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1.  Controlled Release of Cytokines Using Silk-biomaterials for Macrophage Polarization 
Biomaterials  2015;73:272-283.
Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophages has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of β-sheet (crystalline) content enabled a short-term release (4–8 hours) of either cytokine, with smaller amounts released out to 24 hours. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High β-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages.
PMCID: PMC4605898  PMID: 26421484
2.  H9N2 Influenza A Virus Isolated from a Greater White-Fronted Wild Goose (Anser albifrons) in Alaska Has a Mutation in the PB2 Gene, Which Is Associated with Pathogenicity in Human Pandemic 2009 H1N1 
Genome Announcements  2016;4(5):e00869-16.
We report here the genomic sequence of an H9N2 influenza A virus [A/greater white-fronted goose/Alaska/81081/2008 (H9N2)]. This virus shares ≥99.8% identity with a previously reported virus. Both strains contain a G590S mutation in the polymerase basic 2 (PB2) gene, which is a pathogenicity marker in the pandemic 2009 H1N1 virus when combined with R591.
PMCID: PMC5009965  PMID: 27587808
3.  Genome Sequence of a Novel H14N7 Subtype Influenza A Virus Isolated from a Blue-Winged Teal (Anas discors) Harvested in Texas, USA  
Genome Announcements  2016;4(3):e00520-16.
We report here the complete genome sequence of a novel H14N7 subtype influenza A virus (IAV) isolated from a blue-winged teal (Anas discors) harvested in Texas, USA. The genomic characteristics of this IAV strain with a previously undetected subtype combination suggest recent viral evolution within the New World wild-bird IAV reservoir.
PMCID: PMC4901227  PMID: 27284136
4.  Surveillance for Eurasian-origin and intercontinental reassortant highly pathogenic influenza A viruses in Alaska, spring and summer 2015 
Virology Journal  2016;13:55.
Eurasian-origin and intercontinental reassortant highly pathogenic (HP) influenza A viruses (IAVs) were first detected in North America in wild, captive, and domestic birds during November–December 2014. Detections of HP viruses in wild birds in the contiguous United States and southern Canadian provinces continued into winter and spring of 2015 raising concerns that migratory birds could potentially disperse viruses to more northerly breeding areas where they could be maintained to eventually seed future poultry outbreaks.
We sampled 1,129 wild birds on the Yukon-Kuskokwim Delta, Alaska, one of the largest breeding areas for waterfowl in North America, during spring and summer of 2015 to test for Eurasian lineage and intercontinental reassortant HP H5 IAVs and potential progeny viruses. We did not detect HP IAVs in our sample collection from western Alaska; however, we isolated five low pathogenic (LP) viruses. Four isolates were of the H6N1 (n = 2), H6N2, and H9N2 combined subtypes whereas the fifth isolate was a mixed infection that included H3 and N7 gene segments. Genetic characterization of these five LP IAVs isolated from cackling (Branta hutchinsii; n = 2) and greater white-fronted geese (Anser albifrons; n = 3), revealed three viral gene segments sharing high nucleotide identity with HP H5 viruses recently detected in North America. Additionally, one of the five isolates was comprised of multiple Eurasian lineage gene segments.
Our results did not provide direct evidence for circulation of HP IAVs in the Yukon-Kuskokwim Delta region of Alaska during spring and summer of 2015. Prevalence and genetic characteristics of LP IAVs during the sampling period are concordant with previous findings of relatively low viral prevalence in geese during spring, non-detection of IAVs in geese during summer, and evidence for intercontinental exchange of viruses in western Alaska.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-016-0511-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4815243  PMID: 27036114
Alaska; H5N1; H5N2; H5N8; Highly pathogenic; Influenza; Migratory bird; Yukon-Kuskokwim Delta
5.  STAT3 is required for proliferation and maintenance of multipotency in glioblastoma stem cells 
Stem cells (Dayton, Ohio)  2009;27(10):2383-2392.
Signal transducer and activator of transcription 3 (STAT3) regulates diverse cellular processes including cell growth, differentiation, and apoptosis, and is frequently activated during tumorigenesis. Recently, putative glioblastoma stem cells (GBM-SC) have been isolated and characterized. These cells can self-renew indefinitely in culture, are highly tumorigenic, and retain the ability to differentiate in culture. We have found that treatment of GBM-SC with two chemically distinct small molecule inhibitors of STAT3 DNA-binding inhibits cell proliferation and the formation of new neurospheres from single cells. Genetic knockdown of STAT3 using an shSTAT3-containing lentivirus also inhibits GBM-SC proliferation and neurosphere formation, confirming that these effects are specific to STAT3. While STAT3 inhibition can induce apoptosis in serum-derived GBM cell lines, this effect was not observed in GBM-SC grown in stem cell media. Markers of neural stem cell multipotency also decrease upon STAT3 inhibition, suggesting that STAT3 is required for maintenance of the stem-like characteristics of these cells. Strikingly, even a transient inhibition of STAT3 leads to irreversible growth arrest and inhibition of neurosphere formation. These data suggest that STAT3 regulates the growth and self-renewal of GBM-SC and is thus a potential target for cancer stem cell-directed therapy of glioblastoma multiforme.
PMCID: PMC4391626  PMID: 19658181
6.  Genetic Diversity and Host Specificity Varies across Three Genera of Blood Parasites in Ducks of the Pacific Americas Flyway 
PLoS ONE  2015;10(2):e0116661.
Birds of the order Anseriformes, commonly referred to as waterfowl, are frequently infected by Haemosporidia of the genera Haemoproteus, Plasmodium, and Leucocytozoon via dipteran vectors. We analyzed nucleotide sequences of the Cytochrome b (Cytb) gene from parasites of these genera detected in six species of ducks from Alaska and California, USA to characterize the genetic diversity of Haemosporidia infecting waterfowl at two ends of the Pacific Americas Flyway. In addition, parasite Cytb sequences were compared to those available on a public database to investigate specificity of genetic lineages to hosts of the order Anseriformes. Haplotype and nucleotide diversity of Haemoproteus Cytb sequences was lower than was detected for Plasmodium and Leucocytozoon parasites. Although waterfowl are presumed to be infected by only a single species of Leucocytozoon, L. simondi, diversity indices were highest for haplotypes from this genus and sequences formed five distinct clades separated by genetic distances of 4.9%–7.6%, suggesting potential cryptic speciation. All Haemoproteus and Leucocytozoon haplotypes derived from waterfowl samples formed monophyletic clades in phylogenetic analyses and were unique to the order Anseriformes with few exceptions. In contrast, waterfowl-origin Plasmodium haplotypes were identical or closely related to lineages found in other avian orders. Our results suggest a more generalist strategy for Plasmodium parasites infecting North American waterfowl as compared to those of the genera Haemoproteus and Leucocytozoon.
PMCID: PMC4339737  PMID: 25710468
7.  Biotransformation and recovery of the isoflavones genistein and daidzein from industrial antibiotic fermentations 
Applied microbiology and biotechnology  2013;97(14):6427-6437.
The objective of this study was to follow the metabolic fate of isoflavone glucosides from the soybean meal in a model industrial fermentation to determine if commercially useful isoflavones could be harvested as coproducts from the spent broth at the end of the fermentation. The isoflavone aglycones, genistein and daidzein, together make up 0.1 - 0.2% of the soybean meal by weight but serve no known function in the manufacturing process. After feeding genistein to washed cells of the erythromycin-producing organism, Saccharopolyspora erythraea, the first biotransformation product (Gbp1) was determined by x-ray crystallography to be genistein-7-O-α-rhamnoside (rhamnosylgenistein). Subsequent feeding of rhamnosylgenistein to growing cells of S. erythraea led to the production of a second biotransformation product, Gbp2. Chromatographic evidence suggested that Gbp2 accumulated in the spent broth of the erythromycin fermentation. When the spent broth was hydrolyzed with acid or industrial enzyme preparations the isoflavone biotransformation products were returned back to their parental forms, genistein and daidzein, which were then recovered as coproducts. Desirable features of this method are that it does not require modification of the erythromycin manufacturing process or genetic engineering of the producing organism to be put into practice. A preliminary investigation of five additional antibiotic fermentations of industrial importance were also found to have isoflavone coproduct potential.
PMCID: PMC3700627  PMID: 23604533
Isoflavones; genistein; daidzein; coproduct; soybean; fermentation; Saccharopolyspora erythraea; 7-O-rhamnosylgenistein
8.  An erythromycin process improvement using the diethyl methylmalonate responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain 
The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250 – 300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild type (control) strain--a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.
PMCID: PMC3276693  PMID: 22048617
Diethyl methylmalonate; erythromycin; mutB; methylmalonyl-CoA mutase; Dmr; Saccharopolyspora erythraea
9.  Interspecific exchange of avian influenza virus genes in Alaska: the influence of trans-hemispheric migratory tendency and breeding ground sympatry 
Molecular ecology  2010;20(5):1015-1025.
The movement and transmission of avian influenza viral strains via wild migratory birds may vary by host species as a result of migratory tendency and sympatry with other infected individuals. To examine the roles of host migratory tendency and species sympatry on the movement of Eurasian low pathogenic avian influenza (LPAI) genes into North America, we characterized migratory patterns and LPAI viral genomic variation in mallards (Anas platyrhynchos) of Alaska in comparison to LPAI diversity of northern pintails (Anas acuta). A 50-year band recovery data set suggests that unlike northern pintails, mallards rarely make trans-hemispheric migrations between Alaska and Eurasia. Concordantly, fewer (14.5%) of 62 LPAI isolates from mallards contained Eurasian gene segments compared to those from 97 northern pintails (35%), a species with greater intercontinental migratory tendency. Aerial survey and banding data suggest that mallards and northern pintails are largely sympatric throughout Alaska during the breeding season, promoting opportunities for interspecific transmission. Comparisons of full genome isolates confirmed near-complete genetic homology (>99.5%) of seven viruses between mallards and northern pintails. This study found viral segments of Eurasian lineage at a higher frequency in mallards than previous studies, suggesting transmission from other avian species migrating inter-hemispherically or the common occurrence of endemic Alaskan viruses containing segments of Eurasian origin. We conclude that mallards are unlikely to transfer Asian origin viruses directly to North America via Alaska, but that they are likely infected with Asian origin viruses via interspecific transfer from species with regular migrations to the Eastern Hemisphere.
PMCID: PMC3041836  PMID: 21073586
Anas platyrhynchos; avian influenza; migratory; reassortment; transmission
10.  Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72 
Brain  2012;135(3):765-783.
Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset <40 years in six (10%) and >60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (>10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.
PMCID: PMC3286335  PMID: 22366793
frontotemporal dementia; amyotrophic lateral sclerosis; motor neuron disease; TDP-43; neurogenetics; chromosome 9
11.  Clinically relevant doses of chemotherapy agents reversibly block formation of glioblastoma neurospheres 
Cancer letters  2010;296(2):168-177.
Glioblastoma patients have a poor prognosis, even after surgery, radiotherapy, and chemotherapy with temozolomide or 1,3-bis(2-chloroethy)-1-nitrosourea. We developed an in vitro recovery model using neurosphere cultures to analyze the efficacy of chemotherapy treatments, and tested whether glioblastoma neurosphere initiating cells are resistant. Concentrations of chemotherapy drugs that inhibit neurosphere formation are similar to clinically relevant doses. Some lines underwent a transient cell cycle arrest and a robust recovery of neurosphere formation. These results indicate that glioblastoma neurospheres can regrow after treatment with chemotherapy drugs. This neurosphere recovery assay will facilitate studies of chemo-resistant subpopulations and methods to enhance glioblastoma therapy.
PMCID: PMC2914162  PMID: 20435409
chemotherapy; cancer stem cells; DNA damage; temozolomide; BCNU; glioblastoma; neurosphere
12.  Engineering of the methylmalonyl-CoA metabolite node for increased erythromycin production in oil-based fermentations of Saccharopolyspora erythraea 
Metabolic engineering  2007;9(3):293-303.
Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild type strain (FL2267) through duplication of the mmCoA mutase (MCM) operon led to a 51% (range 40%-64%, 0.95 CI, N = 152) increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8 kb DNA fragment in plasmid pFL2212 which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1, none of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in strain FL2385 containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3′ terminus of the mutR coding sequence.
PMCID: PMC2722834  PMID: 17482861
methylmalonyl-CoA mutase (MCM); metabolic engineering; Saccharopolyspora erythraea; erythromycin; precursor; strain improvement
13.  Knockout of the Erythromycin Biosynthetic Cluster Gene, eryBI, Blocks Isoflavone Glucoside Bioconversion during Erythromycin Fermentations in Aeromicrobium erythreum but Not in Saccharopolyspora erythraea▿  
Applied and Environmental Microbiology  2008;74(23):7383-7390.
Isoflavone glucosides are valuable nutraceutical compounds and are present in commercial fermentations, such as the erythromycin fermentation, as constituents of the soy flour in the growth medium. The purpose of this study was to develop a method for recovery of the isoflavone glucosides as value-added coproducts at the end of either Saccharopolyspora erythraea or Aeromicrobium erythreum fermentation. Because the first step in isoflavone metabolism was known to be the conversion of isoflavone glucosides to aglycones by a β-glucosidase, we chose to knock out the only β-glucosidase gene known at the start of the study, eryBI, to see what effect this had on metabolism of isoflavone glucosides in each organism. In the unicellular erythromycin producer A. erythreum, knockout of eryBI was sufficient to block the conversion of isoflavone glucosides to aglycones. In S. erythraea, knockout of eryBI had no effect on this reaction, suggesting that other β-glucosidases are present. Erythromycin production was not significantly affected in either strain as a result of the eryBI knockout. This study showed that isoflavone metabolism could be blocked in A. erythreum by eryBI knockout but that eryBI knockout was not sufficient to block isoflavone metabolism in S. erythraea.
PMCID: PMC2592926  PMID: 18836015
14.  Analysis of an 8.1-kb DNA Fragment Contiguous with the Erythromycin Gene Cluster of Saccharopolyspora erythraea in the eryCI-Flanking Region 
Antimicrobial Agents and Chemotherapy  2002;46(12):3892-3899.
An 8.1-kb region of the Saccharopolyspora erythraea genome, significant for its contiguity to the known genes of the erythromycin biosynthetic gene cluster, was mutationally analyzed and its DNA sequence was determined. The region lies immediately adjacent to eryCI. The newly characterized region is notable for a large, 3.0-kb segment, predicted not to be translated, followed by four probable genes: an acetyltransferase gene, a protease inhibitor gene, a methyltransferase gene, and a transposase gene. Because the probable functions of the genes in this region are not required for erythromycin biosynthesis or resistance and because a deletion of a 6.0-kb portion of this region had no effect on erythromycin biosynthesis, this region marks the outside boundary of the erythromycin gene cluster. Therefore, eryCI represents the end of the cluster. These results complete the analysis of the erythromycin gene cluster and eliminate the possibility that additional sought-after pathway-specific structural or regulatory genes might be found within or adjacent to the cluster.
PMCID: PMC132777  PMID: 12435693
15.  Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of Saccharopolyspora erythraea 
Journal of Bacteriology  1999;181(22):7098-7106.
The transcriptional organization of the erythromycin biosynthetic gene (ery) cluster of Saccharopolyspora erythraea has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a S. erythraea transcriptional terminator within the eryAI, eryAIII, eryBIII, eryBIV, eryBV, eryBVI, eryCIV, and eryCVI genes and additionally by an eryAI −10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream ery biosynthetic genes. Our results demonstrate that the ery gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from eryAI to eryG. Two overlapping polycistronic transcripts extending from eryBIV to eryBVII were identified. In addition, seven ery cluster promoter transcription start sites, one each beginning at eryAI, eryBI, eryBIII, eryBVI, and eryK and two beginning at eryBIV, were determined.
PMCID: PMC94186  PMID: 10559177

Results 1-15 (15)