rα, CD123) is a cell surface receptor which is aberrantly over-expressed on multiple hematologic malignancies including AML. Previous work has demonstrated that CD123 is not expressed on all CD34+/CD38− hematopoietic stem cells and is restricted to cells of the myeloid lineage, making CD123 an attractive target for CAR T cell therapy. We have therefore generated two novel CD123-specific (CD123R) CARs using scFvs from previously characterized antibodies, designated 26292 and 32716, which bind two distinct epitopes on CD123. Here we demonstrate that T cells expressing CARs derived from either 26292 or 32716 effectively redirect T cell specificity against CD123+ cells. Healthy donor T cells (n=3) engineered to express the CD123R CARs efficiently lysed CD123+ cell lines LCL and KG1a while sparing the CD123− cell line K562 as demonstrated by a 4 hour chromium-51 (51Cr) release assay. Additionally, both of the CD123R CAR T cells produced similar levels of IFN-γ and TNF-α and displayed comparable levels of antigen-dependent proliferation following co-culture with CD123+ cell lines. The potent cytolytic activity and activation of our CD123-targeting T cells was not limited to tumor cell lines. Indeed, CD123R CAR T cells, but not donor-matched CD19-specific (CD19R) CAR T cells, robustly lysed panel of primary AML samples (n=6, 3 – persistent, 1 – relapsed, 2 - untreated) (* p<0.05, ** p<0.001 using the unpaired students’ t-test comparing 26292 or 32716 CAR T cells to donor-matched CD19R CAR T cells), and exhibited multiple effector functions for both CD4 and CD8 T cell subsets (ie CD107a degranulation, IFN-γ and TNF-α production, and antigen specific proliferation) when co-cultured with primary AML samples (n=3, 2 – relapsed, 1-persistent). To examine the effect our CD123-specific T cells have on normal and leukemic progenitor cells, we co-cultured CD123R CAR T cells, or donor-matched CD19-targeting T cells, with either CD34-enriched cord blood (CB, n=3) or primary AML samples (n=3, 2 – relapsed, 1 - untreated) for 4 hours (E:T 25:1) prior to plating in semisolid methylcellulose progenitor culture. CD123-targeting T cells did not significantly reduce the number of colony-forming unit granulocyte-macrophage (CFU-GM) or burst-forming unit erythroid (BFU-E) colonies from CB when compared to CD19R CAR T cells. Finally, while CD19-specific T cells had little impact on leukemic colony formation of primary AML samples, CD123-targeting T cells significantly reduced leukemic colony formation in vitro.Collectively, our data demonstrate that CD123-specifc CARs can be expressed in primary healthy donor T cells, distinguish between CD123+ and CD123− cells, and mediate robust anti-leukemic activity against a panel of poor-risk primary AML patient samples. Importantly, we demonstrate that CD123R CAR T cells have little impact on normal progenitor colony formation while significantly reducing the growth of clonogenic myeloid leukemic progentiors in vitro. Thus, CD123R CAR T cells are a promising candidate for future immunotherapy of AML. [Display omitted] Disclosures:Bhatia:Novartis: Consultancy, Honoraria. Jensen:ZetaRx: Equity Ownership, Patents & Royalties.