Pathogenic Microbiology Commons^™

Investigating Antibiotic Resistance Levels Of Salmonella Internalized In Lettuce Leaves, Jason B. Thomas

McNair Scholars Research Journal

Contamination of food crops by the human pathogen Salmonella is a food safety threat worldwide. Though using treated wastewater for irrigation is a sustainable practice, it may introduce trace levels of Salmonella that may contaminate food crops. Salmonella could develop resistance to antibiotics present in wastewater. The overall goal of the project is to increase the understanding of the public health risk associated with the use of treated wastewater to irrigate food crops. The objective of this particular study is to determine the antibiotic resistance level of Salmonella internalized in lettuce leaves. In this experiment, thirty-six plants of the lettuce …

A Glutathione-Independent Glyoxalase Of The Dj-1 Superfamily Plays An Important Role In Managing Metabolically Generated Methylglyoxal In Candida Albicans, Sahar Hasim, Nur Ahmad Hussin, Fadhel Alomar, Keshore R. Bidasee, Kenneth W. Nickerson, Mark A. Wilson

Kenneth Nickerson Papers

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to D-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys¹³⁶- His¹³⁷-Glu¹⁶⁸. Purified Glx3p has an in vitro methylglyoxalase activity (K_m = 5.5 mM andk_cat = 7.8 s^-1) that is significantly greater than that of …

Coccidia Of Gerbils From Mongolia, Ethan T. Jensen

School of Biological Sciences: Dissertations, Theses, and Student Research

In this study, gerbils collected in the Mongolia over the summers of 2009-2012 were examined for coccidia. In total, 171 gerbils of three species from 22 localities were examined for coccidia. Coccidian oocysts were identified from 21 gerbils, but those found in 1 of those gerbils were probably pseudoparasites of the host from which they were recovered. From the remaining 20 gerbils, 7 morphotypes of Eimeria and 1 morphotype of Isospora were identified. Four of the 7 morphotypes of Eimeria were attributed to new species which were described in this study. In addition, 10 previously described species of Eimeria were …

Histone Biotinylation In Candida Albicans, Sahar Hasim, Swetha Tati, Nandakumar Madayiputhiya, Renu Nandakumar, Kenneth W. Nickerson

Kenneth Nickerson Papers

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, …

Candida Albicans Czf1 And Efg1 Coordinate The Response To Farnesol During Quorum Sensing, White-Opaque Thermal Dimorphism, And Cell Death, Melanie L. Langford, Jessica C. Hargarten, Krista D. Patefield, Elizabeth Marta, Jill R. Blankenship, Saranna Fanning, Kenneth W. Nickerson, Audrey L. Atkin

Kenneth Nickerson Papers

Quorum sensing by farnesol in Candida albicans inhibits filamentation and may be directly related to its ability to cause both mucosal and systemic diseases. The Ras1-cyclic AMP signaling pathway is a target for farnesol inhibition. However, a clear understanding of the downstream effectors of the morphological farnesol response has yet to be unraveled. To address this issue, we screened a library for mutants that fail to respond to farnesol. Six mutants were identified, and the czf1/czf1 mutant was selected for further characterization. Czf1 is a transcription factor that regulates filamentation in embedded agar and also whiteto- opaque switching. We found …

Seroprevalence Of Brucellosis In Livestock Within Three Endemic Regions Of The Country Of Georgia, Eliso Mamisashvili, Ian T. Kracalik, Tinatin Onashvili, Lela Kerdzevadze, Ketevan Goginashvili, Tamar Tigilauri, Marina Donduashvili, Marina Nikolaishvili, Irma Beradze, Marina Zakareishvili, Maka Kokhreidze, Makvala Gelashvili, Nino Vepkhvadze, S. Elizabeth Rácz, Philip H. Elzer, Mikeljon P. Nikolich, Jason K. Blackburn

Harold W. Manter Laboratory of Parasitology: Faculty and Staff Publications

Brucellosis is the one of most common livestock zoonoses in Georgia, resulting in significant economic losses. Livestock were sampled in three regions of Georgia (Kakheti, Kvemo Kartli, Imereti). Districts that historically reported high numbers of brucellosis related morbidity were selected for serological, bacteriological and molecular surveys. Surveying efforts yielded samples from 10,819 large and small ruminants. In total, 735 serological tests were positive on Rose Bengal and 33 bacterial isolates were recovered and identified as Brucella melitensis or Brucella abortus by microbiology and AMOS-PCR. A Bayesian framework was implemented to estimate the true prevalence of the disease given an imperfect …

Quorum Sensing And Other Aspects Of The Biology Of Candida Albicans, Kenneth W. Nickerson

Kenneth Nickerson Papers

Most dimorphic fungi exhibit cell density dependent effects in that they grow as yeasts when inoculated at > 10⁶ cells per ml and as mycelia when inoculated at < 10⁶ cells per ml. For Candida albicans, we discovered (2001) that this phenomenon (quorum sensing) is due to the production and secretion of the C₁₅ isoprenoid trans-trans farnesol. Since then, we found that C. albicans cells treated with sublethal levels of zaragozic acid (2003) or fluconazole (2004) produced 10-40X more farnesol and that these fluconazole treated cells were ca. 5X more toxic to mice following tail vein injection. This suggestion …

The Pseudomonas Syringae Type Iii Secretion System: The Translocator Proteins, Their Secretion, And The Restriction Of Translocation By The Plant Immune System, Emerson Crabill

School of Biological Sciences: Dissertations, Theses, and Student Research

Pseudomonas syringae is a Gram-negative plant pathogen whose virulence is dependent upon its type III secretion system (T3SS), a nanosyringe that facilitates translocation, or injection, of type III effector (T3E) proteins into eukaryotic cells. The primary function of P. syringae T3E proteins is suppression of plant immunity. Bacterial proteins called translocators form a translocon that forms a pore in the host plasma membrane which is traversed by T3Es. HrpK1, a putative P. syringae translocator, is a type III-secreted protein important for virulence and T3E injection, but not secretion of T3Es. Harpins are a group of proteins specific to plant pathogens …

Thoughts On Quorum Sensing And Fungal Dimorphism, Kenneth W. Nickerson, Audrey L. Atkin, Jessica C. Hargarten, Ruvini U. Pathirana, Sahar Hasim

Papers in Microbiology

Farnesol has been best studied for its role in regulating fungal dimorphism. However, farnesol is also a lipid and in this review we analyze data relevant to farnesol’s function and synthesis from the perspective of farnesol and bacterial endotoxins acting as membrane active compounds. This analysis implicates the possible roles of: (1) endotoxins in the regulation of farnesol production by C. albicans; (2) farnesol in the interactions between C. albicans and the host during disseminated infections; and (3) ubiquinones in the mechanisms for unusually high resistance to farnesol by some C. albicans cell types. Finally we discuss the implications …

Quorum Sensing Activity In Ophiostoma Ulmi: Effects Of Fusel Oils And Branched Chain Amino Acids On Yeast-Mycelial Dimorphism, A. Berrocal, J. Navarrete, C. Oviedo, K. W. Nickerson

Kenneth Nickerson Papers

Aims: For Ophiostoma (Ceratocystis) ulmi, the ability to undergo morphological change is a crucial factor for its virulence. To gain an understanding of quorum- sensing activity in O. ulmi as it relates to yeast-mycelium dimorphism control, this study examines the effects of branched-chain amino acids as well as their fusel alcohols and fusel acids as quorum sensing molecules.

Methods and Results: In a defined medium containing glucose, proline and salts, O. ulmi grew as yeasts when the culture was inoculated with a high density of spores (2-10⁷ CFU ml^-1) and as mycelia when inoculated …

Zap1 Control Of Cell-Cell Signaling In Candida Albicans Biofilms, Shantanu Ganguly, Andrew C. Bishop, Wenjie Xu, Suman Ghosh, Kenneth W. Nickerson, Frederick Lanni, Jana Patton-Vogt, Aaron P. Mitchell

Kenneth Nickerson Papers

Biofilms of Candida albicans include both yeast cells and hyphae. Prior studies indicated that a zap1/ mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations or from an effect on the production of diffusible quorum-sensing (QS) molecules. Here, we develop biosensor reporter strains that express yeastspecific YWP1-RFP or hypha-specific HWP1-RFP, along with a constitutive TDH3-GFP normalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. A zap1/ biofilm induces the yeast-specific YWP1-RFP reporter in a wild-type …

Group Size And Nest Spacing Affect Buggy Creek Virus (Togaviridae: Alphavirus) Infection In Nestling House Sparrows, Valerie A. Brown, Charles R. Brown

Papers in Ornithology

The transmission of parasites and pathogens among vertebrates often depends on host population size, host species diversity, and the extent of crowding among potential hosts, but little is known about how these variables apply to most vector-borne pathogens such as the arboviruses (arthropod-borne viruses). Buggy Creek virus (BCRV; Togaviridae: Alphavirus) is an RNA arbovirus transmitted by the swallow bug (Oeciacus vicarius) to the cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that has recently invaded swallow nesting colonies. The virus has little impact on cliff swallows, but house sparrows are seriously …

Gata-Family Transcription Factors In Magnaporthe Oryzae, Cristian F. Quispe

Department of Agronomy and Horticulture: Dissertations, Theses, and Student Research

The filamentous fungus, Magnaporthe oryzae, responsible for blast rice disease, destroys around 10-30% of the rice crop annually. Infection begins when the specialized infection structure, the appressorium, generates enormous internal turgor pressure through the accumulation of glycerol. This turgor acts on a penetration peg emerging at the base of the cell, causing it to breach the leaf surface allowing its infection.

The enzyme trehalose-6- phosphate synthase (Tps1) is a central regulator of the transition from appressorium development to infectious hyphal growth. In the first chapter we show that initiation of rice blast disease requires a regulatory mechanism involving an …

Archaeoparasitology Of Chaco Canyon, Rachel Paseka

Department of Environmental Studies: Undergraduate Student Theses

Ancient cultures of the Colorado Plateau have been a focus of archaeoparasitology since its inception, and a vast parasitological history is recorded in coprolites preserved in this arid region. The inhabitants of Chaco Canyon dominated Ancestral Puebloan culture between 1050 and 1120 AD and were responsible for the construction of great towns, road systems, and early agriculture. Analysis of the parasites preserved in fecal remains contributes to an increased knowledge of ancient Chacoan health and culture. Nineteen coprolites from four sites in Chaco Canyon were rehydrated and analyzed microscopically for parasite remains. Rhabditiform and filariform nematode larvae were found from …

Dur3 Is The Major Urea Transporter In Candida Albicans And Is Co-Regulated With The Urea Amidolyase Dur1,2, Dhammika H. M. L. P Navarathna, Aditi Das, Joachim Morschhauser, Kenneth W. Nickerson, David D. Roberts

Kenneth Nickerson Papers

Hemiascomycetes, including the pathogen Candida albicans, acquire nitrogen from urea using the urea amidolyase Dur1,2, whereas all other higher fungi use primarily the nickel-containing urease. Urea metabolism via Dur1,2 is important for resistance to innate host immunity in C. albicans infections. To further characterize urea metabolism in C. albicans we examined the function of seven putative urea transporters. Gene disruption established that Dur3, encoded by orf 19.781, is the predominant transporter. [¹⁴C]Urea uptake was energy-dependent and decreased approximately sevenfold in a dur3D mutant. DUR1,2 and DUR3 expression was strongly induced by urea, whereas the other putative transporter genes …

Farnesol Signaling In Candida Albicans, Melanie L. Langford

School of Biological Sciences: Dissertations, Theses, and Student Research

Candida albicans is a polymorphic fungus that causes a range of disease in humans, from mucosal infections to systemic disease. Its ability to cause disease is linked to conversion between yeast and filamentous forms of growth, and the first quorum-sensing molecule discovered in an eukaryote, farnesol, blocks this transition. In C. albicans, farnesol also kills mating-competent opaque cells, inhibits biofilm formation, protects the cells from oxidative stress, and can be a virulence factor or protective agent in disseminated and mucosal mouse models of infection, respectively. While much emphasis has been placed on determining its effect on C. albicans morphology, …

Candida Albicans Cellwall Components And Farnesol Stimulate The Expression Of Both Inflammatory And Regulatory Cytokines In The Murine Raw264.7 Macrophage Cell Line, Suman Ghosh, Nina Howe, Katie Volk, Swetha Tati, Kenneth W. Nickerson, Thomas M. Petro

Kenneth Nickerson Papers

Candida albicans causes candidiasis, secretes farnesol, and switches from yeast to hyphae to escape from macrophages after phagocytosis. However, before escape, macrophages may respond to C. albicans’ pathogen-associated molecular patterns (PAMPs) through toll-like receptor 2 (TLR2) and dectin-1 receptors by expressing cytokines involved in adaptive immunity, inflammation, and immune regulation. Therefore, macrophages and the RAW264.7 macrophage line were challenged with C. albicans preparations of live wild-type cells, heat-killed cells, a live mutant defective in hyphae formation, a live mutant producing less farnesol, or an isolate producing farnesoic acid instead of farnesol. Interleukin-6 (IL-6), and IL-1b, IL- 10, and tumor …

Evolutionary Aspects Of Urea Utilization By Fungi, Dhammika H. M. L. P Navarathna, Steven D. Harris, David D. Roberts, Kenneth W. Nickerson

Kenneth Nickerson Papers

The higher fungi exhibit a dichotomy with regard to urea utilization. The hemiascomycetes use urea amidolyase (DUR1,2), whereas all other higher fungi use the nickel-containing urease. Urea amidolyase is an energy-dependent biotincontaining enzyme. It likely arose before the Euascomycete/Hemiascomycete divergence c. 350 million years ago by insertion of an unknown gene into one copy of a duplicated methylcrotonyl CoA carboxylase (MccA). The dichotomy between urease and urea amidolyase coincides precisely with that for the Ni/Co transporter (Nic1p), which is present in the higher fungi that use urease and is absent in those that do not. We suggest that …

Escherichia Coli O157:H7 And Other E. Coli Strains Share Physiological Properties Associated With Intestinal Colonization, Lisa Jacobsen, Lisa Durso, Tyrell Conway, Kenneth W. Nickerson

Kenneth Nickerson Papers

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.

An Essay On Evolution: Evolution And The Origin Of Life Are Separate And Distinct Concepts, Kenneth Nickerson

Kenneth Nickerson Papers

Two years ago on a Saturday morning, I was asked quite pointedly by two friends how I could be both spiritual and a scientist. My questioning friends felt that these qualities were incompatible. It turned out their church taught that scientists were ‘the enemy’ because scientists believe in evolution. Since that time, Letters to the Editor regarding evolution have attracted my attention. Many of the published letters stated that the author did not believe in evolution, and argued in support of his/her position that God had created life. The authors thoroughly confuse two concepts which are separate and distinct: Evolution …

Microbiology Of Oil Fly Larvae, Kenneth Nickerson, Bradley A. Plantz

Kenneth Nickerson Papers

One animal beautifully adapted to the viscous asphalt of the La Brea tar pits is the oil fly Helaeomyia petrolei (Syn. Psilopa). As a normal part of its carnivorous existence the oil fly larval guts are filled with tar, with no adverse effects. Surface sterilized larvae 5 contained ca. 2 x 10 heterotrophic bacteria per larva. These bacteria have been identified as a mixture of enteric bacteria, most commonly Providencia rettgeri, and Acinetobacter spp. These bacteria were clearly growing because their numbers in the larval guts were 100 to 1,000 times greater than in free oil/asphalt. There is …

Candida Albicans Tup1 Is Involved In Farnesol-Mediated Inhibition Of Filamentous-Growth Induction, Bessie W. Kebaara, Melanie L. Langford, Dhammika H. M. L. P. Navarathna, Raluca Dumitru, Kenneth W. Nickerson, Audrey L. Atkin

Kenneth Nickerson Papers

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to …

Farnesol Restores Wild-Type Colony Morphology To 96% Of Candida Albicans Colony Morphology Variants Recovered Following Treatment With Mutagens, Ellen C. Jensen, Jacob M. Hornby, Nicole E. Pagliaccetti, Chuleeon M. Wolter, Kenneth Nickerson, Audrey L. Atkin

Kenneth Nickerson Papers

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All …

Determination Of Mrna Half-Lives In Candida Albicans Using Thiolutin As A Transcription Inhibitor, Bessie W. Kebaara, Lindsey E. Nielsen, Kenneth Nickerson, Audrey L. Atkin

Kenneth Nickerson Papers

A method for determining mRNA half-lives in the polymorphic fungus Candida albicans is described. It employs growth in a defined medium, the inhibition of transcription with thiolutin (10–20 μg/mL), and quantitative Northern blotting. The method is effective for the A72, SC5314, and CAI-4 strains of C. albicans, and for mRNAs that have a wide variety of decay rates and steady-state abundances. The range of half-lives detected (from 4–168 min) shows that this method is effective for mRNAs with widely varying half-lives. The mRNA decay rates obtained are compared with those for orthologous mRNAs from Saccharomyces cerevisiae. This procedure …

Myxozoan Genera: Definition And Notes On Taxonomy, Life-Cycle Terminology And Pathogenic Species, Jiří Lom, Iva Dyková

Harold W. Manter Laboratory: Library Materials

A list of myxozoan genera is presented in the current taxonomical scheme. These genera are defined; their type species and most important pathogens along with their hosts are listed. Simultaneously, definitions of actinospore stages representing sexual stages of the myxosporean life cycle are given; altogether, 17 actinospore collective groups with 180 types have been described. Life cycles of the two classes of the phylum Myxozoa, Malacosporea and Myxosporea, are briefly outlined with specification of the appropriate terms. Up to now, 4 malacosporean and 2,180 myxosporean species assigned to a total of 62 genera, have been established. The surviving classification of …

High Phosphate (Up To 600 Mm) Induces Pseudohyphal Development In Five Wild Type Candida Albicans, Jacob M. Hornby, Raluca Dumitru, Kenneth Nickerson

Kenneth Nickerson Papers

A method is described for the formation of nearly 100% pseudohyphae populations of wild-type Candida albicans A72. The method employs fungal growth at 37 °C (ca. 5×10⁶ cells/ml) in a glucose–proline–N-acetyl-glucosamine medium supplemented with up to 600 mM phosphate (KH₂PO₄/K₂HPO₄ 1:1) at pH 6.5. Four other strains of C. albicans (MEN, 10261, SG5314 and CAI-4) also formed pseudohyphae under these conditions, although the phosphate response profiles differed in the concentration required for each strain to form pseudohyphae.

Quorum Sensing In Candida Albicans: Probing Farnesol’S Mode Of Action With 40 Natural And Synthetic Farnesol Analogs, Roman Shchepin, Jacob M. Hornby, Erin Burger, Timothy Niessen, Patrick H. Dussault, Kenneth Nickerson

Kenneth Nickerson Papers

The dimorphic fungus Candida albicans produces extracellular farnesol (3,7, 11-trimethyl-2,6,10-dodecatriene- 1-ol) which acts as a quorum-sensing molecule (QSM) to suppress filamentation. Of four possible geometric isomers of farnesol, only the E,E isomer possesses QSM activity. We tested 40 natural and synthetic analogs of farnesol for their activity in an N-acetylglucosamine-induced differentiation assay for germ tube formation (GTF). Modified structural features include the head group, chain length, presence or absence of the three double bonds, substitution of a backbone carbon by S, O, N, and Se heteroatoms, presence or absence of a 3-methyl branch, and the bulkiness of the hydrophobic …

Eight Gram-Negative Bacteria Are 10,000 Times More Sensitive To Cationic Detergents Than To Anionic Detergents, Soumitra Rajagopal, Nicole Eis, Kenneth Nickerson

Kenneth Nickerson Papers

In liquid culture, eight typical Gram-negative bacteria were ca. 10,000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate. Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01%. Four pseudomonads able to form biofilms were ca. 1000-fold more resistant to CTAB on Luria–Bertani agar plates than they were in liquid culture. A lasI mutant of Pseudomonas aeruginosa was only able to tolerate 0.1% CTAB on Luria–Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants.

En culture liquide, huit bactéries Gram négatif typiques étaient ca. 10,000 fois …

Escherichia Coli O157:H7 In Free-Ranging Deer In Nebraska, David G. Renter, Jan M. Sargeant, Scott E. Hygnstrom, Jeff D. Hoffmann, Jerry R. Gillespie

School of Natural Resources: Faculty Publications

In order to determine the prevalence and distribution of the human pathogen, Escherichia coli O157:H7, in free-ranging deer, hunters were asked to collect and submit fecal samples from deer harvested during a regular firearm season (14–22 November 1998). Prior to the season, 47% of the hunters with permits in the southeastern Nebraska (USA) study area indicated a willingness to participate in the study. Approximately 25% of successful hunters in the area submitted deer fecal samples. Escherichia coli O157:H7 was cultured from four (0.25%) of 1,608 total samples submitted. All of the fecal samples that were properly identified (1,426) and all …

Chlorella Virus Pbcv-1 Encodes A Functional Homospermidine Synthase, Annette Kaiser, Michael Vollmert, Dorothea Tholl, Michael V. Graves, James Gurnon, Weimei Xing, Amber D. Lisec, Kenneth Nickerson, James L. Van Etten

Kenneth Nickerson Papers

Sequence analysis of the 330-kb genome of chlorella virus Paramecium bursaria chlorella virus 1 (PBCV-1) revealed an open reading frame, A237R, that encodes a protein with 34% amino acid identity to homospermidine synthase from Rhodopseudomonas viridis. Expression of the a237r gene product in Escherichia coli established that the recombinant enzyme catalyzes the NAD⁺-dependent formation of homospermidine from two molecules of putrescine. The a237r gene is expressed late in PBCV-1 infection. Both uninfected and PBCV-1-infected chlorella, as well as PBCV-1 virions, contain homospermidine, along with the more common polyamines putrescine, spermidine, and cadaverine. The total number of polyamine …