Comparative In Vitro Dissolution Assessment of Calcined and Uncalcined Hydroxyapatite Using Differences in Bioresorbability and Biomineralization.

This study reports the effect of the not-calcining process on the bioresorption and biomineralization of hydroxyapatite through in vitro dissolution assessment. The prepared calcined hydroxyapatite (c-HAp) and uncalcined hydroxyapatite (unc-HAp) have a particle size of 2 µm and 13 µm, surface areas of 4.47 m2/g and 108.08 m2/g, and a Ca/P ratio of 1.66 and 1.52, respectively. In vitro dissolution assessments of c-HAp and unc-HAp were performed for 20 days at 37 °C in a citric acid buffer according to ISO 10993-14. During the dissolution, the c-HAp and unc-HAp confirmed an increase in weight, and the calcium and phosphorous ions were rapidly released. The calcium ions released from c-HAp formed rod-shaped particles with a longer and thinner morphology, while in unc-HAp, they appeared thicker and shorter. In the ICP-OES results, the concentrations of calcium elements were initially increased and then decreased by this formation. The rod-shaped particles identified as calcium citrate (Ca-citrate) through the XRD pattern. The calcium content of Ca-citrate particles from unc-HAp was higher than that from c-HAp. The unc-HAp demonstrated non-toxic properties in a cytotoxicity evaluation. Therefore, due to its higher bioresorption and biomineralization, unc-HAp exhibits enhanced biocompatibility compared to c-HAp.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Thermally Stable and Reusable Silica and Nano-Fructosome Encapsulated CalB Enzyme Particles for Rapid Enzymatic Hydrolysis and Acylation.

This study reports the preparation of silica-coated and nano-fructosome encapsulated Candida antarctica lipase B particles (CalB@NF@SiO2) and a demonstration of their enzymatic hydrolysis and acylation. CalB@NF@SiO2 particles were prepared as a function of TEOS concentration (3-100 mM). Their mean particle size was 185 nm by TEM. Enzymatic hydrolysis was performed to compare catalytic efficiencies of CalB@NF and CalB@NF@SiO2. The catalytic constants (Km, Vmax, and Kcat) of CalB@NF and CalB@NF@SiO2 were calculated using the Michaelis-Menten equation and Lineweaver-Burk plot. Optimal stability of CalB@NF@SiO2 was found at pH 8 and a temperature of 35 °C. Moreover, CalB@NF@SiO2 particles were reused for seven cycles to evaluate their reusability. In addition, enzymatic synthesis of benzyl benzoate was demonstrated via an acylation reaction with benzoic anhydride. The efficiency of CalB@NF@SiO2 for converting benzoic anhydride to benzyl benzoate by the acylation reaction was 97%, indicating that benzoic anhydride was almost completely converted to benzyl benzoate. Consequently, CalB@NF@SiO2 particles are better than CalB@NF particles for enzymatic synthesis. In addition, they are reusable with high stability at optimal pH and temperature.

Structural Analysis of Spermidine Synthase from Kluyveromyces lactis.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

Molecular Structure of Phosphoserine Aminotransferase from Saccharomyces cerevisiae.

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of Saccharomyces cerevisiae PSAT (ScPSAT) at a resolution of 2.8 Å. The results demonstrated that the ScPSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of ScPSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of ScPSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time.

Effects of Hydrophobic Modification of Linear- and Branch-Structured Fluorinated and Nonfluorinated Silanes on Mesoporous Silica Particles.

This work reports a comparison of hydrophobic surface modification on mesoporous silica particles (MSPs) obtained by large-scale production using a batch reactor with linear and branched fluorinated and nonfluorinated silanes. Fluorinated silanes were used with TDF-TMOS and TFP-TMOS as a linear and branched structure, respectively. Nonfluorinated silanes were used with OD-TEOS and HMDS as a linear and branched structure, respectively. These four silanes were grafted on the surface of the MSPs as the function of the concentrations, and then, the water contact angles (WCAs) were measured. The WCA of the four silane-grafted MSPs was higher in the branch-structured silanes, namely, TFP-TMOS@MSPs and HMDS@MSPs than in linear-structured silanes, namely, TDF-TMOS and OD-TEOS due to the higher hydrophobicity by a lot of -F and -CH3 groups. Furthermore, the relationship between the WCA and BET parameters was demonstrated using the surface areas, pore volumes, and grafted amounts of the four silane-grafted MSPs. The structural characterization was demonstrated by solid-state 29Si MAS NMR to determine the bonding environment of Si atoms between the grafted silane and the surfaces of MSPs using the T 3/T 2 and Q 3/Q 4 ratios of the fluorinated and nonfluorinated silane-grafted MSPs. Among the four silanes, nonfluorinated HMDS@MSPs had a high contact angle of 135° as fluorinated TFP-TMOS@MSPs. When 5 wt % of HMDS@MSPs mixed with gravure ink was coated on a biodegradable polylactic acid (PLA) film, the contact angle was improved to 131.8 from 83.3° of the natural PLA film.

Thermally Stable and Reusable Ceramic Encapsulated and Cross-Linked CalB Enzyme Particles for Rapid Hydrolysis and Esterification.

Candida antarctica lipase B (CalB) enzyme was encapsulated and cross-linked by silica matrix to enhance its thermal stability and reusability, and demonstrated an enzymatic ability for rapid hydrolysis and esterification. Silica encapsulated CalB particles (Si-E-CPs) and silica cross-linked CalB particles (Si-CL-CPs) were prepared as a function of TEOS concentration. The particle size analysis, thermal stability, catalytic activity in different pHs, and reusability of Si-E-CPs and Si-CL-CPs were demonstrated. Furthermore, the determination of the CalB enzyme in Si-E-CPs and Si-CL-CPs was achieved by Bradford assay and TGA analysis. Enzymatic hydrolysis was performed against the p-nitrophenyl butyrate and the catalytic parameters (Km, Vmax, and Kcat) were calculated by the Michaelis-Menten equation and a Lineweaver-Burk plot. Moreover, enzymatic synthesis for benzyl benzoate was demonstrated by esterification with an acyl donor of benzoic acid and two acyl donors of benzoic anhydride. Although the conversion efficiency of Si-CL-CPs was not much higher than that of native CalB, it has an efficiency of 91% compared to native CalB and is expected to be very useful because it has high thermal and pH stability and excellent reusability.

NEAT1 is essential for metabolic changes that promote breast cancer growth and metastasis.

Accelerated glycolysis is the main metabolic change observed in cancer, but the underlying molecular mechanisms and their role in cancer progression remain poorly understood. Here, we show that the deletion of the long noncoding RNA (lncRNA) Neat1 in MMTV-PyVT mice profoundly impairs tumor initiation, growth, and metastasis, specifically switching off the penultimate step of glycolysis. Mechanistically, NEAT1 directly binds and forms a scaffold bridge for the assembly of PGK1/PGAM1/ENO1 complexes and thereby promotes substrate channeling for high and efficient glycolysis. Notably, NEAT1 is upregulated in cancer patients and correlates with high levels of these complexes, and genetic and pharmacological blockade of penultimate glycolysis ablates NEAT1-dependent tumorigenesis. Finally, we demonstrate that Pinin mediates glucose-stimulated nuclear export of NEAT1, through which it exerts isoform-specific and paraspeckle-independent functions. These findings establish a direct role for NEAT1 in regulating tumor metabolism, provide new insights into the Warburg effect, and identify potential targets for therapy.

Hydrophobic Mesoporous Silica Particles Modified With Nonfluorinated Alkyl Silanes.

This work reports the preparation of hydrophobic mesoporous silica particles (MSPs) modified with nonfluorinated alkyl silanes. Alkyl silanes were grafted onto the surface of the MSPs as a function of the length of nonfluorinated alkyl chains such as propyltriethoxysilane (C3), octyltriethoxysilane (C8), dodecyltriethoxysilane (C12), and octadecyltriethoxysilane (C18). Moreover, the grafting of the different alkyl silanes onto the surface of MSPs to make them hydrophobic was demonstrated using different conditions such as by changing the pH (0, 4, 6, 8, and 13), solvent type (protic and aprotic), concentration of silanes (0, 0.12, 0.24, 0.36, 0.48, and 0.60 M), reaction time (1, 2, 3, and 4 days), and reaction temperature (25 and 40 °C). The contact angles of the alkyl silane-modified MSPs were increased as a function of the alkyl chain lengths in the order of C18 > C12 > C8 > C3, and the contact angle of C18-modified MSPs was 4 times wider than that of unmodified MSPs. The unmodified MSPs had a contact angle of 25.3°, but C18-modified MSPs had a contact angle of 102.1°. Furthermore, the hydrophobicity of the nonfluorinated alkyl silane-modified MSPs was also demonstrated by the adsorption of a hydrophobic lecithin compound, which showed the increase of lecithin adsorption as a function of the alkyl chain lengths. The cross-linking ratios of the modified silanes on the MSPs were confirmed by solid-state 29Si-MAS nuclear magnetic resonance (NMR) measurement. Consequently, the hydrophobic modification on MSPs using nonfluorinated alkyl silanes was best preferred in a protic solvent, with a reaction time of ∼24 h at 25 °C and at a high concentration of silanes.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.

The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Fecal Microbiome and Resistome Profiling of Healthy and Diseased Pakistani Individuals Using Next-Generation Sequencing.

In this paper, we aimed to characterize the fecal microbiome and its resistomes of healthy and diseased subjects infected with multidrug-resistant Escherichia coli using next-generation sequencing (NGS). After initial screening, 26 stools samples belonging to healthy (n = 13) and diseased subjects (n = 13) were selected and subjected to NGS. A total of 23 and 42 antibiotic-resistant genes (ARGs) conferring resistance to 6 and 9 classes of antibiotics were identified in the resistomes of healthy and diseased subjects, respectively. Bacteroidetes were found to be the major phylum in both healthy and diseased subjects; however, Proteobacteria was predominantly present in the diseased subjects only. Microbial dysbiosis and predominance of various ARGs in the resistome of diseased subjects reflect the excessive usage of antibiotics in Pakistan and warrants immediate attention to regulate the use of various antimicrobials.

Resistome and microbial profiling of pediatric patient's gut infected with multidrug-resistant diarrhoeagenic Enterobacteriaceae using next-generation sequencing; the first study from Pakistan.

A high prevalence of multidrug-resistant (MDR) pathogens has been reported in adult and pediatric populations of Pakistan. However, data describing the effect of MDR microbes on the gut microbiota is scarce. We designed a cross-sectional pediatric study to investigate the effect of MDR microbes' infection on the gut microbiome and its resistome of children using high-throughput next-generation sequencing (NGS). A cross-sectional study was conducted at a tertiary health care hospital in Peshawar Pakistan, between 5 September 2019 to 15 February 2020. Pediatric patients with acute gastroenteritis (n = 200) were enrolled. All the enrolled pediatric patients underwent initial antimicrobial resistance (AMR) screening using the disk diffusion method. Children with MDR infections were identified and selected for gut microbiome and its resistome profiling using NGS. Out of 200 enrolled pediatric patients, 80 (40%) were found infected with MDR diarrheagenic Enterobacteriaceae consisting of 50 (62.5%) infections caused by extended-spectrum beta-lactamase (ESBL) producing E. coli while 30 (37.5%) by MDR Enterobacter specie. A total of 63 and 17 antibiotic-resistant genes (ARGs) conferring resistance to 7 and 5 classes of antibiotics were identified in the resistomes of MDR diarrheagenic Enterobacteriaceae infected and healthy children, respectively. NGS-based gut microbial profiling of MDR Enterobacter spp., ESBL producing E. coli infected pediatric patients and healthy controls revealed the predominance of Proteobacteria and Actinobacteria, respectively. An increased abundance of several pathogenic gram-negative bacteria namely E. coli, Enterobacter cloacae, and Salmonella enterica was observed in the gut microbiota of children infected with MDR bacterial infections than that of the healthy controls. This work indicates that children with MDR infections have reduced microbial diversity and enriched ARGs than healthy controls. The emergence of MDR bacterial strains and their association with gut dysbiosis needs immediate attention to regulate antibiotics usage in Pakistani children.

Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis.

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

Preparation and In Vitro Cytotoxicity Assessments of Spherical Silica-Encapsulated Liposome Particles for Highly Efficient Drug Carriers.

This work reports the formation of spherical solid silica-encapsulated liposome particles (SLPs) as functions of the concentration of silica precursor, reaction time, temperature, and volume ratios of solvent, respectively. The solid SLPs are more robust and have better drug-loading-efficiency liquid liposomes in carrier formulations. The liquid-state liposomes are hard to handle and have a lower drug-loading efficiency because they are fragile to external stimuli and have narrow hydrophobic phospholipid bilayers. The SLPs were obtained by silicification with tetraethyl orthosilicate (TEOS) in the hydrophilic region of phospholipid bilayers by a sol-gel process. These SLPs were characterized by scanning electron microscopy (SEM), focused ion beam (FIB)-SEM, confocal laser scanning microscopy (CLSM), thermogravimetric analysis (TGA), ζ-potential, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and UV-vis spectroscopy. The obtained SLPs were spherical with an average size of 2-3 µm. The hydrophilic region of the SLP phospholipid bilayers was confirmed using CLSM with green fluorescent fluorescein isothiocyanate (FITC) labeling and FIB-SEM. Furthermore, the drug-loading capacity and in vitro cytotoxicity assessments were performed using several drug compounds and L929 cells. The drug-loading capacity of the SLPs was >95%, and in particular, the hydrophobic drug-loading capacity was 2.3 times higher than that of general liposomes. Moreover, the result of an in vitro cytotoxicity assessment of the SLPs was good, about 99% of cell viability.

Next-Generation Sequencing Based Gut Resistome Profiling of Broiler Chickens Infected with Multidrug-Resistant Escherichia coli.

The study was designed to investigate the fecal microbiome and resistome of broiler chickens infected with multidrug-resistant (MDR) Escherichia coli (E. coli). Fecal samples (n = 410) from broiler chickens were collected from thirteen randomly selected sites of Khyber Pakhtunkhwa and screened for the presence of MDR E. coli. Upon initial screening, thirteen (13) MDR E. coli isolates were then subjected to shotgun metagenome next-generation sequencing (NGS). NGS based resistome analysis identified the multidrug efflux pump system-related genes at the highest prevalence (36%) followed by aminoglycoside (26.1%), tetracycline (15.9%), macrolide-lincosamide-streptogramin (9.6%), beta-lactam (6.6%), rifampin (2%), sulphonamide (1.3%), phenicol (0.91%), vancomycin (0.62%), trimethoprim (0.34%), colistin (0.30%), and quinolone (0.33%). The most abundant virulence-associated genes (VAGs) identified were iroN, iutA, iss, and iucA. NGS based taxonomic profiling at the phylum level revealed the predominance of Proteobacteria (38.9%) followed by Firmicutes (36.4%), Bacteroidetes (15.8%), and Tenericutes (8.9%). Furthermore, pathobionts such as E. coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri belonging to the family Enterobacteriaceae were predominantly found. This study revealed the widespread presence of MDR genes, diverse VAGs, and a dysbiotic gut in the broiler chickens infected with MDR E. coli of Khyber Pakhtunkhwa for the first time using NGS.

Melasolv induces melanosome autophagy to inhibit pigmentation in B16F1 cells.

The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. In contrast to melanosome biogenesis, the processes underlying melanosome degradation remain largely unknown. Autophagy is a process that promotes degradation of intracellular components' cooperative process between autophagosomes and lysosomes, and its role for process of melanosome degradation remains unclear. Here, we assessed the regulation of autophagy and its contributions to depigmentation associated with Melasolv (3,4,5-trimethoxycinnamate thymol ester). B16F1 cells-treated with Melasolv suppressed the α-MSH-stimulated increase of melanin content and resulted in the activation of autophagy. However, introduction of bafilomycin A1 strongly suppressed melanosome degradation in Melasolv-treated cells. Furthermore, inhibition of autophagy by ATG5 resulted in significant suppression of Melasolv-mediated depigmentation in α-MSH-treated cells. Taken together, our results suggest that treatment with Melasolv inhibits skin pigmentation by promoting melanosome degradation via autophagy activation.

Structural insights into the psychrophilic germinal protease PaGPR and its autoinhibitory loop.

In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202-216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation.

Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Transcriptional regulation of Salmochelin glucosyltransferase by Fur in Salmonella.

Pathogenic bacteria acquire the acquisition of iron from the host to ensure their survival. Salmonella spp. utilizes siderophores, including salmochelin, for high affinity aggressive import of iron. Although the iroBCDEN operon is reportedly responsible for the production and the transport of salmochelin, the molecular mechanisms underlying the regulation of its gene expression have not yet been characterized. Here, we analyzed the expression pattern of iroB using the lacZY transcriptional reporter system and determined the transcription start site in response to iron availability using primer extension analysis. We further examined the regulation of iroB expression by the ferric uptake regulator (Fur), a key regulatory protein involved in the maintenance of iron homeostasis in various bacteria, including Salmonella. Using sequence analysis followed by a gel shift assay, we verified that the Fur box lies within the promoter region of iroBCDE. The Fur box contained the consensus sequence (GATATTGGTAATTATTATC) and overlapped with the -10-element region. The expression of iroB was repressed by Fur in the presence of iron, as determined using an in vitro transcription assay. Therefore, we found that the iron acquisition system is regulated in a Fur-dependent manner in Salmonella.