Microbiology Commons^™

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 …

Simplified Microbial Mats: Unraveling A Complex Community One Alga At A Time., Donna A. Dela Calzada, Brad M. Bebout, Angela Detweiler

STAR Program Research Presentations

Microbial mats are the oldest known ecosystems with an over 3.4 billion-year fossil record. Understanding their structure and function will help us better understand the biological evolution of life on early earth. They are rich in diversity (phylogenetically and physiologically) ranking amongst the most complex systems on earth. In this investigation, we aim to develop an effective method to study microbial mat organisms in relatively uncomplicated ecosystems that mimic the way they grow in nature. By growing “simplified microbial mats” on polypropylene cloth, we are able to observe the range of biogeochemical processes of a single species in a biogeochemical …

Regulation Of To Xin Synthesis By Clostridium Difficile, Charles Darkoh

Dissertations and Theses (Open Access)

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce …

Characterization Of Binding And Fusion Efficiencies Mediated By The V1–V5 Env Derived From Transmitted And Non-Transmitted Viruses Isolated From A Perinatal Transmission Cohort From Zambia, Mackenzie Waltke

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

Human Immunodeficiency Virus Type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome (AIDS), which affects over 34 million people worldwide. In sub-Saharan Africa where access to antiretroviral therapies (ART) is limited, mother-to-child transmission (MTCT) rates remain high and represent a major concern in the global HIV/AIDS epidemic. Little is known about the biological properties of viruses that are transmitted perinatally, including how the biological functions of envelope (Env) influence transmissibility. Previously, transmitted viruses were found to have an advantage in replicative fitness mediated by Env V1-V5. In this study viruses derived from transmitting mother infant pairs (MIPs) were …

Line Probe Assay For Detection Of Rifampicin And Isoniazid Resistant Tuberculosis In Pakistan, Joveria Qais Farooqi, Erum Khan, Syed Muhammed Zaheer Alam, Asho Ali, Zahra Hasan, Rumina Hasan

Department of Pathology and Laboratory Medicine

Objective:
To assess the efficacy of a line-probe assay delta (LiPA) as rapid diagnostic test for early detection of drug-resistant tuberculosis compared to conventional susceptibility methods in Pakistan.
Methods:
Resistance to rifampicin (RIF) and isoniazid (INH) in 108 smear-positive pulmonary tuberculosis samples was detected using a line-probe assay [GenoType MTBDRplus (Hain Lifescience, GmbH, Nehren, Germany)] at the clinical microbiology laboratory of Aga Khan University Hospital in May, 2009. Results were compared with susceptibilities performed while using agar proportion.
Results:
In comparison to the agar proportion method, the detection rate and specificity of resistance using MTBDR plus was 92.5% and 98.2% …

Epoxide-Mediated Cifr Repression Of Cif Gene Expression Utilizes Two Binding Sites In Pseudomonas Aeruginosa, Alicia E. Ballok, Christopher D. Bahl, Emily L. Dolben, Allia K. Lindsay, Jessica D. St. Laurent, Deborah Hogan, Dean Madden, George A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR …

Serratia Marcescens: The Miracle Bacillus, Alan L. Gillen, Rebekah Gibbs

Faculty Publications and Presentations

The objectives of this article are to explain the mysterious appearance of crimson-colored bacteria on food and communion bread/wafers, over the centuries, as well as to describe the biological basis

Cholesterol Degradation Pathways As Therapeutic Targets In Mycobacterium Tuberculosis, Veronica Escalante, Johnathan Abou-Fadel, Rhone A. Mendoza, Hugues Ouellet

COURI Symposium Abstracts, Summer 2012

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen responsible for about 2 million deaths worldwide annually. During infection, Mtb faces the nutrient-deficient environment of the macrophage phagosome and switches to lipids, including cholesterol, as its main source or carbon and energy. Extensive work has been done on the cholesterol AB rings degradation, but much less is known about the degradation of the CD rings and aliphatic side-chain, which are thought to be shortened by β-oxidation reactions. In this study, we investigate the role of four fadD (fadD3, fadD17, fadD19 and fadD36) and eight fadE ( …

Identification Of Two Functional Sumo Interacting Motifs (Sims) In The Non-Structural Protein Of Influenza A Virus Ns1a, Lorellie M. Lewis, German Rosas-Acosta

COURI Symposium Abstracts, Summer 2012

We have previously shown that the non-structural protein of Influenza A virus NS1A is post-translationally modified by conjugation of the Small Ubiquitin-like MOdifier (SUMO). Our functional studies have also demonstrated that SUMO conjugation (SUMOylation) affects NS1A's ability to neutralize the cellular interferon response of the host cell and other functions associated to NS1A. Importantly, we have also shown that influenza infections trigger a global increase in cellular SUMOylation and have recently demonstrated that such an increase is mediated by NS1A. This observation led us to investigate the molecular mechanism endowing NS1A with the ability to increase global cellular SUMOylation. Sequence …

Assessment Of A Microbicide Candidate Among A Diverse Cohort Of Urban Southern Us Women And Their Male Sexual Partners, Paula M. Frew, Kimberly Parker, Takeia Horton, Brooke Hixson, Lisa Flowers, Frances Priddy, Lisa Grohskopf, Christine Mauck, Kimberly Workowski

Environmental & Global Health Faculty Research

Background: This mixed methods study reports on product acceptance from a Phase I clinical trial of a candidate non-nucleoside reverse transcriptase inhibitor (NNRTI) vaginal microbicide product (UC781). The product was evaluated in the context of a Phase I clinical trial in an area characterized by high HIV prevalence among minority women. The findings will inform the development of an acceptable microbicide that will address the needs of diverse women and their partners. Methods: This is a mixed methods study of 34 racially and ethnically diverse female participants and 10 male partners in Atlanta, Georgia. Chi-square tests for marginal homogeneity and …

Efficacy Comparison Of Various Pcr Master Mixes And Thermal Cycling Parameters For Detection Of Brucella Abortus In Multiplex Assays, Kevin J. Testo, Jason A. Olivas, Alda C. Carrillo, Pejman Naraghi-Arani

STAR Program Research Presentations

Diagnostic testing for bioterrorism agents in food is essential for the safety and welfare of the general populace. Multiplex assays provide the means to test for multiple pathogen signatures with a single polymerase chain reaction (PCR) test. PCR, Luminex xMAP ® infrared fluorophore bead technology, and flow cytometry, multiplex diagnostic assays are incredibly specific and sensitive to highly conserved regions at various loci in a microbe’s genome. In order to increase the effectiveness of the PCR and the resulting fluorescent response of the beads, different PCR master mix commercial kits were compared to the standard AgPath RT-PCR mix. This study …

Identification Of Persistent Long Range Interactions In GA95 And GB95 Through Thermal Unfolding Simulations, Milen Redai Tesfamariam

Chemistry & Biochemistry Theses & Dissertations

For over five decades, different experiments have been performed to research how proteins attain their native three dimensional structures. However, the folding problem continues to be a puzzle in modern science. The design of two proteins that have maximal sequence identity but different folds and functions is one method that is being used to study the relationship between protein structure and amino acid sequence. In particular, mutant proteins of Streptococcus protein G, G_A and G_B, have 95% sequence identity and a 3a helix fold and β4/a fold, respectively. Molecular dynamics simulations of G_A95 …

Microbial Co-Occurrence Relationships In The Human Microbiome, Karoline Faust, J. Fah Sathirapongsasuti, Jacques Izard, Dirk Gevers, Jeroen Raes, Curtis Huttenhower

Department of Food Science and Technology: Faculty Publications

The healthy microbiota show remarkable variability within and among individuals. In addition to external exposures, ecological relationships (both oppositional and symbiotic) between microbial inhabitants are important contributors to this variation. It is thus of interest to assess what relationships might exist among microbes and determine their underlying reasons. The initial Human Microbiome Project (HMP) cohort, comprising 239 individuals and 18 different microbial habitats, provides an unprecedented resource to detect, catalog, and analyze such relationships. Here, we applied an ensemble method based on multiple similarity measures in combination with generalized boosted linear models (GBLMs) to taxonomic marker (16S rRNA gene) profiles …

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 …

Cyclic Di-Gmp Phosphodiesterases Rmda And Rmdb Are Involved In Regulating Colony Morphology And Development In Streptomyces Coelicolor. Journal Of Bacteriology, Travis D. Hull, Min-Hyung Ryu, Matthew J. Sullivan, Ryan C. Johnson, Nikola T. Klena, Robert M. Gelger, Mark Gomelsky, Jennifer A. Bennett

Biology and Earth Science Faculty Scholarship

Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator ofmorphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. …

The Role And Regulation Of Nsars: A Cell-Envelope Stress Sensing Two-Component System In Staphylococcus Aureus, Stacey Lynn Kolar

USF Tampa Graduate Theses and Dissertations

Abstract

S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals …

Two Boundaries Separate Borrelia Burgdorferi Populations In North America, Gabriele Margos, Jean I. Tsao, Santiago Castillo-Ramirez, Yvette A. Girard, Anne G. Hoen

Dartmouth Scholarship

Understanding the spread of infectious diseases is crucial for implementing effective control measures. For this, it is important to obtain information on the contemporary population structure of a disease agent and to infer the evolutionary processes that may have shaped it. Here, we investigate on a continental scale the population structure of Borrelia burgdorferi, the causative agent of Lyme borreliosis (LB), a tick-borne disease, in North America. We test the hypothesis that the observed d population structure is congruent with recent population expansions and that these were preceded by bottlenecks mostly likely caused by the near extirpation in the 1900s …

Sigma Factor N (Σn): A Novel Regulator Of Extreme Acid Resistance In Enterohemorrhagic E. Coli O157:H7, Pamela Ann Fay

USF Tampa Graduate Theses and Dissertations

Extreme acid resistance contributes to the successful transmission of enterohemorrhagic E. coli (EHEC) through acidic food matrices and the stomach, allowing it to gain access to the intestine and elicit disease in humans. Alternative sigma factor N (σ^N, encoded by rpoN) was previously identified as a novel regulator of extreme acid resistance in EHEC. This study investigated the role for σ^N and co-expressed products of the rpoN operon in the acid resistance phenotype of EHEC. The results revealed that σ^N primarily controls acid resistance through repression of the glutamate-dependent acid resistance (GDAR) system through control …

Lapg, Required For Modulating Biofilm Formation By Pseudomonas Fluorescens Pf0-1, Is A Calcium-Dependent Protease, Chelsea D. Boyd, Debashree Chatterjee, Holger Sondermann, George A. O'Toole

Dartmouth Scholarship

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro …

A Novel Virus Genome Discovered In An Extreme Environment Suggests Recombination Between Unrelated Groups Of Rna And Dna Viruses, Geoffrey S. Diemer, Kenneth M. Stedman

Biology Faculty Publications and Presentations

Background: Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis.

Results: Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar …

Investigation Into The Mechanism Of Salicylate-Associated Genotypic Antibiotic Resistance In Staphylococcus Aureus, Nada Salah Helal

USF Tampa Graduate Theses and Dissertations

Growth of Staphylococcus aureus with the NSAID salicylate increases phenotypic resistance (SAPAR), and the frequency at which heritable resistance occurs to various antibiotics (SAGAR). This study describes the effect of salicylate on heritable and phenotypic resistance to a set of antibiotics for laboratory and multi-drug resistant strains of S. aureus and investigates the link between resistance and SAGAR. Drug gradient plates were used to determine phenotypic resistance to antibiotics targeting DNA replication, transcription, translation and the cell wall in the presence or absence of salicylate. To measure heritable resistance, mutation frequencies were determined for each antibiotic in the presence and …

Effect Of Antioxidants And Oxidative Stress On Different Cancer Cell Types, Gaytri Devi Gupta Elera

Theses and Dissertations

Vaccinium cyanococcus, most commonly known as blueberry, is a fruit native to North America that is known for its unique taste and high antioxidant content. The skin, seed and juice of both organically and conventionally grown blueberry extract were analyzed for antioxidant content using both the Hydrophilic and Lipophilic Oxygen Radical Absorbance Capacity (ORAC) assays. Results from the Hydrophilic ORAC test showed that conventionally grown blue berries had a higher antioxidant capacity across all samples, while the Lipophilic ORAC assay showed that the antioxidant concentration of organically cultivated blueberry juice was highest, but conventionally grown blueberry seed and skin …

Enhanced Hydrocarbon Biodegradation Using Bioaugmentation With Biowish™-Aqua Fog, Michael Robert Lehrer

Master's Theses

This study was done to determine the effectiveness of a commercially available bioaugmentation product, BiOWiSH^TM-Aqua FOG, for remediating petroleum-contaminated sandy soil. Biodegradation enhancement by BiOWiSH^TM-Aqua FOG was evaluated in laboratory microcosms by directly measuring total petroleum hydrocarbon (TPH) and indirectly using respirometry. Attempts were made to enrich hydrocarbon-degrading bacteria in BiOWiSH^TM-Aqua FOG, and the resulting enrichment cultures were screened using respirometry as well. Potential hydrocarbon-degrading bacteria in BiOWiSH^TM-Aqua FOG were isolated. Experiments were performed at bench-scale using microcosm bottles containing sand contaminated with either motor oil or No. 2 diesel fuel. The …

Exposure To Host Ligands Correlates With Colocalization Of Gal/Galnac Lectin Subunits In Lipid Rafts And Phosphatidylinositol (4,5)-Bisphosphate Signaling In Entamoeba Histolytica, Amanda M. Goldston, Rhonda R. Powell, Amrita B. Koushik, Lesly A. Temesvari

Publications

Entamoeba histolytica is an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure …

Virus Infections In Wild Plant Populations Are Both Frequent And Often Unapparent, Holly R. Prendeville, Xiaohong Ye, Thomas Jack Morris, Diana Pilson

Nebraska Center for Virology: Faculty Publications

• Premise of the study: Pathogens are thought to regulate host populations. In agricultural crops, virus infection reduces yield. However, in wild plants little is known about the spatial and temporal patterns of virus prevalence. Thus, pathogen effects on plant population dynamics are unclear. Prevalence data provide necessary background for (1) evaluating the effects of virus infection on plant population size and dynamics and (2) improving risk assessment of virus-resistant transgenic crops.

• Methods: We used ELISA and RT-PCR to survey wild Cucurbita pepo populations over 4 years for five viruses, aphid-transmitted viruses of the genus Potyvirus as a …

Paramecium Bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural And Regulatory Features Of A Giant Virus, David Dunigan, Ronald Cerny, Andrew T. Bauman, Jared C. Roach, Leslie C. Lane, Irina V. Agarkova, Kurt William Wulser, Giane M. Yanai-Balser, James R. Gurnon, Jason C. Vitek, Bernard J. Kronschnabel, Adrien Jeannard, Guillaume Blanc, Chris Upton, Garry Duncan, O. William Mcclung, Fangrui Ma, James L. Van Etten

Nebraska Center for Virology: Faculty Publications

The 331 kilobase pairs chlorovirus PBCV-1 genome was re-sequenced and annotated to correct errors in the original 15 year old sequence; forty codons was considered the minimum protein size of an open reading frame. PBCV-1 encodes 416 predicted protein encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass-spectrometry based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and one host …

The Role Of The Pituitary-Adrenal Axis In G-Csf Therapy After Neonatal Hypoxia-Ischemia, Mélissa Stéphanie Charles

Loma Linda University Electronic Theses, Dissertations & Projects

Several reports indicate that the activity of the hypothalamic-pituitary-adrenal axis (HPA) as measured by the increased level of adrenocorticotropic hormone (ACTH), and corticosterone is increased after a brain insult. These hormones are the effectors secreted respectively by the pituitary and adrenal glands. It has been shown that the down-regulation of corticosterone levels can improve detrimental outcomes associated with ischemic brain injuries. Neonatal hypoxia-ischemia (HI) is a devastating perinatal event with a grim prognosis and limited therapeutic strategies. In recent studies, granulocyte-colony stimulating factor (G-CSF) has shown promise in neonatal HI investigations by improving neuromotor function and reducing apoptosis in the …

Composition Of The Adult Digestive Tract Bacterial Microbiome Based On Seven Mouth Surfaces, Tonsils, Throat And Stool Samples, Nicola Segata, Susan Kinder Haake, Peter Mannon, Katherine P. Lemon, Levi Waldron, Dirk Gevers, Curtis Huttenhower, Jacques Izard

Department of Food Science and Technology: Faculty Publications

Background: To understand the relationship between our bacterial microbiome and health, it is essential to define the microbiome in the absence of disease. The digestive tract includes diverse habitats and hosts the human body’s greatest bacterial density. We describe the bacterial community composition of ten digestive tract sites from more than 200 normal adults enrolled in the Human Microbiome Project, and metagenomically determined metabolic potentials of four representative sites.

Results: The microbiota of these diverse habitats formed four groups based on similar community compositions: buccal mucosa, keratinized gingiva, hard palate; saliva, tongue, tonsils, throat; sub- and supra-gingival plaques; …

Metabolic Reconstruction For Metagenomic Data And Its Application To The Human Microbiome, Sahar Abubucker, Nicola Segata, Johannes Goll, Alyxandria M. Schubert, Jacques Izard, Brandi L. Cantarel, Beltran Rodriguez-Mueller, Jeremy Zucker, Mathangi Thiagarajan, Bernard Henrissat, Owen White, Scott T. Scott, Barbara Methé, Patrick D. Schloss, Dirk Gever, Makedonka Mitreva, Curtis Huttenhower

Department of Food Science and Technology: Faculty Publications

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities’ metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as …

Multifunctional Cd4 Cells Expressing Gamma Interferon And Perforin Mediate Protection Against Lethal Influenza Virus Infection, Deborah M. Brown, Sarah Lee, Maria De La Luz Garcia-Hernandez, Susan L. Swain

Nebraska Center for Virology: Faculty Publications

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in …