Stealth Liposomes Encapsulating a Potent ACAT1/SOAT1 Inhibitor F12511: Pharmacokinetic, Biodistribution, and Toxicity Studies in Wild-Type Mice and Efficacy Studies in Triple Transgenic Alzheimer's Disease Mice.

Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer's Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.

Molecular genetic profiling reveals novel association between FLT3 mutation and survival in glioma.

INTRODUCTION: Recent molecular characterization of gliomas has uncovered somatic gene variation and DNA methylation changes that are associated with etiology, prognosis, and therapeutic response. Here we describe genomic profiling of gliomas assessed for associations between genetic mutations and patient outcomes, including overall survival (OS) and recurrence-free survival (RFS). METHODS: Mutations in a 50-gene cancer panel, 1p19q co-deletion, and MGMT promoter methylation (MGMT methylation) status were obtained from tumor tissue of 293 glioma patients. Multivariable regression models for overall survival (OS) and recurrence-free survival (RFS) were constructed for MGMT methylation, 1p19q co-deletion, and gene mutations controlling for age, treatment status, and WHO grade. RESULTS: Mutational profiles of gliomas significantly differed based on WHO Grade, such as high prevalence of BRAF V600E, IDH1, and PTEN mutations in WHO Grade I, II/III, and IV tumors, respectively. In multivariate regression analysis, MGMT methylation and IDH1 mutations were significantly associated with improved OS (HR = 0.44, p = 0.0004 and HR = 0.21, p = 0.007, respectively), while FLT3 and TP53 mutations were significantly associated with poorer OS (HR = 19.46, p < 0.0001 and HR = 1.67, p = 0.014, respectively). MGMT methylation and IDH1 mutations were the only significant alterations associated with improved RFS in the model (HR = 0.42, p < 0.0001 and HR = 0.37, p = 0.002, respectively). These factors were then included in a combined model, which significantly exceeded the predictive value of the base model alone (age, surgery, radiation, chemo, grade) (likelihood ratio test OS p = 1.64 × 10-8 and RFS p = 3.80 × 10-7). CONCLUSIONS: This study highlights the genomic landscape of gliomas in a single-institution cohort and identifies a novel association between FLT3 mutation and OS in gliomas.

Cervicothoracic epidural hematoma in a toddler with miosis, ptosis, nonspecific symptoms, and no history of major trauma: case report.

INTRODUCTION: Spinal epidural hematomas are uncommon in children. The diagnosis can be elusive as most cases present without a history of trauma, while symptoms can be atypical. CASE REPORT: We encountered a 35-month-old male presenting with nonspecific symptoms and no history of trauma. He later developed unilateral miosis and ptosis; MRI discovered a subacute cervicothoracic epidural which was promptly evacuated. The patient made an excellent recovery. COCLUSIONS: We emphasize the frequent absence of identifiable trauma and the importance of thorough imaging when this entity is suspected. Miosis and ptosis, likely representing a partial Horner syndrome, is an extremely rare presentation, this being one of the only reported cases.

Myeloid Acyl-CoA:Cholesterol Acyltransferase 1 Deficiency Reduces Lesion Macrophage Content and Suppresses Atherosclerosis Progression.

Acyl-CoA:cholesterol acyltransferase 1 (Acat1) converts cellular cholesterol to cholesteryl esters and is considered a drug target for treating atherosclerosis. However, in mouse models for atherosclerosis, global Acat1 knockout (Acat1(-/-)) did not prevent lesion development. Acat1(-/-) increased apoptosis within lesions and led to several additional undesirable phenotypes, including hair loss, dry eye, leukocytosis, xanthomatosis, and a reduced life span. To determine the roles of Acat1 in monocytes/macrophages in atherosclerosis, we produced a myeloid-specific Acat1 knockout (Acat1(-M/-M)) mouse and showed that, in the Apoe knockout (Apoe(-/-)) mouse model for atherosclerosis, Acat1(-M/-M) decreased the plaque area and reduced lesion size without causing leukocytosis, dry eye, hair loss, or a reduced life span. Acat1(-M/-M) enhanced xanthomatosis in apoe(-/-) mice, a skin disease that is not associated with diet-induced atherosclerosis in humans. Analyses of atherosclerotic lesions showed that Acat1(-M/-M) reduced macrophage numbers and diminished the cholesterol and cholesteryl ester load without causing detectable apoptotic cell death. Leukocyte migration analysis in vivo showed that Acat1(-M/-M) caused much fewer leukocytes to appear at the activated endothelium. Studies in inflammatory (Ly6C(hi)-positive) monocytes and in cultured macrophages showed that inhibiting ACAT1 by gene knockout or by pharmacological inhibition caused a significant decrease in integrin ß 1 (CD29) expression in activated monocytes/macrophages. The sparse presence of lesion macrophages without Acat1 can therefore, in part, be attributed to decreased interaction between inflammatory monocytes/macrophages lacking Acat1 and the activated endothelium. We conclude that targeting ACAT1 in a myeloid cell lineage suppresses atherosclerosis progression while avoiding many of the undesirable side effects caused by global Acat1 inhibition.

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.

Structural dynamics of microbial communities in polycyclic aromatic hydrocarbon-contaminated tropical estuarine sediments undergoing simulated aerobic biotreatment.

Coastal sediments contaminated by polycyclic aromatic hydrocarbons (PAHs) can be candidates for remediation via an approach like land farming. Land farming converts naturally anaerobic sediments to aerobic environments, and the response of microbial communities, in terms of community structure alterations and corresponding effects on biodegradative activities, is unknown. A key goal of this study was to determine if different sediments exhibited common patterns in microbial community responses that might serve as indicators of PAH biodegradation. Sediments from three stations in the Lagos Lagoon (Nigeria) were used in microcosms, which were spiked with a mixture of four PAH, then examined for PAH biodegradation and for shifts in microbial community structure by analysis of diversity in PAH degradation genes and Illumina sequencing of 16S rRNA genes. PAH biodegradation was similar in all sediments, yet each exhibited unique microbiological responses and there were no microbial indicators of PAH bioremediation common to all sediments.

Exacerbation of autoimmune neuroinflammation by dietary sodium is genetically controlled and sex specific.

Multiple sclerosis (MS) is a debilitating autoimmune neuroinflammatory disease influenced by genetics and the environment. MS incidence in female subjects has approximately tripled in the last century, suggesting a sex-specific environmental influence. Recent animal and human studies have implicated dietary sodium as a risk factor in MS, whereby high sodium augmented the generation of T helper (Th) 17 cells and exacerbated experimental autoimmune encephalomyelitis (EAE), the principal model of MS. However, whether dietary sodium interacts with sex or genetics remains unknown. Here, we show that high dietary sodium exacerbates EAE in a strain- and sex-specific fashion. In C57BL6/J mice, exposure to a high-salt diet exacerbated disease in both sexes, while in SJL/JCrHsd mice, it did so only in females. In further support of a genetic component, we found that sodium failed to modify EAE course in C57BL6/J mice carrying a 129/Sv-derived interval on chromosome 17. Furthermore, we found that the high-sodium diet did not augment Th17 or Th1 responses, but it did result in increased blood-brain barrier permeability and brain pathology. Our results demonstrate that the effects of dietary sodium on autoimmune neuroinflammation are sex specific, genetically controlled, and CNS mediated.

Early inflammatory mediator gene expression in two models of traumatic brain injury: ex vivo cortical slice in mice and in vivo cortical impact in piglets.

BACKGROUND: The immunological response during the first 24 hours after traumatic brain injury (TBI) may be a critical therapeutic interval for limiting the secondary neuronal damage that is influenced by enhanced inflammatory mediator expression. METHODS: To gain further insight of the early injury response, we examined the expression of several inflammatory genes by real-time qPCR as a function of time or distance from injury in two distinct mammalian models: an ex vivo mouse cortical slice injury system and an in vivo piglet model of brain injury. RESULTS: Interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), chemokine ligands 2 (CCL2), 3 (CCL3), 4 (CCL4), and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs increased within 5 h after injury in mouse cortical slices. Chemokine and PTGS2 mRNAs remained elevated in slices at 24 h, whereas IL-1ß and TNF-α expressions decreased from earlier peak levels. At 24 h after cortical injury in 1-month-old piglets, the expression of CCL2 mRNA was significantly increased in the lesion core and in the penumbra region. The expression of PTGS2, IL-1ß, and TNF-α was variable among the piglets. CONCLUSIONS: These in vitro and large animal models of cortical injury expand our understanding of the early timing and spread of the immunological response and can serve as preclinical systems to facilitate the discovery of therapeutic agents for TBI aimed at regulating inflammatory mediator expression.

Germline SMARCE1 mutations predispose to both spinal and cranial clear cell meningiomas.

We recently reported SMARCE1 mutations as a cause of spinal clear cell meningiomas. Here, we have identified five further cases with non-NF2 spinal meningiomas and six with non-NF2 cranial meningiomas. Three of the spinal cases and three of the cranial cases were clear cell tumours. We screened them for SMARCE1 mutations and investigated copy number changes in all point mutation-negative samples. We identified two novel mutations in individuals with spinal clear cell meningiomas and three mutations in individuals with cranial clear cell meningiomas. Copy number analysis identified a large deletion of the 5' end of SMARCE1 in two unrelated probands with spinal clear cell meningiomas. Testing of affected and unaffected relatives of one of these individuals identified the same deletion in two affected female siblings and their unaffected father, providing further evidence of incomplete penetrance of meningioma disease in males. In addition, we found loss of SMARCE1 protein in three of 10 paraffin-embedded cranial clear cell meningiomas. Together, these results demonstrate that loss of SMARCE1 is relevant to cranial as well as spinal meningiomas. Our study broadens the spectrum of mutations in the SMARCE1 gene and expands the phenotype to include cranial clear cell meningiomas.

Real world utilisation of doravirine among people living with human immunodeficiency virus in England (DRIVE-REAL).

BACKGROUND: Doravirine is a non-nucleoside reverse transcriptase inhibitor recommended for the treatment of virologically suppressed and treatment naïve people living with HIV. The DRIVE-REAL study aimed to describe the characteristics, treatment patterns, and virological outcomes of doravirine users in a real-world cohort in the UK. METHODS: A retrospective, observational, multi-centre chart review was conducted for 300 adults living with HIV initiating doravirine-containing antiretroviral therapy. RESULTS: At baseline 83% of individuals were male, 45% aged ≥50 years, 65% white ethnicity. Median time since HIV diagnosis was 12 years. 96% were antiretroviral therapy-experienced, 87% had a HIV viral load <50 copies/ml, and 15% had resistance to at least one antiretroviral drug. 66% had comorbidities, most commonly depression (26%), and 70% were taking at least one co-medication. At six months, 94% (n = 283/300) were still receiving doravirine. Viral load data were available for n = 266/300 individuals and 95% (n = 253/266) had viral load <50 copies/ml. CONCLUSIONS: Individuals initiating doravirine in this cohort are predominantly treatment-experienced white middle-aged males, with a high frequency of comorbidities and co-medication. The majority of individuals at 6 months remained on doravirine and maintained or achieved HIV viral suppression. This study provides epidemiologic characteristics that can inform clinical care and subsequent hypothesis-testing studies.

ACAT1 gene ablation increases 24(S)-hydroxycholesterol content in the brain and ameliorates amyloid pathology in mice with AD.

Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of amyloid-beta, one of the pathological hallmarks of Alzheimer disease (AD). Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) are two enzymes that convert free cholesterol to cholesteryl esters. ACAT inhibitors have recently emerged as promising drug candidates for AD therapy. However, how ACAT inhibitors act in the brain has so far remained unclear. Here we show that ACAT1 is the major functional isoenzyme in the mouse brain. ACAT1 gene ablation (A1-) in triple transgenic (i.e., 3XTg-AD) mice leads to more than 60% reduction in full-length human APPswe as well as its proteolytic fragments, and ameliorates cognitive deficits. At 4 months of age, A1- causes a 32% content increase in 24-hydroxycholesterol (24SOH), the major oxysterol in the brain. It also causes a 65% protein content decrease in HMG-CoA reductase (HMGR) and a 28% decrease in sterol synthesis rate in AD mouse brains. In hippocampal neurons, A1- causes an increase in the 24SOH synthesis rate; treating hippocampal neuronal cells with 24SOH causes rapid declines in hAPP and in HMGR protein levels. A model is provided to explain our findings: in neurons, A1- causes increases in cholesterol and 24SOH contents in the endoplasmic reticulum, which cause reductions in hAPP and HMGR protein contents and lead to amelioration of amyloid pathology. Our study supports the potential of ACAT1 as a therapeutic target for treating certain forms of AD.

Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria.

The phytopathogen Paracidovorax citrulli possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if P. citrulli formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of npdA orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by P. citrulli AAC00-1 was confirmed by gene deletion mutagenesis (ΔnpdA), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of npdA were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in P. citrulli and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the P. citrulli S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.

Strain-dependent variation in the early transcriptional response to CNS injury using a cortical explant system.

BACKGROUND: While it is clear that inbred strains of mice have variations in immunological responsiveness, the influence of genetic background following tissue damage in the central nervous system is not fully understood. A cortical explant system was employed as a model for injury to determine whether the immediate transcriptional response to tissue resection revealed differences among three mouse strains. METHODS: Immunological mRNAs were measured in cerebral cortex from SJL/J, C57BL/6J, and BALB/cJ mice using real time RT-PCR. Freshly isolated cortical tissue and cortical sections incubated in explant medium were examined. Levels of mRNA, normalized to ß-actin, were compared using one way analysis of variance with pooled samples from each mouse strain. RESULTS: In freshly isolated cerebral cortex, transcript levels of many pro-inflammatory mediators were not significantly different among the strains or too low for comparison. Constitutive, baseline amounts of CD74 and antisecretory factor (ASF) mRNAs, however, were higher in SJL/J and C57BL/6J, respectively. When sections of cortical tissue were incubated in explant medium, increased message for a number of pro-inflammatory cytokines and chemokines occurred within five hours. Message for chemokines, IL-1α, and COX-2 transcripts were higher in C57BL/6J cortical explants relative to SJL/J and BALB/cJ. IL-1ß, IL-12/23 p40, and TNF-α were lower in BALB/cJ explants relative to SJL/J and C57BL/6J. Similar to observations in freshly isolated cortex, CD74 mRNA remained higher in SJL/J explants. The ASF mRNA in SJL/J explants, however, was now lower than levels in both C57BL/6J and BALB/cJ explants. CONCLUSIONS: The short-term cortical explant model employed in this study provides a basic approach to evaluate an early transcriptional response to neurological damage, and can identify expression differences in genes that are influenced by genetic background.

A single nucleotide polymorphism in Tyk2 controls susceptibility to experimental allergic encephalomyelitis.

Genes controlling immunopathologic diseases of differing etiopathology may also influence susceptibility to autoimmune disease. B10.D1-H2(q)/SgJ mice with a 2538 G-->A missense mutation in the tyrosine kinase-2 gene (Tyk2) are susceptible to Toxoplasma gondii yet resistant to autoimmune arthritis, unlike the wild-type B10.Q/Ai substrain. To understand whether Tyk2 is also important in a second autoimmune model, experimental allergic encephalomyelitis (EAE) was induced in B10.D1-H2(q)/SgJ (Tyk2(A)) and B10.Q/Ai (Tyk2(G)) mice with the myelin oligodendrocyte glycoprotein peptide 79-96. B10.D1-H2(q)/SgJ mice were resistant to EAE whereas B10.Q/Ai mice were susceptible, and a single copy of the Tyk2(G) allele conferred EAE susceptibility in F(1) hybrids. Furthermore, EAE resistance in B10.D1-H2(q)/SgJ mice was overridden when pertussis toxin (PTX) was used to mimic the effects of environmental factors derived from infectious agents. Numerous cytokines and chemokines were increased when PTX was included in the immunization protocol. However, only RANTES, IL-6, and IFN-gamma increased significantly with both genetic compensation and PTX treatment. These data indicate that Tyk2 is a shared autoimmune disease susceptibility gene whose genetic contribution to disease susceptibility can be modified by environmental factors. Single nucleotide polymorphisms like the one that distinguishes Tyk2 alleles are of considerable significance given the potential role of gene-by-environment interactions in autoimmune disease susceptibility.

Plasma-borne indicators of inflammasome activity in Parkinson's disease patients.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms and loss of dopaminergic neurons of the substantia nigra. Inflammation and cell death are recognized aspects of PD suggesting that strategies to monitor and modify these processes may improve the management of the disease. Inflammasomes are pro-inflammatory intracellular pattern recognition complexes that couple these processes. The NLRP3 inflammasome responds to sterile triggers to initiate pro-inflammatory processes characterized by maturation of inflammatory cytokines, cytoplasmic membrane pore formation, vesicular shedding, and if unresolved, pyroptotic cell death. Histologic analysis of tissues from PD patients and individuals with nigral cell loss but no diagnosis of PD identified elevated expression of inflammasome-related proteins and activation-related "speck" formation in degenerating mesencephalic tissues compared with controls. Based on previous reports of circulating inflammasome proteins in patients suffering from heritable syndromes caused by hyper-activation of the NLRP3 inflammasome, we evaluated PD patient plasma for evidence of inflammasome activity. Multiple circulating inflammasome proteins were detected almost exclusively in extracellular vesicles indicative of ongoing inflammasome activation and pyroptosis. Analysis of plasma obtained from a multi-center cohort identified elevated plasma-borne NLRP3 associated with PD status. Our findings are consistent with others indicating inflammasome activity in neurodegenerative disorders. Findings suggest mesencephalic inflammasome protein expression as a histopathologic marker of early-stage nigral degeneration and suggest plasma-borne inflammasome-related proteins as a potentially useful class of biomarkers for patient stratification and the detection and monitoring of inflammation in PD.

Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis.

BACKGROUND: The role of neuroinflammation in motor neuron death of amyotrophic lateral sclerosis (ALS) is unclear. The human mutant superoxide dismutase-1 (hmSOD1)-expressing murine transgenic model of ALS has provided some insight into changes in microglia activity during disease progression. The purpose of this study was to gain further knowledge by characterizing the immunological changes during disease progression in the spinal cord and peripheral nerve using the more recently developed hmSOD1 rat transgenic model of ALS. METHODS: Using immunohistochemistry, the extent and intensity of tissue CD11b expression in spinal cord, lumbar nerve roots, and sciatic nerve were evaluated in hmSOD1 rats that were pre-clinical, at clinical onset, and near disease end-stage. Changes in CD11b expression were compared to the detection of MHC class II and CD68 microglial activation markers in the ventral horn of the spinal cord, as well as to the changes in astrocytic GFAP expression. RESULTS: Our study reveals an accumulation of microglia/macrophages both in the spinal cord and peripheral nerve prior to clinical onset based on CD11b tissue expression. The microglia formed focal aggregates in the ventral horn and became more widespread as the disease progressed. Hypertrophic astrocytes were not prominent in the ventral horn until after clinical onset, and the enhancement of GFAP did not have a strong correlation to increased CD11b expression. Detection of MHC class II and CD68 expression was found in the ventral horn only after clinical onset. The macrophages in the ventral nerve root and sciatic nerve of hmSOD1 rats were observed encircling axons. CONCLUSIONS: These findings describe for the first time in the hmSOD1 rat transgenic model of ALS that enhancement of microglia/macrophage activity occurs pre-clinically both in the peripheral nerve and in the spinal cord. CD11b expression is shown to be a superior indicator for early immunological changes compared to other microglia activation markers and astrogliosis. Furthermore, we suggest that the early activity of microglia/macrophages is involved in the early phase of motor neuron degeneration and propose that studies involving immunomodulation in hmSOD1transgenic models need to consider effects on macrophages in peripheral nerves as well as to microglia in the spinal cord.

Molecular analysis of two bacterioferritin genes, bfralpha and bfrbeta, in the model rhizobacterium Pseudomonas putida KT2440.

The model rhizobacterium Pseudomonas putida KT2440 and other fluorescent pseudomonads possess two bacterioferritins, Bfralpha and Bfrbeta. However, the regulatory systems controlling the expression of these genes and the roles of these proteins in iron homeostasis are ill defined. Our studies show that both bfralpha and bfrbeta were monocistronic: promoter motifs and transcriptional start sites were identified, and Fur boxes and sigma(S)-dependent regulatory motifs were absent. The expressions of bfralpha and bfrbeta were enhanced by iron exposure and were maximal in cells rapidly growing in a high-iron environment. Both bfralpha and bfrbeta were positively regulated by Fur, and both were expressed independently of adjoining, functionally related genes. The loss of Bfralpha or Bfrbeta individually resulted in a significant reduction (ca. 17%) in cellular iron levels, and the deletion of both bfralpha and bfrbeta reduced cellular iron levels by 38% relative to those of the wild type. The mutants varied in their abilities to grow in low-iron medium; while growths (rate and final cell density) of single mutants and the wild type were similar, that of the double mutant was reduced significantly. Mutants lacking Bfralpha and/or Bfrbeta showed no change relative to the wild type in sensitivity to reactive oxygen species toxicity. Collectively, the data show that while Bfralpha and Bfrbeta could function independently of each other, an interaction-dependent function cannot be ruled out. Furthermore, regardless of the mechanism, a primary benefit of the bacterioferritins to P. putida KT2440 appears to be the enhancement of its survival in the environment by strengthening its tolerance to iron starvation.

Simultaneous analysis of bacterioferritin gene expression and intracellular iron status in Pseudomonas putida KT2440 by using a rapid dual luciferase reporter assay.

A dual luciferase reporter (DLR) system utilizing firefly and Renilla luciferases was developed and tested in a model rhizobacterium, Pseudomonas putida KT2440. The DLR was applied to simultaneously analyze expression of three putative bacterioferritin genes (bfralpha, bfrbeta, and bfr) and assess the cellular iron status of strain KT2440 by monitoring expression of the Fur-regulated fepA-fes promoter. The DLR proved to be reproducible and sensitive. Expression of bfralpha (PP0482) and bfrbeta (PP1082) was consistent with expectations for bacterioferritin and varied directly with the iron level. However, expression of bfr (PP4856) was inversely related to the iron concentration and it was thus more likely to encode a Dps-like protein rather than a bacterioferritin.

Third ventricle World Health Organization Grade II meningioma presenting with intraventricular hemorrhage and obstructive hydrocephalus: A case report and literature review.

BACKGROUND: Third ventricular meningiomas are exceedingly rare intracranial tumors that may present with intraventricular hemorrhage. CASE DESCRIPTION: The patient is 46-year-old who initially presented with obstructive hydrocephalus from a presumed vascular lesion and who was treated with endoscopic third ventriculostomy. He presented 3 years later with acute intraventricular hemorrhage and hydrocephalus. The hemorrhage was evacuated and the third ventricular tumor was resected, and the patient made an excellent recovery. Histopathological analysis identified the tumor as the World Health Organization Grade II meningioma. CONCLUSION: Third ventricular meningioma is a rare tumor that may present with hemorrhage and obstructive hydrocephalus. Surgical resection can be helpful for this rare presentation of intracranial meningioma.